@article{LiuFriedrichHemmenetal.2023,
  author    = {Liu, Ruiqi and Friedrich, Mike and Hemmen, Katherina and Jansen, Kerstin and Adolfi, Mateus C. and Schartl, Manfred and Heinze, Katrin G.},
  title     = {Dimerization of melanocortin 4 receptor controls puberty onset and body size polymorphism},
  series = {Frontiers in Endocrinology},
  volume    = {14},
  journal   = {Frontiers in Endocrinology},
  issn      = {1664-2392},
  doi       = {10.3389/fendo.2023.1267590},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-354261},
  year      = {2023},
  abstract  = {Xiphophorus fish exhibit a clear phenotypic polymorphism in puberty onset and reproductive strategies of males. In X. nigrensis and X. multilineatus, puberty onset is genetically determined and linked to a melanocortin 4 receptor (Mc4r) polymorphism of wild-type and mutant alleles on the sex chromosomes. We hypothesized that Mc4r mutant alleles act on wild-type alleles by a dominant negative effect through receptor dimerization, leading to differential intracellular signaling and effector gene activation. Depending on signaling strength, the onset of puberty either occurs early or is delayed. Here, we show by F{\"o}rster Resonance Energy Transfer (FRET) that wild-type Xiphophorus Mc4r monomers can form homodimers, but also heterodimers with mutant receptors resulting in compromised signaling which explains the reduced Mc4r signaling in large males. Thus, hetero- vs. homo- dimerization seems to be the key molecular mechanism for the polymorphism in puberty onset and body size in male fish.},
  language  = {en}
}
@article{KressJessenHufnageletal.2023,
  author    = {Kreß, Julia Katharina Charlotte and Jessen, Christina and Hufnagel, Anita and Schmitz, Werner and Da Xavier Silva, Thamara Nishida and Ferreira Dos Santos, Anc{\´e}ly and Mosteo, Laura and Goding, Colin R. and Friedmann Angeli, Jos{\´e} Pedro and Meierjohann, Svenja},
  title     = {The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2},
  series = {Cell Reports},
  volume    = {42},
  journal   = {Cell Reports},
  number    = {7},
  doi       = {10.1016/j.celrep.2023.112724},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350312},
  year      = {2023},
  abstract  = {Highlights • The integrated stress response leads to a general ATF4-dependent activation of NRF2 • ATF4 causes a CHAC1-dependent GSH depletion, resulting in NRF2 stabilization • An elevation of NRF2 transcript levels fosters this effect • NRF2 supports the ISR/ATF4 pathway by improving cystine and antioxidant supply Summary The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.},
  language  = {en}
}
@article{TessmerMargison2023,
  author    = {Tessmer, Ingrid and Margison, Geoffrey P.},
  title     = {The DNA alkyltransferase family of DNA repair proteins: common mechanisms, diverse functions},
  series = {International Journal of Molecular Sciences},
  volume    = {25},
  journal   = {International Journal of Molecular Sciences},
  number    = {1},
  issn      = {1422-0067},
  doi       = {10.3390/ijms25010463},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-355790},
  year      = {2023},
  abstract  = {DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O\(^6\)-alkylguanine and O\(^4\)-alkylthymine bases in DNA. Their mechanism involves binding to the damaged DNA and flipping the base out of the DNA helix into the active site pocket in the protein. Alkyltransferases then directly and irreversibly transfer the alkyl group from the base to the active site cysteine residue. In contrast, alkyltransferase-like proteins recruit nucleotide excision repair components for O\(^6\)-alkylguanine elimination. One or more of these proteins are found in all kingdoms of life, and where this has been determined, their overall DNA repair mechanism is strictly conserved between organisms. Nevertheless, between species, subtle as well as more extensive differences that affect target lesion preferences and/or introduce additional protein functions have evolved. Examining these differences and their functional consequences is intricately entwined with understanding the details of their DNA repair mechanism(s) and their biological roles. In this review, we will present and discuss various aspects of the current status of knowledge on this intriguing protein family.},
  language  = {en}
}
@article{vanUnenStumpfSchmidetal.2016,
  author    = {van Unen, Jakobus and Stumpf, Anette D. and Schmid, Benedikt and Reinhard, Nathalie R. and Hordijk, Peter L. and Hoffmann, Carsten and Gadella, Theodorus W. J. and Goedhart, Joachim},
  title     = {A New Generation of FRET Sensors for Robust Measurement of Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) Activation Kinetics in Single Cells},
  series = {PLoS ONE},
  volume    = {11},
  journal   = {PLoS ONE},
  number    = {1},
  doi       = {10.1371/journal.pone.0146789},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167387},
  pages     = {e0146789},
  year      = {2016},
  abstract  = {G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on F{\"o}rster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα\(_{i}\) subunit, and cp173Venus fused to the Gγ\(_{2}\) subunit as acceptor. The Gα\(_{i}\) FRET biosensors constructs are expressed together with Gβ\(_{1}\) from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα\(_{i}\) FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα\(_{i}\) FRET sensor in single cells upon stimulation of several GPCRs, including the LPA\(_{2}\), M\(_{3}\) and BK\(_{2}\) receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation will be valuable for live-cell measurements that probe Gα\(_{i}\) activation.},
  language  = {en}
}
@article{LiDengXieetal.2018,
  author    = {Li, Cong and Deng, Xiaobing and Xie, Xiaowen and Liu, Ying and Friedmann Angeli, Jos{\´e} Pedro and Lai, Luhua},
  title     = {Activation of Glutathione Peroxidase 4 as a Novel Anti-inflammatory Strategy},
  series = {Frontiers in Pharmacology},
  volume    = {9},
  journal   = {Frontiers in Pharmacology},
  number    = {1120},
  issn      = {1663-9812},
  doi       = {10.3389/fphar.2018.01120},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195985},
  year      = {2018},
  abstract  = {The anti-oxidative enzyme, glutathione peroxidase 4 (GPX4), helps to promote inflammation resolution by eliminating oxidative species produced by the arachidonic acid (AA) metabolic network. Up-regulating its activity has been proposed as a promising strategy for inflammation intervention. In the present study, we aimed to study the effect of GPX4 activator on the AA metabolic network and inflammation related pathways. Using combined computational and experimental screen, we identified a novel compound that can activate the enzyme activity of GPX4 by more than two folds. We further assessed its potential in a series of cellular assays where GPX4 was demonstrated to play a regulatory role. We are able to show that GPX4 activation suppressed inflammatory conditions such as oxidation of AA and NF-κB pathway activation. We further demonstrated that this GPX4 activator can decrease the intracellular ROS level and suppress ferroptosis. Our study suggests that GPX4 activators can be developed as anti-inflammatory or cyto-protective agent in lipid-peroxidation-mediated diseases.},
  language  = {en}
}
@article{KasaragodSchindelin2019,
  author    = {Kasaragod, Vikram Babu and Schindelin, Hermann},
  title     = {Structure of Heteropentameric GABAA Receptors and Receptor-Anchoring Properties of Gephyrin},
  series = {Frontiers in Molecular Neuroscience},
  volume    = {12},
  journal   = {Frontiers in Molecular Neuroscience},
  issn      = {1662-5099},
  doi       = {10.3389/fnmol.2019.00191},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189308},
  pages     = {191},
  year      = {2019},
  abstract  = {γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3-M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.},
  language  = {en}
}
@article{KasaragodSchindelin2019,
  author    = {Kasaragod, Vikram Babu and Schindelin, Hermann},
  title     = {Structure of heteropentameric GABA\(_A\) receptors and receptor-anchoring properties of gephyrin},
  series = {Frontiers in Molecular Neuroscience},
  volume    = {12},
  journal   = {Frontiers in Molecular Neuroscience},
  number    = {191},
  doi       = {10.3389/fnmol.2019.00191},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201886},
  year      = {2019},
  abstract  = {γ-Aminobutyric acid type A receptors (GABA\(_A\)Rs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABA\(_A\)Rs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABA\(_A\)Rs are key drug targets. The majority of synaptic GABA\(_A\)Rs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3-M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABA\(_A\)R provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABA\(_A\)Rs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABA\(_A\)Rs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.},
  language  = {en}
}
@article{LorenzinBenaryBaluapurietal.2016,
  author    = {Lorenzin, Francesca and Benary, Uwe and Baluapuri, Apoorva and Walz, Susanne and Jung, Lisa Anna and von Eyss, Bj{\"o}rn and Kisker, Caroline and Wolf, Jana and Eilers, Martin and Wolf, Elmar},
  title     = {Different promoter affinities account for specificity in MYC-dependent gene regulation},
  series = {eLife},
  volume    = {5},
  journal   = {eLife},
  doi       = {10.7554/eLife.15161},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162913},
  pages     = {e15161},
  year      = {2016},
  abstract  = {Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells.},
  language  = {en}
}
@article{BangaloreHeilMehringeretal.2020,
  author    = {Bangalore, Disha M. and Heil, Hannah S. and Mehringer, Christian F. and Hirsch, Lisa and Hemmen, Katharina and Heinze, Katrin G. and Tessmer, Ingrid},
  title     = {Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases},
  series = {Scientific Reports},
  volume    = {10},
  journal   = {Scientific Reports},
  doi       = {10.1038/s41598-020-72102-7},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231338},
  year      = {2020},
  abstract  = {Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.},
  language  = {en}
}
@article{FazeliBeerGeisenhofetal.2020,
  author    = {Fazeli, Gholamreza and Beer, Katharina B. and Geisenhof, Michaela and Tr{\"o}ger, Sarah and K{\"o}nig, Julia and M{\"u}ller-Reichert, Thomas and Wehman, Ann M.},
  title     = {Loss of the Major Phosphatidylserine or Phosphatidylethanolamine Flippases Differentially Affect Phagocytosis},
  series = {Frontiers in Cell and Developmental Biology},
  volume    = {8},
  journal   = {Frontiers in Cell and Developmental Biology},
  issn      = {2296-634X},
  doi       = {10.3389/fcell.2020.00648},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208771},
  year      = {2020},
  abstract  = {The lipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEth) are normally asymmetrically localized to the cytosolic face of membrane bilayers, but can both be externalized during diverse biological processes, including cell division, cell fusion, and cell death. Externalized lipids in the plasma membrane are recognized by lipid-binding proteins to regulate the clearance of cell corpses and other cell debris. However, it is unclear whether PtdSer and PtdEth contribute in similar or distinct ways to these processes. We discovered that disruption of the lipid flippases that maintain PtdSer or PtdEth asymmetry in the plasma membrane have opposite effects on phagocytosis in Caenorhabditis elegans embryos. Constitutive PtdSer externalization caused by disruption of the major PtdSer flippase TAT-1 led to increased phagocytosis of cell debris, sometimes leading to two cells engulfing the same debris. In contrast, PtdEth externalization caused by depletion of the major PtdEth flippase TAT-5 or its activator PAD-1 disrupted phagocytosis. These data suggest that PtdSer and PtdEth externalization have opposite effects on phagocytosis. Furthermore, externalizing PtdEth is associated with increased extracellular vesicle release, and we present evidence that the extent of extracellular vesicle accumulation correlates with the extent of phagocytic defects. Thus, a general loss of lipid asymmetry can have opposing impacts through different lipid subtypes simultaneously exerting disparate effects.},
  language  = {en}
}
@article{SchulteSoldaSpaenigetal.2022,
  author    = {Schulte, Clemens and Sold{\`a}, Alice and Sp{\"a}nig, Sebastian and Adams, Nathan and Bekić, Ivana and Streicher, Werner and Heider, Dominik and Strasser, Ralf and Maric, Hans Michael},
  title     = {Multivalent binding kinetics resolved by fluorescence proximity sensing},
  series = {Communications Biology},
  volume    = {5},
  journal   = {Communications Biology},
  doi       = {10.1038/s42003-022-03997-3},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301157},
  year      = {2022},
  abstract  = {Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.},
  language  = {en}
}
@article{MielichSuessWagnerMietrachetal.2017,
  author    = {Mielich-S{\"u}ss, Benjamin and Wagner, Rabea M. and Mietrach, Nicole and Hertlein, Tobias and Marincola, Gabriella and Ohlsen, Knut and Geibel, Sebastian and Lopez, Daniel},
  title     = {Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus},
  series = {PLoS Pathogens},
  volume    = {13},
  journal   = {PLoS Pathogens},
  number    = {11},
  doi       = {10.1371/journal.ppat.1006728},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170035},
  pages     = {e1006728},
  year      = {2017},
  abstract  = {Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen.},
  language  = {en}
}
@article{FischerHarrisonRamirezetal.2017,
  author    = {Fischer, Annette and Harrison, Kelly S and Ramirez, Yesid and Auer, Daniela and Chowdhury, Suvagata Roy and Prusty, Bhupesh K and Sauer, Florian and Dimond, Zoe and Kisker, Caroline and Hefty, P Scott and Rudel, Thomas},
  title     = {Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense},
  series = {eLife},
  volume    = {6},
  journal   = {eLife},
  number    = {e21465},
  doi       = {10.7554/eLife.21465},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171073},
  year      = {2017},
  abstract  = {Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.},
  language  = {en}
}
@article{GodboleLygaLohseetal.2017,
  author    = {Godbole, Amod and Lyga, Sandra and Lohse, Martin J. and Calebiro, Davide},
  title     = {Internalized TSH receptors en route to the TGN induce local G\(_{S}\)-protein signaling and gene transcription},
  series = {Nature Communications},
  volume    = {8},
  journal   = {Nature Communications},
  number    = {443},
  doi       = {10.1038/s41467-017-00357-2},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170375},
  year      = {2017},
  abstract  = {A new paradigm of G-protein-coupled receptor (GPCR) signaling at intracellular sites has recently emerged, but the underlying mechanisms and functional consequences are insufficiently understood. Here, we show that upon internalization in thyroid cells, endogenous TSH receptors traffic retrogradely to the trans-Golgi network (TGN) and activate endogenous Gs-proteins in the retromer-coated compartment that brings them to the TGN. Receptor internalization is associated with a late cAMP/protein kinase A (PKA) response at the Golgi/TGN. Blocking receptor internalization, inhibiting PKA II/interfering with its Golgi/TGN localization, silencing retromer or disrupting Golgi/TGN organization all impair efficient TSH-dependent cAMP response element binding protein (CREB) phosphorylation. These results suggest that retrograde trafficking to the TGN induces local G\(_{S}\)-protein activation and cAMP/PKA signaling at a critical position near the nucleus, which appears required for efficient CREB phosphorylation and gene transcription. This provides a new mechanism to explain the functional consequences of GPCR signaling at intracellular sites and reveals a critical role for the TGN in GPCR signaling.},
  language  = {en}
}
@article{OehlerMohammadiPerpinaVicianoetal.2017,
  author    = {Oehler, Beatrice and Mohammadi, Milad and Perpina Viciano, Cristina and Hackel, Dagmar and Hoffmann, Carsten and Brack, Alexander and Rittner, Heike L.},
  title     = {Peripheral interaction of Resolvin D1 and E1 with opioid receptor antagonists for antinociception in inflammatory pain in rats},
  series = {Frontiers in Molecular Neuroscience},
  volume    = {10},
  journal   = {Frontiers in Molecular Neuroscience},
  number    = {242},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158642},
  year      = {2017},
  abstract  = {Antinociceptive pathways are activated in the periphery in inflammatory pain, for instance resolvins and opioid peptides. Resolvins are biosynthesized from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Resolvin D1 (RvD1) and resolvin E1 (RvE1) initiate the resolution of inflammation and control of hypersensitivity via induction of anti-inflammatory signaling cascades. RvD1 binds to lipoxin A4/annexin-A1 receptor/formyl-peptide receptor 2 (ALX/FPR2), RvE1 to chemerin receptor 23 (ChemR23). Antinociception of RvD1 is mediated by interaction with transient receptor potential channels ankyrin 1 (TRPA1). Endogenous opioid peptides are synthesized and released from leukocytes in the tissue and bind to opioid receptors on nociceptor terminals. Here, we further explored peripheral mechanisms of RvD1 and chemerin (Chem), the ligand of ChemR23, in complete Freund's adjuvant (CFA)-induced hindpaw inflammation in male Wistar rats. RvD1 and Chem ameliorated CFA-induced hypersensitivity in early and late inflammatory phases. This was prevented by peripheral blockade of the μ-opioid peptide receptor (MOR) using low dose local naloxone or by local injection of anti-β-endorphin and anti-met-enkephalin (anti-ENK) antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not stimulate the release of β-endorphin from macrophages and neutrophils, nor did RvD1 itself activate G-proteins coupled MOR or initiate β-arrestin recruitment to the membrane. TRPA1 blockade by HC-030031 in inflammation in vivo as well as inhibition of the TRPA1-mediated calcium influx in dorsal root ganglia neurons in vitro was hampered by naloxone. Peripheral application of naloxone alone in vivo already lowered mechanical nociceptive thresholds. Therefore, either a perturbation of the balance of endogenous pro- and antinociceptive mechanisms in early and late inflammation, or an interaction of TRPA1 and opioid receptors weaken the antinociceptive potency of RvD1 and TRPA1 blockers.},
  language  = {en}
}
@article{KoelmelKuperKisker2021,
  author    = {Koelmel, Wolfgang and Kuper, Jochen and Kisker, Caroline},
  title     = {Cesium based phasing of macromolecules: a general easy to use approach for solving the phase problem},
  series = {Scientific Reports},
  volume    = {11},
  journal   = {Scientific Reports},
  number    = {1},
  doi       = {10.1038/s41598-021-95186-1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261644},
  pages     = {17038},
  year      = {2021},
  abstract  = {Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a "quick-soak"-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy.},
  language  = {en}
}
@article{BatschingWolfHeisenberg2016,
  author    = {Batsching, Sophie and Wolf, Reinhard and Heisenberg, Martin},
  title     = {Inescapable Stress Changes Walking Behavior in Flies - Learned Helplessness Revisited},
  series = {PLoS ONE},
  volume    = {11},
  journal   = {PLoS ONE},
  number    = {11},
  doi       = {10.1371/journal.pone.0167066},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178640},
  year      = {2016},
  abstract  = {Like other animals flies develop a state of learned helplessness in response to unescapable aversive events. To show this, two flies, one 'master', one 'yoked', are each confined to a dark, small chamber and exposed to the same sequence of mild electric shocks. Both receive these shocks when the master fly stops walking for more than a second. Behavior in the two animals is differently affected by the shocks. Yoked flies are transiently impaired in place learning and take longer than master flies to exit from the chamber towards light. After the treatment they walk more slowly and take fewer and shorter walking bouts. The low activity is attributed to the fly's experience that its escape response, an innate behavior to terminate the electric shocks, does not help anymore. Earlier studies using heat pulses instead of electric shocks had shown similar effects. This parallel supports the interpretation that it is the uncontrollability that induces the state.},
  language  = {en}
}
@article{MaurerHuppBischoffetal.2017,
  author    = {Maurer, Jana and Hupp, Sabrina and Bischoff, Carolin and Foertsch, Christina and Mitchell, Timothy J. and Chakraborty, Trinad and Iliev, Asparouh I.},
  title     = {Distinct neurotoxicity profile of listeriolysin O from \(Listeria\) \(monocytogenes\)},
  series = {Toxins},
  volume    = {9},
  journal   = {Toxins},
  number    = {1},
  doi       = {10.3390/toxins9010034},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172130},
  year      = {2017},
  abstract  = {Cholesterol-dependent cytolysins (CDCs) are protein toxins that originate from Gram-positive bacteria and contribute substantially to their pathogenicity. CDCs bind membrane cholesterol and build prepores and lytic pores. Some effects of the toxins are observed in non-lytic concentrations. Two pathogens, \(Streptococcus\) \(pneumoniae\) and \(Listeria\) \(monocytogenes\), cause fatal bacterial meningitis, and both produce toxins of the CDC family—pneumolysin and listeriolysin O, respectively. It has been demonstrated that pneumolysin produces dendritic varicosities (dendrite swellings) and dendritic spine collapse in the mouse neocortex, followed by synaptic loss and astrocyte cell shape remodeling without elevated cell death. We utilized primary glial cultures and acute mouse brain slices to examine the neuropathological effects of listeriolysin O and to compare it to pneumolysin with identical hemolytic activity. In cultures, listeriolysin O permeabilized cells slower than pneumolysin did but still initiated non-lytic astrocytic cell shape changes, just as pneumolysin did. In an acute brain slice culture system, listeriolysin O produced dendritic varicosities in an NMDA-dependent manner but failed to cause dendritic spine collapse and cortical astrocyte reorganization. Thus, listeriolysin O demonstrated slower cell permeabilization and milder glial cell remodeling ability than did pneumolysin and lacked dendritic spine collapse capacity but exhibited equivalent dendritic pathology.},
  language  = {en}
}
@article{CaiElMerahbiLoeffleretal.2017,
  author    = {Cai, Kai and El-Merahbi, Rabih and Loeffler, Mona and Mayer, Alexander E. and Sumara, Grzegorz},
  title     = {Ndrg1 promotes adipocyte differentiation and sustains their function},
  series = {Scientific Reports},
  volume    = {7},
  journal   = {Scientific Reports},
  number    = {7191},
  doi       = {10.1038/s41598-017-07497-x},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170565},
  year      = {2017},
  abstract  = {Adipocytes play a central role in maintaining metabolic homeostasis in the body. Differentiation of adipocyte precursor cells requires the transcriptional activity of peroxisome proliferator-activated receptor-γ (Pparγ) and CCAAT/enhancer binding proteins (C/Ebps). Transcriptional activity is regulated by signaling modules activated by a plethora of hormones and nutrients. Mechanistic target of rapamacin complexes (mTORC) 1 and 2 are central for the coordination of hormonal and nutritional inputs in cells and are essential for adipogenesis. Serum glucocorticoid kinase 1 (Sgk1)-dependent phosphorylation of N-Myc downstream-regulated gene 1 (Ndrg1) is a hallmark of mTORC2 activation in cells. Moreover, Pparγ activation promotes Ndrg1 expression. However, the impact of Ndrg1 on adipocyte differentiation and function has not yet been defined. Here, we show that Ndrg1 expression and its Sgk1-dependent phosphorylation are induced during adipogenesis. Consistently, we demonstrate that Ndrg1 promotes adipocyte differentiation and function by inducing Pparγ expression. Additionally, our results indicate that Ndrg1 is required for C/Ebpα phosphorylation. Moreover, we found that Ndrg1 phosphorylation by Sgk1 promotes adipocyte formation. Taken together, we show that induction of Ndrg1 expression by Pparγ and its phosphorylation by Sgk1 kinase are required for the acquisition of adipocyte characteristics by precursor cells.},
  language  = {en}
}
@phdthesis{Schreiber2018,
  author    = {Schreiber, Benjamin},
  title     = {Selective and enhanced fluorescence by biocompatible nanocoatings to monitor G-protein-coupled receptor dynamics},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173923},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Fluorescence microscopy has become one of the most important techniques for the imaging of biological cells and tissue, since the technique allows for selective labeling with fluorescent molecules and is highly suitable for low-light applications down to the single molecule regime. The methodological requirements are well-defined for studying membrane receptors within a highly localized nanometer-thin membrane. For example, G-protein-coupled receptors (GPCRs) are an extensively studied class of membrane receptors that represent one of the most important pharmaceutical targets. Ligand binding and GPCR activation dynamics are suspected to take place at the millisecond scale and may even be far faster. Thus, techniques that are fast, selective, and live-cell compatible are required to monitor GPCR dynamics. Fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRF-M) are methods of choice to monitor the dynamics of GPCRs selectively within the cell membrane. Despite the remarkable success of these modalities, there are limitations. Most importantly, inhomogeneous illumination can induce imaging artifacts, rendering spectroscopic evaluation difficult. Background signal due to scattering processes or imperfect labeling can hamper the signal-to-noise, thus limiting image contrast and acquisition speed. Careful consideration of the internal physiology is required for FRET sensor design, so that ligand binding and cell compatibility are well-preserved despite the fluorescence labeling procedures. This limitation of labeling positions leads to very low signal changes in FRET-based GPCR analysis. In addition, microscopy of these systems becomes even more challenging in single molecule or low-light applications where the accuracy and temporal resolution may become dramatically low. Fluorescent labels should therefore be brighter, protected from photobleaching, and as small as possible to avoid interference with the binding kinetics. The development of new fluorescent molecules and labeling methods is an ongoing process. However, a complete characterization of new labels and sensors takes time. So far, the perfect dye system for GPCR studies has not been found, even though there is high demand. Thus, this thesis explores and applies a different approach based on improved illumination schemes for TIRF-M as well as metal-coated coverslips to enhance fluorescence and FRET efficiency. First, it is demonstrated that a 360° illumination scheme reduces typical TIRF artifacts and produces a much more homogenously illuminated field of view. Second, membrane imaging and FRET spectroscopy are improved by metal coatings that are used to modulate the fluorescent properties of common fluorescent dyes. Computer simulation methods are used to understand the underlying photophysics and to design the coatings. Third, this thesis explores the operational regime and limitations of plasmonic approaches with high sectioning capabilities. The findings are summarized by three publications that are presented in the results section of this work. In addition, the theory of fluorescence and FRET is explained, with particular attention to its emission modulations in the vicinity of metal-dielectric layers. Details of the instrumentation, computer simulations, and cell culture are described in the method section. The work concludes with a discussion of the findings within the framework of recent technological developments as well as perspectives and suggestions for future approaches complete the presented work.},
  subject      = {G-Protein gekoppelte Rezeptoren},
  language  = {en}
}
@article{WeiderWegenerSchmittetal.2015,
  author    = {Weider, Matthias and Wegener, Am{\´e}lie and Schmitt, Christian and K{\"u}spert, Melanie and Hillg{\"a}rtner, Simone and B{\"o}sl, Michael R. and Hermans-Borgmeyer, Irm and Nait-Oumesmar, Brahim and Wegner, Michael},
  title     = {Elevated in vivo levels of a single transcription factor directly convert satellite glia into oligodendrocyte-like cells},
  series = {PLoS Genetics},
  volume    = {11},
  journal   = {PLoS Genetics},
  number    = {2},
  doi       = {10.1371/journal.pgen.1005008},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144123},
  pages     = {e1005008},
  year      = {2015},
  abstract  = {Oligodendrocytes are the myelinating glia of the central nervous system and ensure rapid saltatory conduction. Shortage or loss of these cells leads to severe malfunctions as observed in human leukodystrophies and multiple sclerosis, and their replenishment by reprogramming or cell conversion strategies is an important research aim. Using a transgenic approach we increased levels of the transcription factor Sox10 throughout the mouse embryo and thereby prompted Fabp7-positive glial cells in dorsal root ganglia of the peripheral nervous system to convert into cells with oligodendrocyte characteristics including myelin gene expression. These rarely studied and poorly characterized satellite glia did not go through a classic oligodendrocyte precursor cell stage. Instead, Sox10 directly induced key elements of the regulatory network of differentiating oligodendrocytes, including Olig2, Olig1, Nkx2.2 and Myrf. An upstream enhancer mediated the direct induction of the Olig2 gene. Unlike Sox10, Olig2 was not capable of generating oligodendrocyte-like cells in dorsal root ganglia. Our findings provide proof-of-concept that Sox10 can convert conducive cells into oligodendrocyte-like cells in vivo and delineates options for future therapeutic strategies.},
  language  = {en}
}
@article{BuechnerMaitiDrohatetal.2015,
  author    = {Buechner, Claudia N. and Maiti, Atanu and Drohat, Alexander C. and Tessmer, Ingrid},
  title     = {Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging},
  series = {Nucleic Acids Research},
  volume    = {43},
  journal   = {Nucleic Acids Research},
  number    = {5},
  doi       = {10.1093/nar/gkv139},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148795},
  pages     = {2716-2729},
  year      = {2015},
  abstract  = {The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use singlemolecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG-DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases.},
  language  = {en}
}
@article{ChilloKleinertLautzetal.2016,
  author    = {Chillo, Omary and Kleinert, Eike Christian and Lautz, Thomas and Lasch, Manuel and Pagel, Judith-Irina and Heun, Yvonn and Troidl, Kerstin and Fischer, Silvia and Caballero-Martinez, Amelia and Mauer, Annika and Kurz, Angela R. M. and Assmann, Gerald and Rehberg, Markus and Kanse, Sandip M. and Nieswandt, Bernhard and Walzog, Barbara and Reichel, Christoph A. and Mannell, Hanna and Preissner, Klaus T. and Deindl, Elisabeth},
  title     = {Perivascular Mast Cells Govern Shear Stress-Induced Arteriogenesis by Orchestrating Leukocyte Function},
  series = {Cell Reports},
  volume    = {16},
  journal   = {Cell Reports},
  number    = {8},
  doi       = {10.1016/j.celrep.2016.07.040},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164800},
  pages     = {2197-2207},
  year      = {2016},
  abstract  = {The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit+/CXCR-4+ cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases.},
  language  = {en}
}
@article{KoenigWolfHeisenberg2016,
  author    = {Koenig, Sebastian and Wolf, Reinhard and Heisenberg, Martin},
  title     = {Visual Attention in Flies-Dopamine in the Mushroom Bodies Mediates the After-Effect of Cueing},
  series = {PLoS ONE},
  volume    = {11},
  journal   = {PLoS ONE},
  number    = {8},
  doi       = {10.1371/journal.pone.0161412},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179564},
  year      = {2016},
  abstract  = {Visual environments may simultaneously comprise stimuli of different significance. Often such stimuli require incompatible responses. Selective visual attention allows an animal to respond exclusively to the stimuli at a certain location in the visual field. In the process of establishing its focus of attention the animal can be influenced by external cues. Here we characterize the behavioral properties and neural mechanism of cueing in the fly Drosophila melanogaster. A cue can be attractive, repulsive or ineffective depending upon (e.g.) its visual properties and location in the visual field. Dopamine signaling in the brain is required to maintain the effect of cueing once the cue has disappeared. Raising or lowering dopamine at the synapse abolishes this after-effect. Specifically, dopamine is necessary and sufficient in the αβ-lobes of the mushroom bodies. Evidence is provided for an involvement of the αβ\(_{posterior}\) Kenyon cells.},
  language  = {en}
}
@article{StrittNurdenFavieretal.2016,
  author    = {Stritt, Simon and Nurden, Paquita and Favier, Remi and Favier, Marie and Ferioli, Silvia and Gotru, Sanjeev K. and van Eeuwijk, Judith M.M. and Schulze, Harald and Nurden, Alan T. and Lambert, Michele P. and Turro, Ernest and Burger-Stritt, Stephanie and Matsushita, Masayuki and Mittermeier, Lorenz and Ballerini, Paola and Zierler, Susanna and Laffan, Michael A. and Chubanov, Vladimir and Gudermann, Thomas and Nieswandt, Bernhard and Braun, Attila},
  title     = {Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg\(^{2+}\) homeostasis and cytoskeletal architecture},
  series = {Nature Communications},
  volume    = {7},
  journal   = {Nature Communications},
  doi       = {10.1038/ncomms11097},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173843},
  year      = {2016},
  abstract  = {Mg\(^{2+}\) plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg\(^{2+}\)]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7\(^{fl/fl-Pf4Cre}\)) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7\(^{fl/fl-Pf4Cre}\) MKs, which is rescued by Mg\(^{2+}\) supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice.},
  language  = {en}
}
@phdthesis{GoebneeKlaus2023,
  author    = {G{\"o}b [n{\´e}e Klaus], Vanessa Aline Domenica},
  title     = {Pathomechanisms underlying ischemic stroke},
  doi       = {10.25972/OPUS-28672},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286727},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Every year, stroke affects over 100 million people worldwide and the number of cases continues to grow. Ischemic stroke is the most prevalent form of stroke and rapid restoration of blood flow is the primary therapeutic aim. However, recanalization might fail or reperfusion itself induces detrimental processes leading to infarct progression. Previous studies identified platelets and immune cells as drivers of this so-called ischemia/reperfusion (I/R) injury, establishing the concept of ischemic stroke as thrombo-inflammatory disease. Reduced cerebral blood flow despite recanalization promoted the hypothesis that thrombus formation within the cerebral microcirculation induces further tissue damage. The results presented in this thesis refute this: using complementary methodologies, it was shown that infarct growth precedes the occurrence of thrombi excluding them as I/R injury-underlying cause. Blood brain barrier disruption is one of the hallmarks of ischemic stroke pathology and was confirmed as early event during reperfusion injury in the second part of this study. Abolished platelet α-granule release protects mice from vascular leakage in the early reperfusion phase resulting in smaller infarcts. Using in vitro assays, platelet α-granule-derived PDGF-AB was identified as one factor contributing to blood-brain barrier disruption. In vivo visualization of platelet activation would provide important insights in the spatio-temporal context of platelet activation in stroke pathology. As platelet signaling results in elevated intracellular Ca2+ levels, this is an ideal readout. To overcome the limitations of chemical calcium indicators, a mouse line expressing an endogenous calcium reporter specifically in platelets and megakaryocytes was generated. Presence of the reporter did not interfere with platelet function, consequently these mice were characterized in in vivo and ex vivo models. Upon ischemic stroke, neutrophils are among the first cells that are recruited to the brain. Since for neutrophils both, beneficial and detrimental effects are described, their role was investigated within this thesis. Neither neutrophil depletion nor absence of NADPH-dependent ROS production (Ncf-/- mice) affected stroke outcome. In contrast, abolished NET-formation in Pad4-/- mice resulted in reduced infarct sizes, revealing detrimental effects of NETosis in the context of ischemic stroke, which might become a potential therapeutic target. Cerebral venous (sinus) thrombosis, CV(S)T is a rare type of stroke with mainly idiopathic onset. Whereas for arterial thrombosis a critical contribution of platelets is known and widely accepted, for venous thrombosis this is less clear but considered more and more. In the last part of this thesis, it was shown that fab-fragments of the anti-CLEC-2 antibody INU1 trigger pathological platelet activation in vivo, resulting in foudroyant CVT accompanied by heavy neurological symptoms. Using this novel animal model for CVT, cooperative signaling of the two platelet receptors CLEC-2 and GPIIb/IIIa was revealed as major trigger of CVT and potential target for treatment.},
  subject      = {Schlaganfall},
  language  = {en}
}
@article{SchwanLangSchlosseretal.2022,
  author    = {Schwan, Carsten and Lang, Alexander E. and Schlosser, Andreas and Fujita-Becker, Setsuko and AlHaj, Abdulatif and Schr{\"o}der, Rasmus R. and Faix, Jan and Aktories, Klaus and Mannherz, Hans Georg},
  title     = {Inhibition of Arp2/3 complex after ADP-ribosylation of Arp2 by binary Clostridioides toxins},
  series = {Cells},
  volume    = {11},
  journal   = {Cells},
  number    = {22},
  issn      = {2073-4409},
  doi       = {10.3390/cells11223661},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-297454},
  year      = {2022},
  abstract  = {Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases.},
  language  = {en}
}
@article{BruennertSeupelGoyaletal.2023,
  author    = {Br{\"u}nnert, Daniela and Seupel, Raina and Goyal, Pankaj and Bach, Matthias and Schraud, Heike and Kirner, Stefanie and K{\"o}ster, Eva and Feineis, Doris and Bargou, Ralf C. and Schlosser, Andreas and Bringmann, Gerhard and Chatterjee, Manik},
  title     = {Ancistrocladinium A induces apoptosis in proteasome inhibitor-resistant multiple myeloma cells: a promising therapeutic agent candidate},
  series = {Pharmaceuticals},
  volume    = {16},
  journal   = {Pharmaceuticals},
  number    = {8},
  issn      = {1424-8247},
  doi       = {10.3390/ph16081181},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-362887},
  year      = {2023},
  abstract  = {The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent.},
  language  = {en}
}
@unpublished{HennigPrustyKauferetal.2022,
  author    = {Hennig, Thomas and Prusty, Archana B. and Kaufer, Benedikt and Whisnant, Adam W. and Lodha, Manivel and Enders, Antje and Thomas, Julius and Kasimir, Francesca and Grothey, Arnhild and Herb, Stefanie and J{\"u}rges, Christopher and Meister, Gunter and Erhard, Florian and D{\"o}lken, Lars and Prusty, Bhupesh K.},
  title     = {Selective inhibition of miRNA 1 processing by a herpesvirus encoded miRNA},
  edition   = {accepted version},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-267862},
  year      = {2022},
  abstract  = {Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a thus far unknown cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the lytic-latent switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily drugable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.},
  language  = {en}
}
@article{ElMeseryRosenthalRauertWunderlichetal.2019,
  author    = {El-Mesery, Mohamed and Rosenthal, Tina and Rauert-Wunderlich, Hilka and Schreder, Martin and St{\"u}hmer, Thorsten and Leich, Ellen and Schlosser, Andreas and Ehrenschwender, Martin and Wajant, Harald and Siegmund, Daniela},
  title     = {The NEDD8-activating enzyme inhibitor MLN4924 sensitizes a TNFR1+ subgroup of multiple myeloma cells for TNF-induced cell death},
  series = {Cell Death \& Disease},
  volume    = {10},
  journal   = {Cell Death \& Disease},
  doi       = {10.1038/s41419-019-1860-2},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226666},
  year      = {2019},
  abstract  = {The NEDD8-activating enzyme (NAE) inhibitor MLN4924 inhibits cullin-RING ubiquitin ligase complexes including the SKP1-cullin-F-box E3 ligase βTrCP. MLN4924 therefore inhibits also the βTrCP-dependent activation of the classical and the alternative NFĸB pathway. In this work, we found that a subgroup of multiple myeloma cell lines (e.g., RPMI-8226, MM.1S, KMS-12BM) and about half of the primary myeloma samples tested are sensitized to TNF-induced cell death by MLN4924. This correlated with MLN4924-mediated inhibition of TNF-induced activation of the classical NFκB pathway and reduced the efficacy of TNF-induced TNFR1 signaling complex formation. Interestingly, binding studies revealed a straightforward correlation between cell surface TNFR1 expression in multiple myeloma cell lines and their sensitivity for MLN4924/TNF-induced cell death. The cell surface expression levels of TNFR1 in the investigated MM cell lines largely correlated with TNFR1 mRNA expression. This suggests that the variable levels of cell surface expression of TNFR1 in myeloma cell lines are decisive for TNF/MLN4924 sensitivity. Indeed, introduction of TNFR1 into TNFR1-negative TNF/MLN4924-resistant KMS-11BM cells, was sufficient to sensitize this cell line for TNF/MLN4924-induced cell death. Thus, MLN4924 might be especially effective in myeloma patients with TNFR1+ myeloma cells and a TNFhigh tumor microenvironment.},
  language  = {en}
}
@phdthesis{Neagoe2024,
  author    = {Neagoe, Raluca Alexandra Iulia},
  title     = {Development of techniques for studying the platelet glycoprotein receptors GPVI and GPIb localisation and signalling},
  doi       = {10.25972/OPUS-31306},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313064},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Platelets play an important role in haemostasis by mediating blood clotting at sites of blood vessel damage. Platelets, also participate in pathological conditions including thrombosis and inflammation. Upon vessel damage, two glycoprotein receptors, the GPIb-IX-V complex and GPVI, play important roles in platelet capture and activation. GPIb-IX-V binds to von Willebrand factor and GPVI to collagen. This initiates a signalling cascade resulting in platelet shape change and spreading, which is dependent on the actin cytoskeleton. This thesis aimed to develop and implement different super-resolution microscopy techniques to gain a deeper understanding of the conformation and location of these receptors in the platelet plasma membrane, and to provide insights into their signalling pathways. We suggest direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM) as the best candidates for imaging single platelets, whereas expansion microscopy (ExM) is ideal for imaging platelets aggregates. Furthermore, we highlighted the role of the actin cytoskeleton, through Rac in GPVI signalling pathway. Inhibition of Rac, with EHT1864 in human platelets induced GPVI and GPV, but not GPIbα shedding. Furthermore, EHT1864 treatment did not change GPVI dimerisation or clustering, however, it decreased phospholipase Cγ2 phosphorylation levels, in human, but not murine platelets, highlighting interspecies differences. In summary, this PhD thesis demonstrates that; 1) Rac alters GPVI signalling pathway in human but not mouse platelets; 2) our newly developed ExM protocol can be used to image platelet aggregates labelled with F(ab') fragments},
  subject      = {Platelet-Membranglykoprotein p62},
  language  = {en}
}
@article{GoebelPankratzAsaridouetal.2016,
  author    = {G{\"o}bel, Kerstin and Pankratz, Susann and Asaridou, Chloi-Magdalini and Herrmann, Alexander M. and Bittner, Stefan and Merker, Monika and Ruck, Tobias and Glumm, Sarah and Langhauser, Friederike and Kraft, Peter and Krug, Thorsten F. and Breuer, Johanna and Herold, Martin and Gross, Catharina C. and Beckmann, Denise and Korb-Pap, Adelheid and Schuhmann, Michael K. and Kuerten, Stefanie and Mitroulis, Ioannis and Ruppert, Clemens and Nolte, Marc W. and Panousis, Con and Klotz, Luisa and Kehrel, Beate and Korn, Thomas and Langer, Harald F. and Pap, Thomas and Nieswandt, Bernhard and Wiendl, Heinz and Chavakis, Triantafyllos and Kleinschnitz, Christoph and Meuth, Sven G.},
  title     = {Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells},
  series = {Nature Communications},
  volume    = {7},
  journal   = {Nature Communications},
  number    = {11626},
  doi       = {10.1038/ncomms11626},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165503},
  year      = {2016},
  abstract  = {Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein-kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders.},
  language  = {en}
}
@phdthesis{ElMerahbi2021,
  author    = {El Merahbi, Rabih},
  title     = {Adrenergic-induced ERK3 pathway drives lipolysis and suppresses energy dissipation},
  doi       = {10.25972/OPUS-21751},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217510},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Obesity-induced diabetes affects over 400 million people worldwide. Obesity is a complex metabolic disease and is associated with several co-morbidities, all of which negatively affect the individual's quality of life. It is commonly considered that obesity is a result of a positive energy misbalance, as increased food intake and lower expenditure eventually lead to the development of this disease. Moreover, the pathology of obesity is attributed to several genetic and epigenetic factors that put an individual at high risk compared to another. Adipose tissue is the main site of the organism's energy storage. During the time when the nutrients are available in excess, adipocytes acquire triglycerides, which are released during the time of food deprivation in the process of lipolysis (free fatty acids and glycerol released from adipocytes). Uncontrolled lipolysis is the consequent event that contributes to the development of diabetes and paradoxically obesity. To identify the genetic factors aiming for future therapeutic avenues targeting this pathway, we performed a high-throughput screen and identified the Extracellular-regulated kinase 3 (ERK3) as a hit. We demonstrate that β-adrenergic stimulation stabilizes ERK3 leading to the formation of a complex with the co-factor MAP kinase-activated protein kinase 5 (MK5) thereby driving lipolysis. Mechanistically, we identify a downstream target of the ERK3/MK5 pathway, the transcription factor FOXO1, which promotes the expression of the major lipolytic enzyme ATGL. Finally, we provide evidence that targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, but elevates energy dissipation, promoting lean phenotype and ameliorating diabetes. Moreover, we shed the light on our pharmacological approach in targeting ERK3/MK5 pathways using MK5 specific inhibitor. Already after 1 week of administering the inhibitor, mice showed signs of improvement of their metabolic fitness as showed here by a reduction in induced lipolysis and the elevation in the expression of thermogenic genes. Taken together, our data suggest that targeting the ERK3/MK5 pathway, a previously unrecognized signaling axis in adipose tissue, could be an attractive target for future therapies aiming to combat obesity-induced diabetes.},
  subject      = {Metabolism},
  language  = {en}
}
@article{MakbulKraftGriessmannetal.2021,
  author    = {Makbul, Cihan and Kraft, Christian and Grießmann, Matthias and Rasmussen, Tim and Katzenberger, Kilian and Lappe, Melina and Pfarr, Paul and Stoffer, Cato and St{\"o}hr, Mara and Wandinger, Anna-Maria and B{\"o}ttcher, Bettina},
  title     = {Binding of a pocket factor to Hepatitis B virus capsids changes the rotamer conformation of Phenylalanine 97},
  series = {Viruses},
  volume    = {13},
  journal   = {Viruses},
  number    = {11},
  issn      = {1999-4915},
  doi       = {10.3390/v13112115},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248565},
  year      = {2021},
  abstract  = {(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.},
  language  = {en}
}
@phdthesis{Toepfer2018,
  author    = {Toepfer, Franziska Helene},
  title     = {Component selectivity and multistability in a \(Drosophila\) orientation paradigm using incoherent motion stimuli},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153346},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Visual information is essential for Drosophila to navigate its environment. The visual system of the fly has been studied for many decades and has yielded many insights about vision in general. However, visual information can be ambiguous and the system processing it needs to be able to cope with that. In this study, the visual orientation behavior of Drosophila is challenged by panoramic incoherent motion stimuli to which the fly can respond in three different, equally adaptive ways. The study is conducted in a well-established setup, the so-called flight simulator (Heisenberg and Wolf, 1993), where the fly can control its visual surroundings in stationary flight with its yaw torque, which is simultaneously recorded. The fly can either use one of two incoherently moving panorama patterns or the integrated motion of both as its reference for straight flight. It is observed that flies use all three of these behavioral alternatives for orientation. Previous models of fly motion vision do not predict a bimodal tuning to incoherent wide-field motion stimuli (Joesch et al., 2008, Borst et al., 1995), however, a recent study on blowflies could suggests that they show component selectivity to the individual moving gratings in a compound plaid stimulus (Saleem et al., 2012). Here, it can be shown that the same bimodal tuning manifests in Drosophila, although the stimuli used are different and most of the experiments are conducted in closed loop. It is found that the extent to which the Drosophila expresses this component selectivity in its orientation behavior, i.e. how often it stabilizes a single panorama pattern instead of the integrated motion of both, depends on two properties of the panorama stimuli, pattern contrast and horizontal pattern element distance. Single pattern stabilization decreases with increasing contrast and increasing pattern element distance. In the latter case, it increases again when there are very few horizontal pattern elements, although that appears to be the result of a lack of rivalry between the patterns due to the low number of pattern elements. Both increased pattern contrast and pattern element distance increase the salience of the single pattern elements. A single element in a compound visual stimulus, like a dot within a dot pattern, can be interpreted as a standalone figure or a part of a bigger unit. Previous studies on Drosophila vision have concentrated on how the fly discriminates a figure from the background (Heisenberg and Wolf, 1984, Bahl et al., 2013, Aptekar et al., 2012), but have hardly touched the question of what qualifies a figure or a background (i.e. a panorama) stimulus as such. In the present study, it is observed that, when exposed to incoherent panoramic motion stimuli, the flies prefer to orient themselves towards the average of the two motions when the panorama stimuli possess strong figure features and towards the single patterns when they do not and single pattern elements are therefore less salient. The above-mentioned plaid stimuli are a well-known multistable percept in human psychophysics. Multistability is a property of higher visual systems and considered an indicator of endogenous activity in vision. As Drosophila expresses behavioral multistability in the IPMP, it is evaluated in this respect. The results show several parallels to human multistable perception. For one, the frequency and duration with which a behavior occurs, can be influenced, but the occurrence of the behaviors is non-deterministic and not coupled to the stimulus. It can also be shown that the switches between behaviors do not stem from a rivalry of the two visual hemispheres of the fly, although monocularity does also influence the likelihood with which the behaviors occur. Secondly, like in human perceptual rivalry, individual flies exhibit strong idiosyncrasies regarding the overall durations they spend with the different behaviors and the frequencies with which they switch between them. Finally, the distribution of the durations between the behavioral switches can be fit to the same function as the distribution of percept durations in human multistable perception, the gamma function, although it has a different shape and therefore also differing parameters. The Drosophila mutant radish, which has been shown to have attention-like deficits (van Swinderen and Brembs, 2010, Koenig et al., 2016a), does also express an altered behavior in the IPMP compared to wildtype flies. As these behavioral alterations resemble effects on multistable perception found in humans suffering from ADHD (Amador-Campos et al., 2015) and perceptual multistability is generally considered to be closely related to attention (Leopold and Logothetis, 1999), attentional processes are also very likely to play a role in the flies' behavior in the IPMP. In conclusion, the visual system of Drosophila is capable disentangle incoherent motion stimuli even if they overlap and cover the entire visual field, i.e. it shows component selectivity of wide-field motion. Whether it uses a single wide-field motion component or the average of two as its reference for straight flight depends on pattern contrast and horizontal pattern element density, which indicates an involvement of a figure-background rivalry. This rivalry and the one between the two wide-field motion components elicit a multistability in the orientation behavior of the fly the temporal dynamics of which partially resemble the temporal dynamics of human multistable perception and which also suggests the involvement of attentional processes.},
  subject      = {Drosophila},
  language  = {en}
}
@article{HerrmannMuellerOrthetal.2020,
  author    = {Herrmann, Andreas B. and M{\"u}ller, Martha-Lena and Orth, Martin F. and M{\"u}ller, J{\"o}rg P. and Zernecke, Alma and Hochhaus, Andreas and Ernst, Thomas and Butt, Elke and Frietsch, Jochen J.},
  title     = {Knockout of LASP1 in CXCR4 expressing CML cells promotes cell persistence, proliferation and TKI resistance},
  series = {Journal of Cellular and Molecular Medicine},
  volume    = {24},
  journal   = {Journal of Cellular and Molecular Medicine},
  number    = {5},
  doi       = {10.1111/jcmm.14910},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214122},
  pages     = {2942 -- 2955},
  year      = {2020},
  abstract  = {Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR-ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR-ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1-mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell-mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.},
  language  = {en}
}
@article{ShaikhVargasMokhtarietal.2021,
  author    = {Shaikh, Haroon and Vargas, Juan Gamboa and Mokhtari, Zeinab and Jarick, Katja J. and Ulbrich, Maria and Mosca, Josefina Pe{\~n}a and Viera, Estibaliz Arellano and Graf, Caroline and Le, Duc-Dung and Heinze, Katrin G. and B{\"u}ttner-Herold, Maike and Rosenwald, Andreas and Pezoldt, Joern and Huehn, Jochen and Beilhack, Andreas},
  title     = {Mesenteric Lymph Node Transplantation in Mice to Study Immune Responses of the Gastrointestinal Tract},
  series = {Frontiers in Immunology},
  volume    = {12},
  journal   = {Frontiers in Immunology},
  issn      = {1664-3224},
  doi       = {10.3389/fimmu.2021.689896},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244869},
  year      = {2021},
  abstract  = {Mesenteric lymph nodes (mLNs) are sentinel sites of enteral immunosurveillance and immune homeostasis. Immune cells from the gastrointestinal tract (GIT) are constantly recruited to the mLNs in steady-state and under inflammatory conditions resulting in the induction of tolerance and immune cells activation, respectively. Surgical dissection and transplantation of lymph nodes (LN) is a technique that has supported seminal work to study LN function and is useful to investigate resident stromal and endothelial cell biology and their cellular interactions in experimental disease models. Here, we provide a detailed protocol of syngeneic mLN transplantation and report assays to analyze effective mLN engraftment in congenic recipients. Transplanted mLNs allow to study T cell activation and proliferation in preclinical mouse models. Donor mLNs proved viable and functional after surgical transplantation and regenerated blood and lymphatic vessels. Immune cells from the host completely colonized the transplanted mLNs within 7-8 weeks after the surgical intervention. After allogeneic hematopoietic cell transplantation (allo-HCT), adoptively transferred allogeneic CD4+ T cells from FVB/N (H-2q) mice homed to the transplanted mLNs in C57BL/6 (H-2b) recipients during the initiation phase of acute graft-versus-host disease (aGvHD). These CD4+ T cells retained full proliferative capacity and upregulated effector and gut homing molecules comparable to those in mLNs from unmanipulated wild-type recipients. Wild type mLNs transplanted into MHCII deficient syngeneic hosts sufficed to activate alloreactive T cells upon allogeneic hematopoietic cell transplantation, even in the absence of MHCII+ CD11c+ myeloid cells. These data support that orthotopically transplanted mLNs maintain physiological functions after transplantation. The technique of LN transplantation can be applied to study migratory and resident cell compartment interactions in mLNs as well as immune reactions from and to the gut under inflammatory and non-inflammatory conditions.},
  language  = {en}
}
@article{KnappBenz2020,
  author    = {Knapp, Oliver and Benz, Roland},
  title     = {Membrane activity and channel formation of the adenylate cyclase toxin (CyaA) of Bordetella pertussis in lipid bilayer membranes},
  series = {Toxins},
  volume    = {12},
  journal   = {Toxins},
  number    = {3},
  issn      = {2072-6651},
  doi       = {10.3390/toxins12030169},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203362},
  year      = {2020},
  abstract  = {The Gram-negative bacterium Bordetella pertussis is the cause of whooping cough. One of its pathogenicity factors is the adenylate cyclase toxin (CyaA) secreted by a Type I export system. The 1706 amino acid long CyaA (177 kDa) belongs to the continuously increasing family of repeat in toxin (RTX) toxins because it contains in its C-terminal half a high number of nine-residue tandem repeats. The protein exhibits cytotoxic and hemolytic activities that target primarily myeloid phagocytic cells expressing the αMβ2 integrin receptor (CD11b/CD18). CyaA represents an exception among RTX cytolysins because the first 400 amino acids from its N-terminal end possess a calmodulin-activated adenylate cyclase (AC) activity. The entry of the AC into target cells is not dependent on the receptor-mediated endocytosis pathway and penetrates directly across the cytoplasmic membrane of a variety of epithelial and immune effector cells. The hemolytic activity of CyaA is rather low, which may have to do with its rather low induced permeability change of target cells and its low conductance in lipid bilayer membranes. CyaA forms highly cation-selective channels in lipid bilayers that show a strong dependence on aqueous pH. The pore-forming activity of CyaA but not its single channel conductance is highly dependent on Ca\(^{2+}\) concentration with a half saturation constant of about 2 to 4 mM.},
  language  = {en}
}
@article{ButtStempfleListeretal.2020,
  author    = {Butt, Elke and Stempfle, Katrin and Lister, Lorenz and Wolf, Felix and Kraft, Marcella and Herrmann, Andreas B. and Viciano, Cristina Perpina and Weber, Christian and Hochhaus, Andreas and Ernst, Thomas and Hoffmann, Carsten and Zernecke, Alma and Frietsch, Jochen J.},
  title     = {Phosphorylation-dependent differences in CXCR4-LASP1-AKT1 interaction between breast cancer and chronic myeloid leukemia},
  series = {Cells},
  volume    = {9},
  journal   = {Cells},
  number    = {2},
  issn      = {2073-4409},
  doi       = {10.3390/cells9020444},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200638},
  year      = {2020},
  abstract  = {The serine/threonine protein kinase AKT1 is a downstream target of the chemokine receptor 4 (CXCR4), and both proteins play a central role in the modulation of diverse cellular processes, including proliferation and cell survival. While in chronic myeloid leukemia (CML) the CXCR4 is downregulated, thereby promoting the mobilization of progenitor cells into blood, the receptor is highly expressed in breast cancer cells, favoring the migratory capacity of these cells. Recently, the LIM and SH3 domain protein 1 (LASP1) has been described as a novel CXCR4 binding partner and as a promoter of the PI3K/AKT pathway. In this study, we uncovered a direct binding of LASP1, phosphorylated at S146, to both CXCR4 and AKT1, as shown by immunoprecipitation assays, pull-down experiments, and immunohistochemistry data. In contrast, phosphorylation of LASP1 at Y171 abrogated these interactions, suggesting that both LASP1 phospho-forms interact. Finally, findings demonstrating different phosphorylation patterns of LASP1 in breast cancer and chronic myeloid leukemia may have implications for CXCR4 function and tyrosine kinase inhibitor treatment.},
  language  = {en}
}
@article{Benz2020,
  author    = {Benz, Roland},
  title     = {RTX-Toxins},
  series = {Toxins},
  volume    = {12},
  journal   = {Toxins},
  number    = {6},
  issn      = {2072-6651},
  doi       = {10.3390/toxins12060359},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205860},
  year      = {2020},
  abstract  = {No abstract available.},
  language  = {en}
}
@article{SegererHadamekZundleretal.2016,
  author    = {Segerer, Gabriela and Hadamek, Kerstin and Zundler, Matthias and Fekete, Agnes and Seifried, Annegrit and Mueller, Martin J. and Koentgen, Frank and Gessler, Manfred and Jeanclos, Elisabeth and Gohla, Antje},
  title     = {An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation},
  series = {Scientific Reports},
  volume    = {6},
  journal   = {Scientific Reports},
  doi       = {10.1038/srep35160},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181094},
  year      = {2016},
  abstract  = {Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive Pgp\(^{D34N}\) mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation.},
  language  = {en}
}
@article{RaduSchoenwetterBraunetal.2017,
  author    = {Radu, Laura and Schoenwetter, Elisabeth and Braun, Cathy and Marcoux, Julien and Koelmel, Wolfgang and Schmitt, Dominik R. and Kuper, Jochen and Cianf{\´e}rani, Sarah and Egly, Jean M. and Poterszman, Arnaud and Kisker, Caroline},
  title     = {The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH},
  series = {Nucleic Acids Research},
  volume    = {45},
  journal   = {Nucleic Acids Research},
  number    = {18},
  doi       = {10.1093/nar/gkx743},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170173},
  pages     = {10872-10883},
  year      = {2017},
  abstract  = {The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.},
  language  = {en}
}
@article{MostosiSchindelinKollmannsbergeretal.2020,
  author    = {Mostosi, Philipp and Schindelin, Hermann and Kollmannsberger, Philip and Thorn, Andrea},
  title     = {Haruspex: A Neural Network for the Automatic Identification of Oligonucleotides and Protein Secondary Structure in Cryo-Electron Microscopy Maps},
  series = {Angewandte Chemie International Edition},
  volume    = {59},
  journal   = {Angewandte Chemie International Edition},
  number    = {35},
  doi       = {10.1002/anie.202000421},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214763},
  pages     = {14788 -- 14795},
  year      = {2020},
  abstract  = {In recent years, three-dimensional density maps reconstructed from single particle images obtained by electron cryo-microscopy (cryo-EM) have reached unprecedented resolution. However, map interpretation can be challenging, in particular if the constituting structures require de-novo model building or are very mobile. Herein, we demonstrate the potential of convolutional neural networks for the annotation of cryo-EM maps: our network Haruspex has been trained on a carefully curated set of 293 experimentally derived reconstruction maps to automatically annotate RNA/DNA as well as protein secondary structure elements. It can be straightforwardly applied to newly reconstructed maps in order to support domain placement or as a starting point for main-chain placement. Due to its high recall and precision rates of 95.1 \% and 80.3 \%, respectively, on an independent test set of 122 maps, it can also be used for validation during model building. The trained network will be available as part of the CCP-EM suite.},
  language  = {en}
}
@article{VogelsangEichlerHuntemannetal.2021,
  author    = {Vogelsang, Anna and Eichler, Susann and Huntemann, Niklas and Masanneck, Lars and B{\"o}hnlein, Hannes and Sch{\"u}ngel, Lisa and Willison, Alice and Loser, Karin and Nieswandt, Bernhard and Kehrel, Beate E. and Zarbock, Alexander and G{\"o}bel, Kerstin and Meuth, Sven G.},
  title     = {Platelet inhibition by low-dose acetylsalicylic acid reduces neuroinflammation in an animal model of multiple sclerosis},
  series = {International Journal of Molecular Sciences},
  volume    = {22},
  journal   = {International Journal of Molecular Sciences},
  number    = {18},
  issn      = {1422-0067},
  doi       = {10.3390/ijms22189915},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284535},
  year      = {2021},
  abstract  = {Aside from the established immune-mediated etiology of multiple sclerosis (MS), compelling evidence implicates platelets as important players in disease pathogenesis. Specifically, numerous studies have highlighted that activated platelets promote the central nervous system (CNS)-directed adaptive immune response early in the disease course. Platelets, therefore, present a novel opportunity for modulating the neuroinflammatory process that characterizes MS. We hypothesized that the well-known antiplatelet agent acetylsalicylic acid (ASA) could inhibit neuroinflammation by affecting platelets if applied at low-dose and investigated its effect during experimental autoimmune encephalomyelitis (EAE) as a model to study MS. We found that oral administration of low-dose ASA alleviates symptoms of EAE accompanied by reduced inflammatory infiltrates and less extensive demyelination. Remarkably, the percentage of CNS-infiltrated CD4\(^+\) T cells, the major drivers of neuroinflammation, was decreased to 40.98 ± 3.28\% in ASA-treated mice compared to 56.11 ± 1.46\% in control animals at the disease maximum as revealed by flow cytometry. More interestingly, plasma levels of thromboxane A\(_2\) were decreased, while concentrations of platelet factor 4 and glycoprotein VI were not affected by low-dose ASA treatment. Overall, we demonstrate that low-dose ASA could ameliorate the platelet-dependent neuroinflammatory response in vivo, thus indicating a potential treatment approach for MS.},
  language  = {en}
}
@article{NavarroStegnerNieswandtetal.2021,
  author    = {Navarro, Stefano and Stegner, David and Nieswandt, Bernhard and Heemskerk, Johan W. M. and Kuijpers, Marijke J. E.},
  title     = {Temporal roles of platelet and coagulation pathways in collagen- and tissue factor-induced thrombus formation},
  series = {International Journal of Molecular Sciences},
  volume    = {23},
  journal   = {International Journal of Molecular Sciences},
  number    = {1},
  issn      = {1422-0067},
  doi       = {10.3390/ijms23010358},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284219},
  year      = {2021},
  abstract  = {In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.},
  language  = {en}
}
@article{KollikowskiPhamMaerzetal.2022,
  author    = {Kollikowski, Alexander M. and Pham, Mirko and M{\"a}rz, Alexander G. and Papp, Lena and Nieswandt, Bernhard and Stoll, Guido and Schuhmann, Michael K.},
  title     = {Platelet Activation and Chemokine Release Are Related to Local Neutrophil-Dominant Inflammation During Hyperacute Human Stroke},
  series = {Translational Stroke Research},
  volume    = {13},
  journal   = {Translational Stroke Research},
  number    = {3},
  issn      = {1868-601X},
  doi       = {10.1007/s12975-021-00938-w},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270194},
  pages     = {364-369},
  year      = {2022},
  abstract  = {Experimental evidence has emerged that local platelet activation contributes to inflammation and infarct formation in acute ischemic stroke (AIS) which awaits confirmation in human studies. We conducted a prospective observational study on 258 consecutive patients undergoing mechanical thrombectomy (MT) due to large-vessel-occlusion stroke of the anterior circulation (08/2018-05/2020). Intraprocedural microcatheter aspiration of 1 ml of local (occlusion condition) and systemic arterial blood samples (self-control) was performed according to a prespecified protocol. The samples were analyzed for differential leukocyte counts, platelet counts, and plasma levels of the platelet-derived neutrophil-activating chemokine C-X-C-motif ligand (CXCL) 4 (PF-4), the neutrophil attractant CXCL7 (NAP-2), and myeloperoxidase (MPO). The clinical-biological relevance of these variables was corroborated by specific associations with molecular-cellular, structural-radiological, hemodynamic, and clinical-functional parameters. Seventy consecutive patients fulfilling all predefined criteria entered analysis. Mean local CXCL4 (+ 39\%: 571 vs 410 ng/ml, P = .0095) and CXCL7 (+ 9\%: 693 vs 636 ng/ml, P = .013) concentrations were higher compared with self-controls. Local platelet counts were lower (- 10\%: 347,582 vs 383,284/µl, P = .0052), whereas neutrophil counts were elevated (+ 10\%: 6022 vs 5485/µl, P = 0.0027). Correlation analyses revealed associations between local platelet and neutrophil counts (r = 0.27, P = .034), and between CXCL7 and MPO (r = 0.24, P = .048). Local CXCL4 was associated with the angiographic degree of reperfusion following recanalization (r =  - 0.2523, P = .0479). Functional outcome at discharge correlated with local MPO concentrations (r = 0.3832, P = .0014) and platelet counts (r = 0.288, P = .0181). This study provides human evidence of cerebral platelet activation and platelet-neutrophil interactions during AIS and points to the relevance of per-ischemic thrombo-inflammatory mechanisms to impaired reperfusion and worse functional outcome following recanalization.},
  language  = {en}
}
@article{SchanbacherBieberReindersetal.2022,
  author    = {Schanbacher, Constanze and Bieber, Michael and Reinders, Yvonne and Cherpokova, Deya and Teichert, Christina and Nieswandt, Bernhard and Sickmann, Albert and Kleinschnitz, Christoph and Langhauser, Friederike and Lorenz, Kristina},
  title     = {ERK1/2 activity is critical for the outcome of ischemic stroke},
  series = {International Journal of Molecular Sciences},
  volume    = {23},
  journal   = {International Journal of Molecular Sciences},
  number    = {2},
  issn      = {1422-0067},
  doi       = {10.3390/ijms23020706},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-283991},
  year      = {2022},
  abstract  = {Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2\(^{wt}\)) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood-brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP\(^{wt}\)) and its phosphorylation-deficient mutant RKIP\(^{S153A}\), known inhibitors of the ERK1/2 signaling cascade. RKIP\(^{wt}\) and RKIP\(^{S153A}\) attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.},
  language  = {en}
}
@article{MarzoccoFazeliDiMiccoetal.2018,
  author    = {Marzocco, Stefania and Fazeli, Gholamreza and Di Micco, Lucia and Autore, Giuseppina and Adesso, Simona and Dal Piaz, Fabrizio and Heidland, August and Di Iorio, Biagio},
  title     = {Supplementation of short-chain fatty acid, sodium propionate, in patients on maintenance hemodialysis: beneficial effects on inflammatory parameters and gut-derived uremic toxins, a pilot study (PLAN Study)},
  series = {Journal of Clinical Medicine},
  volume    = {7},
  journal   = {Journal of Clinical Medicine},
  number    = {10},
  issn      = {2077-0383},
  doi       = {10.3390/jcm7100315},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-197626},
  pages     = {315},
  year      = {2018},
  abstract  = {Background: In end-stage renal disease (ESRD), gut-derived uremic toxins play a crucial role in the systemic inflammation and oxidative stress promoting the excess morbidity and mortality. The biochemical derangement is in part a consequence of an insufficient generation of short-chain fatty acids (SCFA) due to the dysbiosis of the gut and an insufficient consumption of the fermentable complex carbohydrates. Aim of the study: The primary end-point was to evaluate the potential efficacy of SCFA (specifically, sodium propionate (SP)) for patients on maintenance hemodialysis (MHD) on systemic inflammation. Secondary end-points included potential attenuation of oxidative stress markers, insulin resistance and production of gut-derived uremic toxins indoxyl sulfate and p-cresol sulfate, as well as health status after SP supplementation. Study design: We performed a single-center non-randomized pilot study in 20 MHD patients. They received the food additive SP with a daily intake of 2 × 500 mg in the form of capsules for 12 weeks. Pre-dialysis blood samples were taken at the beginning, after six weeks and at the end of the administration period, as well as four weeks after withdrawal of the treatment. Results: The subjects revealed a significant decline of inflammatory parameters C-reactive protein (-46\%), interleukin IL-2 (-27\%) and IL-17 (-15\%). The inflammatory parameters IL-6 and IFN-gamma showed a mild non-significant reduction and the anti-inflammatory cytokine IL-10 increased significantly (+71\%). While the concentration of bacterial endotoxins and TNF-α remained unchanged, the gut-derived uremic toxins, indoxyl sulfate (-30\%) and p-cresyl sulfate (-50\%), revealed a significant decline. The SP supplementation reduced the parameters of oxidative stress malondialdehyde (-32\%) and glutathione peroxidase activity (-28\%). The serum insulin levels dropped by 30\% and the HOMA-index by 32\%. The reduction of inflammatory parameters was associated with a lowering of ferritin and a significant increase in transferrin saturation (TSAT). Four weeks after the end of the treatment phase, all improved parameters deteriorated again. Evaluation of the psycho-physical performance with the short form 36 (SF-36) questionnaire showed an enhancement in the self-reported physical functioning, general health, vitality and mental health. The SP supplementation was well tolerated and without important side effects. No patient had left the study due to intolerance to the medication. The SP supplementation in MHD patients reduced pro-inflammatory parameters and oxidative stress and improved insulin resistance and iron metabolism. Furthermore, SP effectively lowered the important gut-derived uremic toxins indoxyl and p-cresol sulfate. These improvements were associated with a better quality of life. Further controlled studies are required in a larger cohort to evaluate the clinical outcome.},
  language  = {en}
}
@article{FusiPaudelMederetal.2022,
  author    = {Fusi, Lorenza and Paudel, Rupesh and Meder, Katharina and Schlosser, Andreas and Schrama, David and Goebeler, Matthias and Schmidt, Marc},
  title     = {Interaction of transcription factor FoxO3 with histone acetyltransferase complex subunit TRRAP modulates gene expression and apoptosis},
  series = {Journal of Biological Chemistry},
  volume    = {298},
  journal   = {Journal of Biological Chemistry},
  number    = {3},
  doi       = {10.1016/j.jbc.2022.101714},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-299820},
  year      = {2022},
  abstract  = {Forkhead box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promotes nuclear localization and activation of FoxO proteins, which—depending on the cellular context—can lead to cell cycle arrest or apoptosis. In endothelial cells (ECs), they further regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxOs exert a tumor-suppressive function by regulating proliferation and survival. We and others have previously shown that FoxOs can regulate these processes via two different mechanisms: by direct binding to forkhead-responsive elements at the promoter of target genes or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here, we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners that might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3-interacting proteins revealed transformation/transcription domain-associated protein (TRRAP), a member of multiple histone acetyltransferase complexes, as a novel binding partner of FoxO family proteins. We demonstrate that TRRAP is required to support FoxO3 transactivation and FoxO3-dependent G1 arrest and apoptosis in ECs via transcriptional activation of the cyclin-dependent kinase inhibitor p27\(^{kip1}\) and the proapoptotic B-cell lymphoma 2 family member, BIM. Moreover, FoxO-TRRAP interaction could explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking forkhead-responsive element sequences.},
  language  = {en}
}
@phdthesis{Slotta2019,
  author    = {Slotta, Anja Maria},
  title     = {The Role of Protein Kinase D 1 in the regulation of murine adipose tissue function under physiological and pathophysiological conditions},
  doi       = {10.25972/OPUS-17911},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179112},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Adipocytes are specialized cells found in vertebrates to ensure survival in terms of adaption to food deficit and abundance. However, their dysfunction accounts for the pathophysiology of metabolic diseases such as T2DM. Preliminary data generated by Mona L{\"o}ffler suggested that PKD1 is involved in adipocyte function. Here, I show that PKD1 expression and activity is linked to lipid metabolism of murine adipocytes. PKD1 gene expression and activity was reduced in murine white adipose tissue upon fasting, a physiological condition which induces lipolysis. Isoproterenol-stimulated lipolysis in adipose tissue and 3T3-L1 adipocytes reduced PKD1 gene expression. Silencing ATGL in adipocytes inhibited isoproterenol-stimulated lipolysis, however, the β-adrenergic stimulation of ATGL-silenced adipocytes lowered PKD1 expression levels as well. Adipose tissue of obese mice exhibited high PKD1 RNA levels but paradoxically lower protein levels of phosphorylated PKD1-Ser916. However, HFD generated a second PKD1 protein product of low molecular weight in mouse adipose tissue. Furthermore, constitutively active PKD1 predominantly displayed nuclear localization in 3T3-L1 adipocytes containing many fat vacuoles. However, adipocytes overexpressing non-functional PKD1 contained fewer lipid droplets and PKD1-KD was distributed in cytoplasm. Most importantly, deficiency of PKD1 in mouse adipose tissue caused expression of genes involved in adaptive thermogenesis such as UCP-1 and thus generated brown-like phenotype adipocytes. Thus, PKD1 is implicated in adipose tissue function and presents an interesting target for therapeutic approaches in the prevention of obesity and associated diseases.},
  subject      = {adipocyte},
  language  = {en}
}
@phdthesis{MonyNair2021,
  author    = {Mony Nair, Rahul},
  title     = {Elucidating ubiquitin recognition by the HECT-type ubiquitin ligase HUWE1},
  doi       = {10.25972/OPUS-22103},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221030},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The small protein modifier ubiquitin is at the heart of an immensely versatile posttranslational modification system that orchestrates countless physiological and disease-associated cellular processes. Key to this versatility are the manifold modifications that can be assembled from ubiquitin "building blocks" and are associated with specific functional outcomes for the modified substrates. In particular, ubiquitin molecules can form polymeric chains of distinct lengths and linkage types that give rise to distinct chain conformations, thereby providing recognition sites for specific signaling receptors/effectors. The class of E3 enzymes (ubiquitin ligases) provides critical specificity determinants in ubiquitin linkage formation; it is therefore crucial to unravel precisely how E3 enzymes operate in order to understand the structural basis of ubiquitin signaling and exploit these insights for therapeutic benefit. Overexpression and deregulation of the HECT-type ubiquitin ligase HUWE1 is implicated in several different cancer types and neurodegenerative disorders. It is largely unknown which factors control the ubiquitin modifications formed by HUWE1, how the catalytic HECT domain interacts with functionally distinct ubiquitin molecules (donor, acceptor and regulatory ubiquitin molecules) and which conformational transitions enable these interactions during ubiquitin chain formation. One aim of this study was to structurally elucidate the recognition of donor ubiquitin by the HECT domain of HUWE1. To this end I utilized a ubiquitin activity-based probe to reconstitute a proxy for a donor ubiquitin-linked conjugate of the HECT domain of HUWE1 and determined its structure by X-ray crystallography. This structure reveals that the donor ubiquitin binds to the C-lobe of HUWE1 in the same way as NEDD4-type ligases, corroborating the idea that HECT ligases utilize a conserved mode of donor ubiquitin recognition. independent of their linkage and substrate specificities. With the help of biochemical analyses, I also validated specific features of the structure, in particular the positioning of the C-terminal tail of the ligase, which was known to be critical for activity. In the newly determined structure, which reflects an "L-shaped", active state of the HECT domain, this tail is fully resolved and coordinated at the N-lobe-C-lobe interface. I defined residues that are critical for this coordination and showed that they are also essential for the activity of HUWE1, including auto-ubiquitination, free ubiquitin chain formation, and substrate ubiquitination. Furthermore, I discovered that the N-lobe of HUWE1 harbors a ubiquitin-binding exosite similar to NEDD4-type ligases and E6AP. My in-vitro activity and binding assays show that HUWE1 uses the exosite for isopeptide bond formation, but that it is dispensable for thioester bond formation. The binding assays further show that the donor ubiquitin loaded HECT domain binds an additional ubiquitin molecule at the exosite more tightly than the apo HECT domain, which possibly suggests allosteric communication between the two sites. Finally, I showed that the ubiquitin activity-based probe (ubiquitin-propargylamine) can label the catalytic cysteine of HUWE1 and NEDD4-type with close to quantitative turn- over, while it does not react with the HECT domain of the evolutionarily more divergent E6AP. The determinants underlying these differential reactivities remain to be explored. Taken, together my results significantly enhance our mechanistic understanding of the catalytic domain of HUWE1 and pinpoint linchpins for therapeutic interventions with the activity of this disease-relevant enzyme.},
  language  = {en}
}
@article{TruongvanLiMisraetal.2022,
  author    = {Truongvan, Ngoc and Li, Shurong and Misra, Mohit and Kuhn, Monika and Schindelin, Hermann},
  title     = {Structures of UBA6 explain its dual specificity for ubiquitin and FAT10},
  series = {Nature Communications},
  volume    = {13},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-022-32040-6},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301161},
  year      = {2022},
  abstract  = {The covalent modification of target proteins with ubiquitin or ubiquitin-like modifiers is initiated by E1 activating enzymes, which typically transfer a single modifier onto cognate conjugating enzymes. UBA6 is an unusual E1 since it activates two highly distinct modifiers, ubiquitin and FAT10. Here, we report crystal structures of UBA6 in complex with either ATP or FAT10. In the UBA6-FAT10 complex, the C-terminal domain of FAT10 binds to where ubiquitin resides in the UBA1-ubiquitin complex, however, a switch element ensures the alternate recruitment of either modifier. Simultaneously, the N-terminal domain of FAT10 interacts with the 3-helix bundle of UBA6. Site-directed mutagenesis identifies residues permitting the selective activation of either ubiquitin or FAT10. These results pave the way for studies investigating the activation of either modifier by UBA6 in physiological and pathophysiological settings.},
  language  = {en}
}
@article{PaulsHamaratTrufasuetal.2019,
  author    = {Pauls, Dennis and Hamarat, Yasmin and Trufasu, Luisa and Schendzielorz, Tim M. and Gramlich, Gertrud and Kahnt, J{\"o}rg and Vanselow, Jens and Schlosser, Andreas and Wegener, Christian},
  title     = {Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate},
  series = {European Journal of Neuroscience},
  volume    = {50},
  journal   = {European Journal of Neuroscience},
  number    = {9},
  doi       = {10.1111/ejn.14516},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204863},
  pages     = {3502-3519},
  year      = {2019},
  abstract  = {Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well-characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE ), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD ) in global neuropeptide processing and selected peptide-regulated behaviours in Drosophila . We found that a deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD -encoding gene silver in the larva causes lethality, and leads to deficits in starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide-regulated behaviour in Drosophila . dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE.},
  language  = {en}
}
@article{BalakrishnanHemmenChoudhuryetal.2022,
  author    = {Balakrishnan, Ashwin and Hemmen, Katherina and Choudhury, Susobhan and Krohn, Jan-Hagen and Jansen, Kerstin and Friedrich, Mike and Beliu, Gerti and Sauer, Markus and Lohse, Martin J. and Heinze, Katrin G.},
  title     = {Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence},
  series = {Communications Biology},
  volume    = {5},
  journal   = {Communications Biology},
  number    = {1},
  doi       = {10.1038/s42003-022-03106-4},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301140},
  year      = {2022},
  abstract  = {G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β\(_{2}\)-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm\(^{2}\)/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbr{\"u}ck model.},
  language  = {en}
}
@phdthesis{Balakrishnan2021,
  author    = {Balakrishnan, Ashwin},
  title     = {Fast molecular mobility of β\(_2\)-adrenergic receptor revealed by time-resolved fluorescence spectroscopy},
  doi       = {10.25972/OPUS-25085},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250856},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {G-protein- coupled receptors (GPCRs) are the largest family of membrane confined receptors and they transduce ligand binding to downstream effects. Almost 40\% of the drugs in the world target GPCRs due to their function, albeit knowing less about their activation. Understanding their dynamic behaviour in basal and activated state could prove key to drug development in the future. GPCRs are known to exhibit complex molecular mobility patterns. A plethora of studies have been and are being conducted to understand the mobility of GPCRs. Due to limitations of imaging and spectroscopic techniques commonly used, the relevant timescales are hard to access. The most commonly used techniques are electron paramagnetic resonance or double electronelectron resonance, nuclear magnetic resonance, time-resolved fluorescence, single particle tracking and fluorescence recovery after photobleaching. Among these techniques only fluorescence has the potential to probe live cells. In this thesis, I use different time-resolved fluorescence spectroscopic techniques to quantify diffusion dynamics / molecular mobility of β2-adrenergic receptor (β2-AR) in live cells. The thesis shows that β2-AR exhibits mobility over an exceptionally broad temporal range (nanosecond to second) that can be linked to its respective physiological scenario. I explain how β2-AR possesses surprisingly fast lateral mobility (~10 μm²/s) associated with vesicular transport in contrast to the prior reports of it originating from fluorophore photophysics and free fluorophores in the cytosol. In addition, β2-AR has rotational mobility (~100 μs) that makes it conform to the Saffman-Delbr{\"u}ck model of membrane diffusion unlike earlier studies. These contrasts are due to the limitations of the methodologies used. The limitations are overcome in this thesis by using different time-resolved fluorescence techniques of fluorescence correlation spectroscopy (FCS), time-resolved anisotropy (TRA) and polarisation resolved fullFCS (fullFCS). FCS is limited to microsecond to the second range and TRA is limited to the nanosecond range. fullFCS complements the two techniques by covering the blind spot of FCS and TRA in the microsecond range. Finally, I show how ligand stimulation causes a decrease in lateral mobility which could be a hint at cluster formation due to internalisation and how β2-AR possesses a basal oligomerisation that does not change on activation. Thus, through this thesis, I show how different complementary fluorescence techniques are necessary to overcome limitations of each technique and to thereby elucidate functional dynamics of GPCR activation and how it orchestrates downstream signalling.},
  language  = {en}
}
@article{KlenkHommersLohse2022,
  author    = {Klenk, Christoph and Hommers, Leif and Lohse, Martin J.},
  title     = {Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor},
  series = {Frontiers in Endocrinology},
  volume    = {13},
  journal   = {Frontiers in Endocrinology},
  issn      = {1664-2392},
  doi       = {10.3389/fendo.2022.839351},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-262055},
  year      = {2022},
  abstract  = {Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G\(_s\) and increased activation of G\(_q\) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.},
  language  = {en}
}
@phdthesis{Stetter2021,
  author    = {Stetter, Maurice},
  title     = {LC3-associated phagocytosis seals the fate of the second polar body in \(Caenorhabditis\) \(elegans\)},
  doi       = {10.25972/OPUS-23198},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231981},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {This work investigates the death and degradation of the second polar body of the nematode C. elegans in order to improve our understanding how pluripotent undifferentiated cells deal with dying cells. With the use of fluorescence microscopy this work demonstrates that both polar bodies loose membrane integrity early. The second polar body has contact to embryonic cells and gets internalized, dependent on the Rac1-ortholog CED-10. The polar body gets degraded via LC3-associated phagocytosis. While lysosome recruitment depends on RAB-7, LC3 does not improve lysosome recruitment but still accelerates polar body degradation. This work establishes the second polar body as a genetic model to study cell death and LC3-associated phagocytosis and has revealed further aspects of phagosome maturation and degradation.},
  subject      = {Polk{\"o}rper},
  language  = {en}
}
@article{KaiserSauerKisker2017,
  author    = {Kaiser, Sebastian and Sauer, Florian and Kisker, Caroline},
  title     = {The structural and functional characterization of human RecQ4 reveals insights into its helicase mechanism},
  series = {Nature Communications},
  volume    = {8},
  journal   = {Nature Communications},
  number    = {15907},
  doi       = {10.1038/ncomms15907},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170769},
  year      = {2017},
  abstract  = {RecQ4 is a member of the RecQ helicase family, an evolutionarily conserved class of enzymes, dedicated to preserving genomic integrity by operating in telomere maintenance, DNA repair and replication. While reduced RecQ4 activity is associated with cancer predisposition and premature aging, RecQ4 upregulation is related to carcinogenesis and metastasis. Within the RecQ family, RecQ4 assumes an exceptional position, lacking several characteristic RecQ domains. Here we present the crystal structure of human RecQ4, encompassing the conserved ATPase core and a novel C-terminal domain that lacks resemblance to the RQC domain observed in other RecQ helicases. The new domain features a zinc-binding site and two distinct types of winged-helix domains, which are not involved in canonical DNA binding or helicase activity. Based on our structural and functional analysis, we propose that RecQ4 exerts a helicase mechanism, which may be more closely related to bacterial RecQ helicases than to its human family members.},
  language  = {en}
}
@article{KronhardtBeitzingerBarthetal.2016,
  author    = {Kronhardt, Angelika and Beitzinger, Christoph and Barth, Holger and Benz, Roland},
  title     = {Chloroquine Analog Interaction with C2-and Iota-Toxin in Vitro and in Living Cells},
  series = {Toxins},
  volume    = {8},
  journal   = {Toxins},
  number    = {8},
  doi       = {10.3390/toxins8080237},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168286},
  pages     = {237},
  year      = {2016},
  abstract  = {C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells' receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa.},
  language  = {en}
}
@phdthesis{Beer2021,
  author    = {Beer, Katharina Beate},
  title     = {Identification and characterization of TAT-5 interactors that regulate extracellular vesicle budding},
  doi       = {10.25972/OPUS-20672},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206724},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Cells from bacteria to man release extracellular vesicles (EV) such as microvesicles (MV) that carry signaling molecules like morphogens and miRNAs to control intercellular communication during health and disease. MV release also sculpts membranes, e.g. repairing damaged membranes to avoid cell death. HIV viruses also bud from the plasma membrane in a similar fashion. In order to determine the in vivo functions of MVs and regulate their release, we need to understand the mechanisms of MV release by plasma membrane budding (ectocytosis). The conserved phospholipid flippase TAT-5 maintains the asymmetric localization of phosphatidylethanolamine (PE) in the plasma membrane and was the only known inhibitor of ESCRT-mediated ectocytosis in C. elegans. Loss of TAT-5 lipid flipping activity increased the externalization of PE and accumulation of MVs. However, it was unclear how cells control TAT-5 activity to release the right amount of MVs at the right time, since no upstream regulators of TAT-5 were known. To identify conserved TAT-5 regulators we looked for new proteins that inhibit MV release. To do so, we first developed a degradation-based technique to specifically label MVs. We tagged a plasma membrane reporter with the endogenous ZF1 degradation tag (degron) and expressed it in C. elegans embryos. This reporter is protected from degradation inside MVs, but is degraded inside the cell. Thus, the fluorescence is selectively maintained inside MVs, creating the first MV-specific reporter. We identified four MV release inhibitors associated with retrograde recycling, including the class III PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. We found that VPS-34, BEC-1, RME-8, and redundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit MV release. Although we confirmed that PAD-1 and the GEF-like protein MON-2 are required for endosomal recycling, they only traffic TAT-5 in the absence of sorting nexin-mediated recycling. Instead, PAD-1 is specifically required for the lipid flipping activity of TAT-5 that inhibits MV release. Thus, our work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis. In addition, we uncovered redundant intracellular trafficking pathways, which affect organelle size and revealed new regulators of TAT-5 flippase activity. These newly identified ectocytosis inhibitors provide a toolkit to test the in vivo roles of MVs. In the long term, our work will help to identify the mechanisms that govern MV budding, furthering our understanding of the mechanisms that regulate disease-mediated EV release, membrane sculpting and viral budding.},
  subject      = {Caenorhabditis elegans},
  language  = {en}
}
@article{GrabarczykBerks2017,
  author    = {Grabarczyk, Daniel B. and Berks, Ben C.},
  title     = {Intermediates in the Sox sulfur oxidation pathway are bound to a sulfane conjugate of the carrier protein SoxYZ},
  series = {PLoS ONE},
  volume    = {12},
  journal   = {PLoS ONE},
  number    = {3},
  doi       = {10.1371/journal.pone.0173395},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171147},
  pages     = {e0173395},
  year      = {2017},
  abstract  = {The Sox pathway found in many sulfur bacteria oxidizes thiosulfate to sulfate. Pathway intermediates are covalently bound to a cysteine residue in the carrier protein SoxYZ. We have used biochemical complementation by SoxYZ-conjugates to probe the identity of the intermediates in the Sox pathway. We find that unconjugated SoxYZ and SoxYZ-S-sulfonate are unlikely to be intermediates during normal turnover in disagreement with current models. By contrast, conjugates with multiple sulfane atoms are readily metabolised by the Sox pathway. The most parsimonious interpretation of these data is that the true carrier species in the Sox pathway is a SoxYZ-S-sulfane adduct.},
  language  = {en}
}
@phdthesis{Imam2023,
  author    = {Imam, Nasir},
  title     = {Molecular basis of collybistin conformational activation},
  doi       = {10.25972/OPUS-31145},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-311458},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The nervous system relies on an orchestrated assembly of complex cellular entities called neurons, which are specifically committed to information management and transmission. Inter-neuronal communication takes place via synapses, membrane-membrane junctions which ensure efficient signal transfer. Synaptic neurotransmission involves release of presynaptic neurotransmitters and their reception by cognate receptors at postsynaptic terminals. Inhibitory neurotransmission is primarily mediated by the release of neurotransmitters GABA (γ-Aminobutyric acid) and glycine, which are precisely sensed by GABA type-A receptors (GABAARs) and glycine receptors (GlyRs), respectively. GABAAR assembly and maintenance is coordinated by various postsynaptic neuronal factors including the scaffolding protein gephyrin, the neuronal adaptor collybistin (CB) and cell adhesion proteins of the neuroligin (NL) family, specifically NL2 and NL4. At inhibitory postsynaptic specializations, gephyrin has been hypothesized to form extended structures underneath the plasma membrane, where its interaction with the receptors leads to their stabilization and impedes their lateral movement. Gephyrin mutations have been associated with various brain disorders, including autism, schizophrenia, Alzheimer's disease, and epilepsy. Furthermore, gephyrin loss is lethal and causes mice to die within the first post-natal day. Gephyrin recruitment from intracellular deposits to postsynaptic membranes primarily relies on the adaptor protein CB. As a moonlighting protein, CB, a guanine nucleotide exchange factor (GEF), also catalyzes a nucleotide exchange reaction, thereby regenerating the GTP-bound state of the small GTPase Cdc42 from its GDP-bound form. The CB gene undergoes alternative splicing with the majority of CB splice variants featuring an N-terminal SH3 domain followed by tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains. Previous studies demonstrated that the most widely expressed, SH3-domain containing splice variant (CB2SH3+) preferentially adopts a closed conformation, in which the N-terminally located SH3 domain forms intra-molecular interaction with the DH-PH domain tandem. Previous cell-based studies indicated that SH3 domain-encoding CB variants remain untargeted and colocalize with intracellular gephyrin deposits and hence require additional factors which interact with the SH3 domain, thus inducing an open or active conformation. The SH3 domain-deficient CB isoform (CB2SH3-), on the contrary, adopts an open conformation, which possess enhanced postsynaptic gephyrin-clustering and also effectively replenishes the GTP-bound small GTPase-Cdc42 from its GDP-bound state. Despite the fundamental role of CB as a neuronal adaptor protein maintaining the proper function of inhibitory GABAergic synapses, its interactions with the neuronal scaffolding protein gephyrin and other post synaptic neuronal factors remain poorly understood. Moreover, CB interaction studies with the small GTPase Cdc42 and TC10, a closely related member of Cdc42 subfamily, remains poorly characterized. Most importantly, the roles of the neuronal factors and small GTPases in CB conformational activation have not been elucidated. This PhD dissertation primarily focuses on delineating the molecular basis of the interactions between CB and postsynaptic neuronal factors. During the course of my PhD dissertation, I engineered a series of CB FRET (F{\"o}rster Resonance Energy Transfer) sensors to characterize the CB interaction with its binding partners along with outlining their role in CB conformational activation. Through the aid of these CB FRET sensors, I analyzed the gephyrin-CB interaction, which, due to technical limitations remained unaddressed for more than two decades (refer Chapter 2 for more details). Subsequently, I also unraveled the molecular basis of the interactions between CB and the neuronal cell adhesion factor neuroligin 2 (refer chapter 2) and the small GTPases Cdc42 and TC10 (refer chapter 3) and describe how these binding partners induce a conformational activation of CB. In summary, this PhD dissertation provides strong evidence of a closely knit CB communication network with gephyrin, neuroligin and the small GTPase TC10, wherein CB activation from closed/inactive to open/active states is effectively triggered by these ligands.},
  language  = {en}
}
@article{KuhlemannBeliuJanzenetal.2021,
  author    = {Kuhlemann, Alexander and Beliu, Gerti and Janzen, Dieter and Petrini, Enrica Maria and Taban, Danush and Helmerich, Dominic A. and Doose, S{\"o}ren and Bruno, Martina and Barberis, Andrea and Villmann, Carmen and Sauer, Markus and Werner, Christian},
  title     = {Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy},
  series = {Frontiers in Synaptic Neuroscience},
  volume    = {13},
  journal   = {Frontiers in Synaptic Neuroscience},
  issn      = {1663-3563},
  doi       = {10.3389/fnsyn.2021.727406},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251035},
  year      = {2021},
  abstract  = {Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.},
  language  = {en}
}
@article{JeanclosKnoblochHoffmannetal.2020,
  author    = {Jeanclos, Elisabeth and Knobloch, Gunnar and Hoffmann, Axel and Fedorchenko, Oleg and Odersky, Andrea and Lamprecht, Anna-Karina and Schindelin, Hermann and Gohla, Antje},
  title     = {Ca\(^{2+}\) functions as a molecular switch that controls the mutually exclusive complex formation of pyridoxal phosphatase with CIB1 or calmodulin},
  series = {FEBS Letters},
  volume    = {594},
  journal   = {FEBS Letters},
  number    = {13},
  doi       = {10.1002/1873-3468.13795},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217963},
  pages     = {2099 -- 2115},
  year      = {2020},
  abstract  = {Pyridoxal 5′-phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ-aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)- and integrin-binding protein 1 (CIB1) as a PDXP interactor by yeast two-hybrid screening and find a calmodulin (CaM)-binding motif that overlaps with the PDXP-CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP-CIB1 complex may functionally differ from the PDXP-Ca\(^{2+}\)-CaM complex.},
  language  = {en}
}
@article{SchuhmannKraftBieberetal.2019,
  author    = {Schuhmann, Michael K. and Kraft, Peter and Bieber, Michael and Kollikowski, Alexander M. and Schulze, Harald and Nieswandt, Bernhard and Pham, Mirko and Stegner, David and Stoll, Guido},
  title     = {Targeting platelet GPVI plus rt-PA administration but not α2β1-mediated collagen binding protects against ischemic brain damage in mice},
  series = {International Journal of Molecular Science},
  volume    = {20},
  journal   = {International Journal of Molecular Science},
  number    = {8},
  issn      = {1422-0067},
  doi       = {10.3390/ijms20082019},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201700},
  year      = {2019},
  abstract  = {Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{-/-}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.},
  language  = {en}
}
@article{MakbulKhayenkoMaricetal.2021,
  author    = {Makbul, Cihan and Khayenko, Vladimir and Maric, Hans Michael and B{\"o}ttcher, Bettina},
  title     = {Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype},
  series = {Microorganisms},
  volume    = {9},
  journal   = {Microorganisms},
  number    = {5},
  issn      = {2076-2607},
  doi       = {10.3390/microorganisms9050956},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236720},
  year      = {2021},
  abstract  = {Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an "LLGRMKG" motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide "GSLLGRMKGA" binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies "SLLGRM" as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.},
  language  = {en}
}
@article{KitzenmaierSchaeferKasaragodetal.2019,
  author    = {Kitzenmaier, Alexandra and Schaefer, Natascha and Kasaragod, Vikram Babu and Polster, Tilman and Hantschmann, Ralph and Schindelin, Hermann and Villmann, Carmen},
  title     = {The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia},
  series = {European Journal of Neuroscience},
  volume    = {50},
  journal   = {European Journal of Neuroscience},
  number    = {12},
  doi       = {10.1111/ejn.14533},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206158},
  pages     = {3906-3920},
  year      = {2019},
  abstract  = {Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α-helix 5, presumably disrupting the integrity of this α-helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild-type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non-symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild-type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α-helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium-binding site of the transporter.},
  language  = {en}
}
@article{KalledaAmichArslanetal.2016,
  author    = {Kalleda, Natarajaswamy and Amich, Jorge and Arslan, Berkan and Poreddy, Spoorthi and Mattenheimer, Katharina and Mokhtari, Zeinab and Einsele, Hermann and Brock, Matthias and Heinze, Katrin Gertrud and Beilhack, Andreas},
  title     = {Dynamic Immune Cell Recruitment After Murine Pulmonary Aspergillus fumigatus Infection under Different Immunosuppressive Regimens},
  series = {Frontiers in Microbiology},
  volume    = {7},
  journal   = {Frontiers in Microbiology},
  number    = {1107},
  doi       = {10.3389/fmicb.2016.01107},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165368},
  year      = {2016},
  abstract  = {Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4\(^+\) or CD8\(^+\) T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b\(^+\) myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b\(^+\) myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions.},
  language  = {en}
}
@article{KooMatthewsHarrisonetal.2022,
  author    = {Koo, Chek Ziu and Matthews, Alexandra L. and Harrison, Neale and Szyroka, Justyna and Nieswandt, Bernhard and Gardiner, Elizabeth E. and Poulter, Natalie S. and Tomlinson, Michael G.},
  title     = {The platelet collagen receptor GPVI is cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 molecular scissors},
  series = {International Journal of Molecular Sciences},
  volume    = {23},
  journal   = {International Journal of Molecular Sciences},
  number    = {5},
  issn      = {1422-0067},
  doi       = {10.3390/ijms23052440},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284468},
  year      = {2022},
  abstract  = {The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a 'molecular scissor' that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.},
  language  = {en}
}
@article{StegnerKlausNieswandt2019,
  author    = {Stegner, David and Klaus, Vanessa and Nieswandt, Bernhard},
  title     = {Platelets as modulators of cerebral ischemia/reperfusion injury},
  series = {Frontiers in Immunology},
  volume    = {10},
  journal   = {Frontiers in Immunology},
  number    = {2505},
  issn      = {1664-3224},
  doi       = {10.3389/fimmu.2019.02505},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195748},
  year      = {2019},
  abstract  = {Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed "reperfusion injury." Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury.},
  language  = {en}
}
@article{DuettingGaitsIacovoniStegneretal.2017,
  author    = {D{\"u}tting, Sebastian and Gaits-Iacovoni, Frederique and Stegner, David and Popp, Michael and Antkowiak, Adrien and van Eeuwijk, Judith M.M. and Nurden, Paquita and Stritt, Simon and Heib, Tobias and Aurbach, Katja and Angay, Oguzhan and Cherpokova, Deya and Heinz, Niels and Baig, Ayesha A. and Gorelashvili, Maximilian G. and Gerner, Frank and Heinze, Katrin G. and Ware, Jerry and Krohne, Georg and Ruggeri, Zaverio M. and Nurden, Alan T. and Schulze, Harald and Modlich, Ute and Pleines, Irina and Brakebusch, Cord and Nieswandt, Bernhard},
  title     = {A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis},
  series = {Nature Communications},
  volume    = {8},
  journal   = {Nature Communications},
  number    = {15838},
  doi       = {10.1038/ncomms15838},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170797},
  year      = {2017},
  abstract  = {Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard-Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.},
  language  = {en}
}
@article{StegnervanEeuwijkAngayetal.2017,
  author    = {Stegner, David and van Eeuwijk, Judith M.M. and Angay, Oğuzhan and Gorelashvili, Maximilian G. and Semeniak, Daniela and Pinnecker, J{\"u}rgen and Schmithausen, Patrick and Meyer, Imke and Friedrich, Mike and D{\"u}tting, Sebastian and Brede, Christian and Beilhack, Andreas and Schulze, Harald and Nieswandt, Bernhard and Heinze, Katrin G.},
  title     = {Thrombopoiesis is spatially regulated by the bone marrow vasculature},
  series = {Nature Communications},
  volume    = {8},
  journal   = {Nature Communications},
  number    = {127},
  doi       = {10.1038/s41467-017-00201-7},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170591},
  year      = {2017},
  abstract  = {In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.},
  language  = {en}
}
@article{KollikowskiSchuhmannNieswandtetal.2020,
  author    = {Kollikowski, Alexander M. and Schuhmann, Michael K. and Nieswandt, Bernhard and M{\"u}llges, Wolfgang and Stoll, Guido and Pham, Mirko},
  title     = {Local Leukocyte Invasion during Hyperacute Human Ischemic Stroke},
  series = {Annals of Neurology},
  volume    = {87},
  journal   = {Annals of Neurology},
  number    = {3},
  doi       = {10.1002/ana.25665},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212168},
  pages     = {466-479},
  year      = {2020},
  abstract  = {Objective Bridging the gap between experimental stroke and patients by ischemic blood probing during the hyperacute stage of vascular occlusion is crucial to assess the role of inflammation in human stroke and for the development of adjunct treatments beyond recanalization. Methods We prospectively observed 151 consecutive ischemic stroke patients with embolic large vessel occlusion of the anterior circulation who underwent mechanical thrombectomy. In all these patients, we attempted microcatheter aspiration of 3 different arterial blood samples: (1) within the core of the occluded vascular compartment and controlled by (2) carotid and (3) femoral samples obtained under physiological flow conditions. Subsequent laboratory analyses comprised leukocyte counting and differentiation, platelet counting, and the quantification of 13 proinflammatory human chemokines/cytokines. Results Forty patients meeting all clinical, imaging, interventional, and laboratory inclusion criteria could be analyzed, showing that the total number of leukocytes significantly increased under the occlusion condition. This increase was predominantly driven by neutrophils. Significant increases were also apparent for lymphocytes and monocytes, accompanied by locally elevated plasma levels of the T-cell chemoattractant CXCL-11. Finally, we found evidence that short-term clinical outcome (National Institute of Health Stroke Scale at 72 hours) was negatively associated with neutrophil accumulation. Interpretation We provide the first direct human evidence that neutrophils, lymphocytes, and monocytes, accompanied by specific chemokine upregulation, accumulate in the ischemic vasculature during hyperacute stroke and may affect outcome. These findings strongly support experimental evidence that immune cells contribute to acute ischemic brain damage and indicate that ischemic inflammation initiates already during vascular occlusion. Ann Neurol 2020;87:466-479},
  language  = {en}
}
@article{GoebVollZimmermannetal.2021,
  author    = {G{\"o}b, Vanessa and Voll, Maximilian G. and Zimmermann, Lena and Hemmen, Katharina and Stoll, Guido and Nieswandt, Bernhard and Schuhmann, Michael K. and Heinze, Katrin G. and Stegner, David},
  title     = {Infarct growth precedes cerebral thrombosis following experimental stroke in mice},
  series = {Scientific Reports},
  volume    = {11},
  journal   = {Scientific Reports},
  number    = {1},
  doi       = {10.1038/s41598-021-02360-6},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265791},
  year      = {2021},
  abstract  = {Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, successful recanalization of occluded vessels is the primary therapeutic aim, but even if it is achieved, not all patients benefit. Although blockade of platelet aggregation did not prevent infarct progression, cerebral thrombosis as cause of secondary infarct growth has remained a matter of debate. As cerebral thrombi are frequently observed after experimental stroke, a thrombus-induced impairment of the brain microcirculation is considered to contribute to tissue damage. Here, we combine the model of transient middle cerebral artery occlusion (tMCAO) with light sheet fluorescence microscopy and immunohistochemistry of brain slices to investigate the kinetics of thrombus formation and infarct progression. Our data reveal that tissue damage already peaks after 8 h of reperfusion following 60 min MCAO, while cerebral thrombi are only observed at later time points. Thus, cerebral thrombosis is not causative for secondary infarct growth during ischemic stroke.},
  language  = {en}
}
@article{BeckStegnerLorochetal.2021,
  author    = {Beck, Sarah and Stegner, David and Loroch, Stefan and Baig, Ayesha A. and G{\"o}b, Vanessa and Schumbutzki, Cornelia and Eilers, Eva and Sickmann, Albert and May, Frauke and Nolte, Marc W. and Panousis, Con and Nieswandt, Bernhard},
  title     = {Generation of a humanized FXII knock-in mouse-A powerful model system to test novel anti-thrombotic agents},
  series = {Journal of Thrombosis and Haemostasis},
  volume    = {19},
  journal   = {Journal of Thrombosis and Haemostasis},
  number    = {11},
  doi       = {10.1111/jth.15488},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259567},
  pages     = {2835-2840},
  year      = {2021},
  abstract  = {Background Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII. Objective The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model. Methods A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models. Results These hF12\(^{KI}\) mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12\(^{KI}\) mice in an arterial thrombosis model without affecting bleeding times. Conclusion These data establish the newly generated hF12\(^{KI}\) mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.},
  language  = {en}
}
@phdthesis{Aigner2023,
  author    = {Aigner, Max},
  title     = {Establishing successful protocols and imaging pipelines for Expansion Microscopy in murine blood platelets},
  doi       = {10.25972/OPUS-30900},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-309003},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Platelets play an important role in the body, since they are part of the hemostasis system, preventing and stopping blood loss. Nevertheless, when platelet or coagulation system function are impaired, uncontrolled bleedings but also irreversible vessel occlusion followed by ischemic tissue damage can occur. Therefore, understanding platelet function and activation, mechanisms which are controlled by a variety of platelet membrane receptors and other factors is important to advance out knowledge of hemostasis and platelet malfunction. For a complete picture of platelet function and their modulating behavior it is desired to be able to quantify receptor distributions and interactions of these densely packed molecular ensembles in the membrane. This challenges scientists for several reasons. Most importantly, platelets are microscopically small objects, challenging the spatial resolution of conventional light microscopy. Moreover, platelet receptors are highly abundant on the membrane so even super-resolution microscopy struggles with quantitative receptor imaging on platelets. With Expansion microscopy (ExM), a new super-resolution technique was introduced, allowing resolutions to achieve super-resolution without using a super-resolution microscope, but by combining a conventional confocal microscopy with a highly processed sample that has been expanded physically. In this doctoral thesis, I evaluated the potential of this technique for super-resolution platelet imaging by optimizing the sample preparation process and establishing an imaging and image processing pipeline for dual-color 3D images of different membrane receptors. The analysis of receptor colocalization using ExM demonstrated a clear superiority compared to conventional microscopy. Furthermore, I identified a library of fluorescently labeled antibodies against different platelet receptors compatible with ExM and showed the possibility of staining membrane receptors and parts of the cytoskeleton at the same time.},
  subject      = {Mikroskopie},
  language  = {en}
}
@article{MammadovaBachBraun2019,
  author    = {Mammadova-Bach, Elmina and Braun, Attila},
  title     = {Zinc homeostasis in platelet-related diseases},
  series = {International Journal of Molecular Sciences},
  volume    = {20},
  journal   = {International Journal of Molecular Sciences},
  number    = {21},
  issn      = {1422-0067},
  doi       = {10.3390/ijms20215258},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285554},
  year      = {2019},
  abstract  = {Zn\(^{2+}\) deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn\(^{2+}\)-deficient diets, accounting for 1-4\% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn\(^{2+}\) deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn\(^{2+}\) status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn\(^{2+}\) uptake in the gut using different nutritional supplementation of Zn\(^{2+}\) could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn\(^{2+}\) diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn\(^{2+}\) in hemostasis. Storage protein metallothionein maintains or releases Zn\(^{2+}\) in the cytoplasm, and the dynamic change of this cytoplasmic Zn\(^{2+}\) pool is regulated by the redox status of the cell. An increase of labile Zn\(^{2+}\) pool can be toxic for the cells, and therefore cytoplasmic Zn\(^{2+}\) levels are tightly regulated by several Zn\(^{2+}\) transporters located on the cell surface and also on the intracellular membrane of Zn\(^{2+}\) storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn\(^{2+}\) is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn\(^{2+}\) transport and the physiological role of Zn\(^{2+}\) store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn\(^{2+}\) to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn\(^{2+}\) homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.},
  language  = {en}
}
@article{RiesLiessFeileretal.2020,
  author    = {Ries, Lena K. and Liess, Anna K. L. and Feiler, Christian G. and Spratt, Donald E. and Lowe, Edward D. and Lorenz, Sonja},
  title     = {Crystal structure of the catalytic C-lobe of the HECT-type ubiquitin ligase E6AP},
  series = {Protein Science},
  volume    = {29},
  journal   = {Protein Science},
  number    = {6},
  doi       = {10.1002/pro.3832},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214812},
  pages     = {1550 -- 1554},
  year      = {2020},
  abstract  = {The HECT-type ubiquitin ligase E6AP (UBE3A) is critically involved in several neurodevelopmental disorders and human papilloma virus-induced cervical tumorigenesis; the structural mechanisms underlying the activity of this crucial ligase, however, are incompletely understood. Here, we report a crystal structure of the C-terminal lobe ("C-lobe") of the catalytic domain of E6AP that reveals two molecules in a domain-swapped, dimeric arrangement. Interestingly, the molecular hinge that enables this structural reorganization with respect to the monomeric fold coincides with the active-site region. While such dimerization is unlikely to occur in the context of full-length E6AP, we noticed a similar domain swap in a crystal structure of the isolated C-lobe of another HECT-type ubiquitin ligase, HERC6. This may point to conformational strain in the active-site region of HECT-type ligases with possible implications for catalysis. Significance Statement The HECT-type ubiquitin ligase E6AP has key roles in human papilloma virus-induced cervical tumorigenesis and certain neurodevelopmental disorders. Here, we present a crystal structure of the C-terminal, catalytic lobe of E6AP, providing basic insight into the conformational properties of this functionally critical region of HECT-type ligases.},
  language  = {en}
}
@article{SchuhmannBieberFrankeetal.2021,
  author    = {Schuhmann, Michael K. and Bieber, Michael and Franke, Maximilian and Kollikowski, Alexander M. and Stegner, David and Heinze, Katrin G. and Nieswandt, Bernhard and Pham, Mirko and Stoll, Guido},
  title     = {Platelets and lymphocytes drive progressive penumbral tissue loss during middle cerebral artery occlusion in mice},
  series = {Journal of Neuroinflammation},
  volume    = {18},
  journal   = {Journal of Neuroinflammation},
  number    = {1},
  doi       = {10.1186/s12974-021-02095-1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259172},
  pages     = {46},
  year      = {2021},
  abstract  = {Background In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood. Methods To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1\(^{-/-}\) mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion. Results We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion. Conclusions Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization.},
  language  = {en}
}
@phdthesis{Mietrach2020,
  author    = {Mietrach, Nicole Aline},
  title     = {Structural and functional elucidation of the Type VIIb secretion system from Staphylococcus aureus},
  doi       = {10.25972/OPUS-21482},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214824},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The Type VII secretion system (T7SS) is linked to virulence and long-term pathogenesis in a broad range of Gram-positive bacteria, including the human commensal and pathogen Staphylococcus aureus. The Type VIIb secretion system (T7SSb) is responsible for the export of small toxic proteins, which induce antibacterial immune responses and mediate bacterial persistence in the host. In addition, it is also involved in bacterial competition. The T7SSb requires several proteins to build up the secretion machinery. This work focuses on the structural and functional investigation of the motor ATPase EssC and the putative pore forming, multi-pass membrane component EsaA. Both proteins are indispensable for substrate secretion. EssC belongs to the FtsK/SpoIIIE ATPase family and is conserved among the T7SSs. It contains three C-terminal, cytosolic ATPase domains, designated as EssC- D1, -D2 and -D3, whereby EssC-D3 is the most distal one. In this thesis, I am presenting the crystal structure of the EssC-D3 at 1.7 {\AA} resolution. As the deletion of EssC-D3 abrogates substrate export, I have demonstrated that this domain comprises a hydrophobic, surface-exposed pocket, which is required for substrate secretion. More specifically, I have identified two amino acids involved in the secretion process. In addition, my results indicate that not only EssC-D3 is important for substrate interaction but also EssC-D2 and/or EssC-D1. Unlike in the related Yuk T7SSb of Bacillus subtilis, the ATPase activity of D3 domain contributes to substrate secretion. Mutation of the modified Walker B motif in EssC-D3 diminishes substrate secretion completely. The membrane protein EsaA encompasses an extracellular segment spanning through the cell wall of S. aureus. I was able to reveal that this part folds into a stable domain, which was crystallized and diffracted up to 4 {\AA}. The first attempts to dissolve the structure failed due to a lack of homologues structures. Therefore, crystals for single-wavelength anomalous dispersion, containing selenomethionyl-substitutes, were produced and the structure solution is still in progress. Preliminary experiments addressing the function of the extracellular domain indicate an important role in substrate secretion and bacterial competition.},
  subject      = {Secretion},
  language  = {en}
}
@article{PetrusevaNaumenkoKuperetal.2021,
  author    = {Petruseva, Irina and Naumenko, Natalia and Kuper, Jochen and Anarbaev, Rashid and Kappenberger, Jeannette and Kisker, Caroline and Lavrik, Olga},
  title     = {The Interaction Efficiency of XPD-p44 With Bulky DNA Damages Depends on the Structure of the Damage},
  series = {Frontiers in Cell and Developmental Biology},
  volume    = {9},
  journal   = {Frontiers in Cell and Developmental Biology},
  issn      = {2296-634X},
  doi       = {10.3389/fcell.2021.617160},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231806},
  year      = {2021},
  abstract  = {The successful elimination of bulky DNA damages via the nucleotide excision repair (NER) system is largely determined by the damage recognition step. This step consists of primary recognition and verification of the damage. The TFIIH helicase XPD plays a key role in the verification step during NER. To date, the mechanism of damage verification is not sufficiently understood and requires further detailed research. This study is a systematic investigation of the interaction of ctXPD (Chaetomium thermophilum) as well as ctXPD-ctp44 with model DNAs, which contain structurally different bulky lesions with previously estimated NER repair efficiencies. We have used ATPase and DNA binding studies to assess the interaction of ctXPD with damaged DNA. The result of the analysis of ctXPD-ctp44 binding to DNA containing fluorescent and photoactivatable lesions demonstrates the relationship between the affinity of XPD for DNAs containing bulky damages and the ability of the NER system to eliminate the damage. Photo-cross-linking of ctXPD with DNA probes containing repairable and unrepairable photoactivatable damages reveals differences in the DNA interaction efficiency in the presence and absence of ctp44. In general, the results obtained indicate the ability of ctXPD-ctp44 to interact with a damage and suggest a significant role for ctp44 subunit in the verification process.},
  language  = {en}
}
@article{PeissertSauerGrabarczyketal.2020,
  author    = {Peissert, Stefan and Sauer, Florian and Grabarczyk, Daniel B. and Braun, Cathy and Sander, Gudrun and Poterszman, Arnaud and Egly, Jean-Marc and Kuper, Jochen and Kisker, Caroline},
  title     = {In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair},
  series = {Nature Communications},
  volume    = {11},
  journal   = {Nature Communications},
  number    = {1},
  doi       = {10.1038/s41467-020-15241-9},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229857},
  year      = {2020},
  abstract  = {The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold.},
  language  = {en}
}
@article{WagnerMottUpcinetal.2021,
  author    = {Wagner, Nicole and Mott, Kristina and Upcin, Berin and Stegner, David and Schulze, Harald and Erg{\"u}n, S{\"u}leyman},
  title     = {CXCL12-abundant reticular (CAR) cells direct megakaryocyte protrusions across the bone marrow sinusoid wall},
  series = {Cells},
  volume    = {10},
  journal   = {Cells},
  number    = {4},
  issn      = {2073-4409},
  doi       = {10.3390/cells10040722},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-234180},
  year      = {2021},
  abstract  = {Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.},
  language  = {en}
}
@article{HarnošCanizalJuraseketal.2019,
  author    = {Harnoš, Jakub and Ca{\~n}izal, Maria Consuelo Alonso and Jur{\´a}sek, Miroslav and Kumar, Jitender and Holler, Cornelia and Schambony, Alexandra and Han{\´a}kov{\´a}, Kateřina and Bernat{\´i}k, Ondřej and Zdr{\´a}hal, Zbyn{\^e}k and G{\"o}m{\"o}ryov{\´a}, Krist{\´i}na and Gybeľ, Tom{\´a}š and Radaszkiewicz, Tomasz Witold and Kravec, Marek and Trant{\´i}rek, Luk{\´a}š and Ryneš, Jan and Dave, Zankruti and Fern{\´a}ndez-Llamazares, Ana Iris and V{\´a}cha, Robert and Tripsianes, Konstantinos and Hoffmann, Carsten and Bryja, V{\´i}tězslav},
  title     = {Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1},
  series = {Nature Communications},
  volume    = {10},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-019-09651-7},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227837},
  year      = {2019},
  abstract  = {Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.},
  language  = {en}
}
@article{HeilSchreiberGoetzetal.2018,
  author    = {Heil, Hannah S. and Schreiber, Benjamin and G{\"o}tz, Ralph and Emmerling, Monika and Dabauvalle, Marie-Christine and Krohne, Georg and H{\"o}fling, Sven and Kamp, Martin and Sauer, Markus and Heinze, Katrin G.},
  title     = {Sharpening emitter localization in front of a tuned mirror},
  series = {Light: Science \& Applications},
  volume    = {7},
  journal   = {Light: Science \& Applications},
  doi       = {10.1038/s41377-018-0104-z},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-228080},
  year      = {2018},
  abstract  = {Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2,3,4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.},
  language  = {en}
}
@article{JarickMokhtariSchelleretal.2018,
  author    = {Jarick, Katja J. and Mokhtari, Zeinab and Scheller, Lukas and Hartweg, Julia and Thusek, Sina and Le, Duc-Dung and Ranecky, Maria and Shaikh, Haroon and Qureischi, Musga and Heinze, Katrin G. and Beilhack, Andreas},
  title     = {Photoconversion of Alloreactive T Cells in Murine Peyer's Patches During Acute Graft-Versus-Host Disease: Tracking the Homing Route of Highly Proliferative Cells In Vivo},
  series = {Frontiers in Immunology},
  volume    = {9},
  journal   = {Frontiers in Immunology},
  doi       = {10.3389/fimmu.2018.01468},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323309},
  year      = {2018},
  abstract  = {The regulation of immune cell migration throughout the body is essential to warrant immunosurveillance and to maintain immune homeostasis. Marking and tracking of these cells has proven important to study mechanisms of immune cell trafficking and cell interaction in vivo. Photoconversion is a well-suited technique for intravital application because it enables contactless time- and location-specific marking of cells in the tissue without surgically manipulating the microenvironment of the cells in question. However, in dividing cells the converted fluorescent protein may decline quickly. Here, we provide a detailed description of the photoconversion technique and its applicability to tracking highly proliferating T cells from the priming site of T cell activation to peripheral target organs of effector function in a preclinical model. Dendra2+ T cells were photoconverted in the Peyer's patches during the initiation phase of acute graft-versus-host disease (GvHD) and tracked through the mesenteric lymph nodes and the peripheral blood to the small intestine with flow cytometry and intravital two-photon microscopy. Photoconverted alloreactive T cells preserved the full proliferative capacity, homing, and migration of alloreactive T cells in the intestinal lamina propria. We conclusively proved that photoconversion of highly proliferative alloreactive T cells in the Peyer's patches is an effective tool to study trafficking of alloreactive T cells under physiologic conditions and to GvHD target tissues. This technique can also be applied to the study of immune cell tracking under inflammatory and non-inflammatory conditions.},
  language  = {en}
}
@article{HersterBittnerCodreaetal.2019,
  author    = {Herster, Franziska and Bittner, Zsofia and Codrea, Marius Cosmin and Archer, Nathan K. and Heister, Martin and L{\"o}ffler, Markus W. and Heumos, Simon and Wegner, Joanna and Businger, Ramona and Schindler, Michael and Stegner, David and Sch{\"a}kel, Knut and Grabbe, Stephan and Ghoreschi, Kamran and Miller, Lloyd S. and Weber, Alexander N. R.},
  title     = {Platelets Aggregate With Neutrophils and Promote Skin Pathology in Psoriasis},
  series = {Frontiers in Immunology},
  volume    = {10},
  journal   = {Frontiers in Immunology},
  doi       = {10.3389/fimmu.2019.01867},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-320175},
  year      = {2019},
  abstract  = {Psoriasis is a frequent systemic inflammatory autoimmune disease characterized primarily by skin lesions with massive infiltration of leukocytes, but frequently also presents with cardiovascular comorbidities. Especially polymorphonuclear neutrophils (PMNs) abundantly infiltrate psoriatic skin but the cues that prompt PMNs to home to the skin are not well-defined. To identify PMN surface receptors that may explain PMN skin homing in psoriasis patients, we screened 332 surface antigens on primary human blood PMNs from healthy donors and psoriasis patients. We identified platelet surface antigens as a defining feature of psoriasis PMNs, due to a significantly increased aggregation of neutrophils and platelets in the blood of psoriasis patients. Similarly, in the imiquimod-induced experimental in vivo mouse model of psoriasis, disease induction promoted PMN-platelet aggregate formation. In psoriasis patients, disease incidence directly correlated with blood platelet counts and platelets were detected in direct contact with PMNs in psoriatic but not healthy skin. Importantly, depletion of circulating platelets in mice in vivo ameliorated disease severity significantly, indicating that both PMNs and platelets may be relevant for psoriasis pathology and disease severity.},
  language  = {en}
}
@phdthesis{WeigelverhHoffmann2024,
  author    = {Weigel [verh. Hoffmann], Mathis Leonard},
  title     = {Thrombozytenfunktionsanalyse als potenzielles Instrument zur Fr{\"u}herkennung von Sepsis},
  doi       = {10.25972/OPUS-35819},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358193},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Sepsis ist ein h{\"a}ufiges und akut lebensbedrohliches Syndrom, das eine Organfunktionsst{\"o}rung in Folge einer dysregulierten Immunantwort auf eine Infektion beschreibt. Eine fr{\"u}hzeitige Diagnosestellung und Therapieeinleitung sind von zentraler Bedeutung f{\"u}r das {\"U}berleben der Patient:innen. In einer Pilotstudie konnte unsere Forschungsgruppe mittels Durchflusszytometrie eine ausgepr{\"a}gte Hyporeaktivit{\"a}t der Thrombozyten bei Sepsis nachweisen, die einen potenziell neuen Biomarker zur Sepsis-Fr{\"u}herkennung darstellt. Zur Evaluation des Ausmaßes und Entstehungszeitpunktes der detektierten Thrombozytenfunktionsst{\"o}rung wurden im Rahmen der vorliegenden Arbeit zus{\"a}tzlich zu Patient:innen mit Sepsis (SOFA-Score ≥ 2; n=13) auch hospitalisierte Patient:innen mit einer Infektion ohne Sepsis (SOFA-Score < 2; n=12) rekrutiert. Beide Kohorten wurden zu zwei Zeitpunkten (t1: <24h; t2: Tag 5-7) im Krankheitsverlauf mittels Durchflusszytometrie und PFA-200 untersucht und mit einer gesunden Kontrollgruppe (n=28) verglichen. Ph{\"a}notypische Auff{\"a}lligkeiten der Thrombozyten bei Sepsis umfassten: (i) eine ver{\"a}nderte Expression verschiedener Untereinheiten des GPIb-IX-V-Rezeptorkomplexes, die auf ein verst{\"a}rktes Rezeptor-Shedding hindeutet; (ii) ein ausgepr{\"a}gtes Mepacrin-Beladungsdefizit, das auf eine zunehmend reduzierte Anzahl von δ-Granula entlang des Infektion-Sepsis Kontinuums hinweist; (iii) eine Reduktion endst{\"a}ndig gebundener Sialins{\"a}ure im Sinne einer verst{\"a}rkten Desialylierung. Die funktionelle Analyse der Thrombozyten bei Sepsis ergab bei durchflusszytometrischer Messung der Integrin αIIbβ3-Aktivierung (PAC-1-Bindung) eine ausgepr{\"a}gte generalisierte Hyporeaktivit{\"a}t gegen{\"u}ber multiplen Agonisten, die abgeschw{\"a}cht bereits bei Infektion nachweisbar war und gem{\"a}ß ROC-Analysen gut zwischen Infektion und Sepsis diskriminierte (AUC >0.80 f{\"u}r alle Agonisten). Im Gegensatz dazu zeigten Thrombozyten bei Sepsis und Analyse mittels PFA-200 unter Einfluss physiologischer Scherkr{\"a}fte eine normale bis gar beschleunigte Aggregation. Die Reaktivit{\"a}tsmessung von Thrombozyten mittels Durchflusszytometrie stellt weiterhin einen vielversprechenden Biomarker f{\"u}r die Sepsis-Fr{\"u}herkennung dar. F{\"u}r weitere Schlussfolgerungen ist jedoch eine gr{\"o}ßere Kohorte erforderlich. In nachfolgenden Untersuchungen sollten zudem mechanistische Ursachen der beschriebenen ph{\"a}notypischen und funktionellen Auff{\"a}lligkeiten von Thrombozyten bei Infektion und Sepsis z.B. mittels Koinkubationsexperimenten untersucht werden.},
  subject      = {Sepsis},
  language  = {de}
}
@article{GotruvanGeffenNagyetal.2019,
  author    = {Gotru, Sanjeev Kiran and van Geffen, Johanna P. and Nagy, Magdolna and Mammadova-Bach, Elmina and Eilenberger, Julia and Volz, Julia and Manukjan, Georgi and Schulze, Harald and Wagner, Leonard and Eber, Stefan and Schambeck, Christian and Deppermann, Carsten and Brouns, Sanne and Nurden, Paquita and Greinacher, Andreas and Sachs, Ulrich and Nieswandt, Bernhard and Hermanns, Heike M. and Heemskerk, Johan W. M. and Braun, Attila},
  title     = {Defective Zn2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases},
  series = {Scientific Reports},
  volume    = {9},
  journal   = {Scientific Reports},
  doi       = {10.1038/s41598-019-44751-w},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227455},
  year      = {2019},
  abstract  = {Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d-/- mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2-/- mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2-/- and Unc13d-/- mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.},
  language  = {en}
}
@article{HinesMaricHinesetal.2018,
  author    = {Hines, Rochelle M. and Maric, Hans Michael and Hines, Dustin J. and Modgil, Amit and Panzanelli, Patrizia and Nakamura, Yasuko and Nathanson, Anna J. and Cross, Alan and Deeb, Tarek and Brandon, Nicholas J. and Davies, Paul and Fritschy, Jean-Marc and Schindelin, Hermann and Moss, Stephen J.},
  title     = {Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin},
  series = {Nature Communications},
  volume    = {9},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-018-05481-1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-320719},
  year      = {2018},
  abstract  = {Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2-1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.},
  language  = {en}
}
@article{FazeliStetterLisacketal.2018,
  author    = {Fazeli, Gholamreza and Stetter, Maurice and Lisack, Jaime N. and Wehman, Ann M.},
  title     = {C. elegans Blastomeres Clear the Corpse of the Second Polar Body by LC3-Associated Phagocytosis},
  series = {Cell Reports},
  volume    = {23},
  journal   = {Cell Reports},
  doi       = {10.1016/j.celrep.2018.04.043},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227651},
  pages     = {2070-2082},
  year      = {2018},
  abstract  = {To understand how undifferentiated pluripotent cells cope with cell corpses, we examined the clearance of polar bodies born during female meiosis. We found that polar bodies lose membrane integrity and expose phosphatidylserine in Caenorhabditis elegans. Polar body signaling recruits engulfment receptors to the plasma membrane of embryonic blastomeres using the PI3K VPS-34, RAB-5 GTPase and the sorting nexin SNX-6. The second polar body is then phagocytosed using receptor-mediated engulfment pathways dependent on the Rac1 ortholog CED-10 but undergoes non-apoptotic programmed cell death independent of engulfment. RAB-7 GTPase is required for lysosome recruitment to the polar body phagosome, while LC3 lipidation is required for degradation of the corpse membrane after lysosome fusion. The polar body phagolysosome vesiculates in an mTOR- and ARL-8-dependent manner, which assists its timely degradation. Thus, we established a genetic model to study clearance by LC3-associated phagocytosis and reveal insights into the mechanisms of phagosome maturation and degradation.},
  language  = {en}
}
@phdthesis{Karwen2024,
  author    = {Karwen, Till},
  title     = {Platelets promote insulin secretion of pancreatic β-cells},
  doi       = {10.25972/OPUS-31393},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313933},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The pancreas is the key organ for the maintenance of euglycemia. This is regulated in particular by α-cell-derived glucagon and β-cell-derived insulin, which are released in response to nutrient deficiency and elevated glucose levels, respectively. Although glucose is the main regulator of insulin secretion, it is significantly enhanced by various potentiators. Platelets are anucleate cell fragments in the bloodstream that are essential for hemostasis to prevent and stop bleeding events. Besides their classical role, platelets were implemented to be crucial for other physiological and pathophysiological processes, such as cancer progression, immune defense, and angiogenesis. Platelets from diabetic patients often present increased reactivity and basal activation. Interestingly, platelets store and release several substances that have been reported to potentiate insulin secretion by β-cells. For these reasons, the impact of platelets on β-cell functioning was investigated in this thesis. Here it was shown that both glucose and a β-cell-derived substance/s promote platelet activation and binding to collagen. Additionally, platelet adhesion specifically to the microvasculature of pancreatic islets was revealed, supporting the hypothesis of their influence on glucose homeostasis. Genetic or pharmacological ablation of platelet functioning and platelet depletion consistently resulted in reduced insulin secretion and associated glucose intolerance. Further, the platelet-derived lipid fraction was found to enhance glucose-stimulated insulin secretion, with 20-hydroxyeicosatetraenoic acid (20-HETE) and possibly also lyso-precursor of platelet-activating factor (lysoPAF) being identified as crucial factors. However, the acute platelet-stimulated insulin secretion was found to decline with age, as did the levels of platelet-derived 20-HETE. In addition to their direct stimulatory effect on insulin secretion, specific defects in platelet activation have also been shown to affect glucose homeostasis by potentially influencing islet vascular development. Taking together, the results of this thesis suggest a direct and indirect mechanism of platelets in the regulation of insulin secretion that ensures glucose homeostasis, especially in young individuals.},
  subject      = {Thrombozyt},
  language  = {en}
}
@article{SchurrSpindlerKurzetal.2019,
  author    = {Schurr, Yvonne and Spindler, Markus and Kurz, Hendrikje and Bender, Markus},
  title     = {The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis},
  series = {Scientific Reports},
  volume    = {9},
  journal   = {Scientific Reports},
  doi       = {10.1038/s41598-019-44183-6},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-234966},
  year      = {2019},
  abstract  = {Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.},
  language  = {en}
}
@article{LeeLiRuanetal.2019,
  author    = {Lee, Hong-Jen and Li, Chien-Feng and Ruan, Diane and He, Jiabei and Montal, Emily D. and Lorenz, Sonja and Girnun, Geoffrey D. and Chan, Chia-Hsin},
  title     = {Non-proteolytic ubiquitination of Hexokinase 2 by HectH9 controls tumor metabolism and cancer stem cell expansion},
  series = {Nature Communications},
  volume    = {10},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-019-10374-y},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236445},
  year      = {2019},
  abstract  = {Enormous efforts have been made to target metabolic dependencies of cancer cells for developing new therapies. However, the therapeutic efficacy of glycolysis inhibitors is limited due to their inability to elicit cell death. Hexokinase 2 (HK2), via its mitochondrial localization, functions as a central nexus integrating glycolysis activation and apoptosis resilience. Here we identify that K63-linked ubiquitination by HectH9 regulates the mitochondrial localization and function of HK2. Through stable isotope tracer approach and functional metabolic analyses, we show that HectH9 deficiency impedes tumor glucose metabolism and growth by HK2 inhibition. The HectH9/HK2 pathway regulates cancer stem cell (CSC) expansion and CSC-associated chemoresistance. Histological analyses show that HectH9 expression is upregulated and correlated with disease progression in prostate cancer. This work uncovers that HectH9 is a novel regulator of HK2 and cancer metabolism. Targeting HectH9 represents an effective strategy to achieve long-term tumor remission by concomitantly disrupting glycolysis and inducing apoptosis.},
  language  = {en}
}
@article{BaluapuriHofstetterDudvarskiStankovicetal.2019,
  author    = {Baluapuri, Apoorva and Hofstetter, Julia and Dudvarski Stankovic, Nevenka and Endres, Theresa and Bhandare, Pranjali and Vos, Seychelle Monique and Adhikari, Bikash and Schwarz, Jessica Denise and Narain, Ashwin and Vogt, Markus and Wang, Shuang-Yan and D{\"u}ster, Robert and Jung, Lisa Anna and Vanselow, Jens Thorsten and Wiegering, Armin and Geyer, Matthias and Maric, Hans Michael and Gallant, Peter and Walz, Susanne and Schlosser, Andreas and Cramer, Patrick and Eilers, Martin and Wolf, Elmar},
  title     = {MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation},
  series = {Molecular Cell},
  volume    = {74},
  journal   = {Molecular Cell},
  doi       = {10.1016/j.molcel.2019.02.031},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221438},
  pages     = {674-687},
  year      = {2019},
  abstract  = {The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.},
  language  = {en}
}
@article{ChenGehringerLorenz2018,
  author    = {Chen, Dan and Gehringer, Matthias and Lorenz, Sonja},
  title     = {Developing Small-Molecule Inhibitors of HECT-Type Ubiquitin Ligases for Therapeutic Applications: Challenges and Opportunities},
  series = {ChemBioChem},
  volume    = {19},
  journal   = {ChemBioChem},
  doi       = {10.1002/cbic.201800321},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-222412},
  pages     = {2123-2135},
  year      = {2018},
  abstract  = {The ubiquitin system regulates countless physiological and disease-associated processes and has emerged as an attractive entryway for therapeutic efforts. With over 600 members in the human proteome, ubiquitin ligases are the most diverse class of ubiquitylation enzymes and pivotal in encoding specificity in ubiquitin signaling. Although considerable progress has been made in the identification of small molecules targeting RING ligases, relatively little is known about the "druggability" of HECT (homologous to E6AP C terminus) ligases, many of which are critically implicated in human pathologies. A major obstacle to optimizing the few available ligands is our incomplete understanding of their inhibitory mechanisms and the structural basis of catalysis in HECT ligases. Here, we survey recent approaches to manipulate the activities of HECT ligases with small molecules to showcase the particular challenges and opportunities these enzymes hold as therapeutic targets.},
  language  = {en}
}
@phdthesis{Nair2024,
  author    = {Nair, Radhika Karal},
  title     = {Structural and biochemical characterization of USP28 inhibition by small molecule inhibitors},
  doi       = {10.25972/OPUS-28174},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-281742},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes. Deubiquitinases (DUBs) are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate. Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases, including cancer. Ubiquitin specific proteases (USPs), which constitute the largest family of DUBs in humans, have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment. Two related members of the USP family, USP28 and USP25, share high sequence identities but play diverse biological roles. USP28 regulates cell proliferation, oncogenesis, DNA damage repair and apoptosis, whereas USP25 is involved in the anti-viral response, innate immunity and ER-associated degradation in addition to carcinogenesis. USP28 and USP25 also exhibit different oligomeric states - while USP28 is a constitutively active dimer, USP25 assumes an auto-inhibited tetrameric structure. The catalytic domains of both USP28 and USP25 comprise the canonical, globular USP-domain but contain an additional, extended insertion site called USP25/28 catalytic domain inserted domain (UCID) that mediates oligomerization of the proteins. Disruption of the USP25 tetramer leads to the formation of an activated dimeric protein. However, it is still not clear what triggers its activation. Due to their role in maintaining and stabilizing numerous oncoproteins, USP28 and USP25 have emerged as interesting candidates for anti-cancer therapy. Recent advances in small-molecular inhibitor development have led to the discovery of relatively potent inhibitors of USP28 and USP25. This thesis focuses on the structural elucidation of USP28 and the biochemical characterization of USP28/USP25, both in complex with representatives of three out of the eight compound classes reported as USP28/USP25-specific inhibitors. The crystal structures of USP28 in complex with the AZ compounds, Vismodegib and FT206 reveal that all three inhibitor classes bind into the same allosteric pocket distant from the catalytic center, located between the palm and the thumb subdomains (the S1-site). Intriguingly, this binding pocket is identical to the UCID-tip binding interface in the USP25 tetramer, rendering the protein in a locked, inactive conformation. Formation of the binding pocket in USP28 requires a shift in the helix α5, which induces conformational changes and local distortion of the binding channel that typically accommodates the C-terminal tail of Ubiquitin, thus preventing catalysis and abrogating USP28 activity. The key residues of the USP28-inhibitor binding pocket are highly conserved in USP25. Mutagenesis studies of these residues accompanied by biochemical and biophysical assays confirm the proposed mechanism of inhibition and similar binding to USP25. This work provides valuable insights into the inhibition mechanism of the small molecule compounds specifically for the DUBs USP28 and USP25. The USP28-inhibitor complex structures offer a framework to develop more specific and potent inhibitors.},
  subject      = {Unique Selling Proposition},
  language  = {en}
}
@article{AngayFriedrichPinneckeretal.2018,
  author    = {Angay, Oguzhan and Friedrich, Mike and Pinnecker, J{\"u}rgen and Hintzsche, Henning and Stopper, Helga and Hempel, Klaus and Heinze, Katrin G.},
  title     = {Image-based modeling and scoring of Howell-Jolly Bodies in human erythrocytes},
  series = {Cytometry Part A},
  volume    = {93},
  journal   = {Cytometry Part A},
  doi       = {10.1002/cyto.a.23123},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221140},
  pages     = {305-313},
  year      = {2018},
  abstract  = {The spleen selectively removes cells with intracellular inclusions, for example, detached nuclear fragments in circulating erythrocytes, called Howell-Jolly Bodies (HJBs). With absent or deficient splenic function HJBs appear in the peripheral blood and can be used as a simple and non-invasive risk-indicator for fulminant potentially life-threatening infection after spleenectomy. However, it is still under debate whether counting of the rare HJBs is a reliable measure of splenic function. Investigating HJBs in premature erythrocytes from patients during radioiodine therapy gives about 10 thousand times higher HJB counts than in blood smears. However, we show that there is still the risk of false-positive results by unspecific nuclear remnants in the prepared samples that do not originate from HJBs, but from cell debris residing above or below the cell. Therefore, we present a method to improve accuracy of image-based tests that can be performed even in non-specialized medical institutions. We show how to selectively label HJB-like clusters in human blood samples and how to only count those that are undoubtedly inside the cell. We found a "critical distance" dcrit referring to a relative HJB-Cell distance that true HJBs do not exceed. To rule out false-positive counts we present a simple inside-outside-rule based on dcrit—a robust threshold that can be easily assessed by combining conventional 2D imaging and straight-forward image analysis. Besides data based on fluorescence imaging, simulations of randomly distributed HJB-like objects on realistically modelled cell objects demonstrate the risk and impact of biased counting in conventional analysis. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.},
  language  = {en}
}