TY - JOUR A1 - Segerer, Gabriela A1 - Hadamek, Kerstin A1 - Zundler, Matthias A1 - Fekete, Agnes A1 - Seifried, Annegrit A1 - Mueller, Martin J. A1 - Koentgen, Frank A1 - Gessler, Manfred A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation JF - Scientific Reports N2 - 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. KW - cell proliferation KW - DNA metabolism KW - lipidomics Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181094 VL - 6 ER - TY - JOUR A1 - Radu, Laura A1 - Schoenwetter, Elisabeth A1 - Braun, Cathy A1 - Marcoux, Julien A1 - Koelmel, Wolfgang A1 - Schmitt, Dominik R. A1 - Kuper, Jochen A1 - Cianférani, Sarah A1 - Egly, Jean M. A1 - Poterszman, Arnaud A1 - Kisker, Caroline T1 - The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH JF - Nucleic Acids Research N2 - 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. KW - general transcription factor IIH (TFIIH) KW - DNA repair KW - protein–protein interaction KW - p44 KW - p34 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170173 VL - 45 IS - 18 ER - TY - JOUR A1 - Mostosi, Philipp A1 - Schindelin, Hermann A1 - Kollmannsberger, Philip A1 - Thorn, Andrea T1 - Haruspex: A Neural Network for the Automatic Identification of Oligonucleotides and Protein Secondary Structure in Cryo‐Electron Microscopy Maps JF - Angewandte Chemie International Edition N2 - 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. KW - DNA structures KW - electron microscopy KW - neural networks KW - protein structures KW - RNA structures Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214763 VL - 59 IS - 35 SP - 14788 EP - 14795 ER - TY - JOUR A1 - Vogelsang, Anna A1 - Eichler, Susann A1 - Huntemann, Niklas A1 - Masanneck, Lars A1 - Böhnlein, Hannes A1 - Schüngel, Lisa A1 - Willison, Alice A1 - Loser, Karin A1 - Nieswandt, Bernhard A1 - Kehrel, Beate E. A1 - Zarbock, Alexander A1 - Göbel, Kerstin A1 - Meuth, Sven G. T1 - Platelet inhibition by low-dose acetylsalicylic acid reduces neuroinflammation in an animal model of multiple sclerosis JF - International Journal of Molecular Sciences N2 - 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. KW - acetylsalicylic acid KW - experimental autoimmune encephalomyelitis KW - platelets KW - multiple sclerosis KW - thromboxane KW - glycoprotein VI KW - platelet factor 4 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284535 SN - 1422-0067 VL - 22 IS - 18 ER - TY - JOUR A1 - Navarro, Stefano A1 - Stegner, David A1 - Nieswandt, Bernhard A1 - Heemskerk, Johan W. M. A1 - Kuijpers, Marijke J. E. T1 - Temporal roles of platelet and coagulation pathways in collagen- and tissue factor-induced thrombus formation JF - International Journal of Molecular Sciences N2 - 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. KW - coagulation KW - fibrin KW - glycoprotein VI KW - platelet receptors KW - spatiotemporal thrombus KW - thrombin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284219 SN - 1422-0067 VL - 23 IS - 1 ER - TY - JOUR A1 - Kollikowski, Alexander M. A1 - Pham, Mirko A1 - März, Alexander G. A1 - Papp, Lena A1 - Nieswandt, Bernhard A1 - Stoll, Guido A1 - Schuhmann, Michael K. T1 - Platelet Activation and Chemokine Release Are Related to Local Neutrophil-Dominant Inflammation During Hyperacute Human Stroke JF - Translational Stroke Research N2 - 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. KW - chemokines KW - CXCL4 KW - PF4 KW - CXCL7 KW - NAP-2 KW - ischemic stroke Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270194 SN - 1868-601X VL - 13 IS - 3 ER - TY - JOUR A1 - Schanbacher, Constanze A1 - Bieber, Michael A1 - Reinders, Yvonne A1 - Cherpokova, Deya A1 - Teichert, Christina A1 - Nieswandt, Bernhard A1 - Sickmann, Albert A1 - Kleinschnitz, Christoph A1 - Langhauser, Friederike A1 - Lorenz, Kristina T1 - ERK1/2 activity is critical for the outcome of ischemic stroke JF - International Journal of Molecular Sciences N2 - 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. KW - ERK1/2 KW - tMCAO KW - ischemic stroke KW - RKIP Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-283991 SN - 1422-0067 VL - 23 IS - 2 ER - TY - JOUR A1 - Marzocco, Stefania A1 - Fazeli, Gholamreza A1 - Di Micco, Lucia A1 - Autore, Giuseppina A1 - Adesso, Simona A1 - Dal Piaz, Fabrizio A1 - Heidland, August A1 - Di Iorio, Biagio T1 - 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) JF - Journal of Clinical Medicine N2 - 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. KW - propionic acid KW - chronic kidney disease KW - hemodialysis KW - gut microbiome KW - systemic micro-inflammation oxidative stress KW - indoxyl sulfate KW - p-cresyl sulfate Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197626 SN - 2077-0383 VL - 7 IS - 10 ER - TY - JOUR A1 - Fusi, Lorenza A1 - Paudel, Rupesh A1 - Meder, Katharina A1 - Schlosser, Andreas A1 - Schrama, David A1 - Goebeler, Matthias A1 - Schmidt, Marc T1 - Interaction of transcription factor FoxO3 with histone acetyltransferase complex subunit TRRAP modulates gene expression and apoptosis JF - Journal of Biological Chemistry N2 - 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. KW - FoxO3 KW - TRRAP KW - transcription factors Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299820 VL - 298 IS - 3 ER - TY - THES A1 - Slotta, Anja Maria T1 - The Role of Protein Kinase D 1 in the regulation of murine adipose tissue function under physiological and pathophysiological conditions T1 - Die Bedeutung von Protein Kinase D 1 in der Funktion von murinem Fettgewebe unter physiologischen und pathophysiologischen Bedingungen N2 - 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 Löffler 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. N2 - Adipozyten sind spezialisierte Zellen der Wirbeltiere, die das Überleben durch Anpassung an Nahrungsmangel und Nahrungsüberfluss gewährleisten. Eine Dysfunktion von Adipozyten bedingt jedoch die Pathophysiologie von Stoffwechselerkrankungen wie dem T2DM. Vorläufige Ergebnisse von Mona Löfflers Versuchen zeigten, dass PKD1 in der Funktion von Adipozyten involviert ist. Innerhalb dieser Arbeit konnte dargestellt werden, dass die Expression und Aktivität von PKD1 in murinen Adipozyten an den Lipidmetabolismus gekoppelt ist. Beim Hungern von murinem weißen Fettgewebe, einem physiologischen Zustand, der Lipolyse induziert, war die Genexpression von PKD1 reduziert. Isoproterenol-stimulierte Lipolyse führte ebenfalls zu verminderter Expression von PKD1 in murinen weißen Fettgewebe und 3T3-L1 Adipozyten. In ATGL-silenced Adipozyten war die Isoproterenol-stimulierte Lipolyse zwar inhibiert, allerdings wurde die Genexpression von PKD1 durch die β-adrenerge Stimulation ebenfalls vermindert. Fettgewebe von adipösen Mäusen hingegen wiesen hohe PKD1 RNA Level sowie einen niedrigen Proteingehalt der phosphorylierten Form PKD1-Ser916 auf. Fettreiche Ernährung von Mäusen generierte in Fettgewebe jedoch ein weiteres Produkt von PKD1 mit niedrigem Molekulargewicht im Western Blot. Des Weiteren wurde dargestellt, dass konstitutiv aktives PKD1 in 3T3-L1 Adipozyten vorwiegend nuklear lokalisiert war und diese Adipozyten einen hohen Gehalt von Fettvakuolen aufwiesen. Adipozyten, die funktionsloses PKD1 exprimierten, enthielten wenige Lipidtropfen und PKD1-KD war im Cytoplasma verteilt. Vor allem zeigte diese Arbeit, dass die Deletion von PKD1 spezifisch in murinem Fettgewebe die Expression von Genen wie UCP-1 verursachte, die eine Rolle in adaptiver Thermogenese spielen, und dadurch einen brown-like Phänotypen generierte. Zusammenfassend ist PKD1 in die Funktionen von Adipozyten verwickelt und stellt ein attraktives Ziel für therapeutische Ansätze in der Prävention von Übergewicht und damit assoziierten Erkrankungen dar. KW - adipocyte KW - murine KW - pkd KW - Protein Kinase D KW - adipose KW - Protein Kinase D 1 KW - PKD1 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179112 ER - TY - THES A1 - Mony Nair, Rahul T1 - Elucidating ubiquitin recognition by the HECT-type ubiquitin ligase HUWE1 T1 - Studien zur Ubiquitinerkennung durch die HECT-Typus Ubiquitinligase HUWE1 N2 - 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. N2 - Der kleine Proteinmodifikator Ubiquitin ist das Herzstück eines immens vielseitigen posttranslationalen Modifikationssystems, das unzählige physiologische und krankheitsassoziierte zelluläre Prozesse orchestriert. Der Schlüssel zu dieser Vielseitigkeit sind die vielfältigen Modifikationen, die sich aus Ubiquitin-"Bausteinen" zusammensetzen lassen und mit spezifischen funktionellen Ergebnissen für die modifizierten Substrate verbunden sind. Insbesondere können Ubiquitin-Moleküle Ketten unterschiedlicher Länge und Verknüpfungstypen bilden, die zu unterschiedlichen Kettenkonformationen führen und dadurch Erkennungsstellen für spezifische Signalrezeptoren/-effektoren bieten. Die Klasse der E3-Enzyme (Ubiquitin-Ligasen) liefert kritische Spezifitätsdeterminanten für die Bildung von Ubiquitin-Bindungen; daher ist es entscheidend, die genaue Funktionsweise der E3-Enzyme zu entschlüsseln, um die strukturelle Grundlage der Ubiquitin-Signalisierung zu verstehen und diese Erkenntnisse für therapeutische Anwendungen zu nutzen. Die Überexpression und Deregulierung der Ubiquitin-Ligase HUWE1 aus der Klasse der HECT-E3-Ligasen ist an mehreren verschiedenen Krebsarten und neurodegenerativen Erkrankungen beteiligt. Es ist weitgehend unbekannt, welche Faktoren durch die von HUWE1 gebildeten Ubiquitin-Modifikationen kontrolliert werden, wie die katalytische HECT-Domäne mit funktionell unterschiedlichen Ubiquitin-Molekülen (Donor-, Akzeptor- und regulatorische Ubiquitin-Moleküle) interagiert und welche Konformationsübergänge diese Interaktionen während der Ubiquitin-Kettenbildung ermöglichen. Ein Ziel dieser Studie war es, die Erkennung des Donor-Ubiquitin-Moleküls durch die HECT-Domäne von HUWE1 strukturell aufzuklären. Zu diesem Zweck verwendete ich eine ´ubiquitin activity-based probe´, um ein Konjugat der HUWE1-HECT-Domäne mit einem Donor-Ubiquitin-Molekül zu rekonstitutieren und die Struktur mittels Röntgenkristallographie zu bestimmen. Diese Struktur zeigte, dass das Donor-Ubiquitin-Molekül auf die gleiche Weise an den C-lobe von HUWE1 bindet wie die Klasse der NEDD4-Ligasen, was die Idee bestätigt, dass HECT-Ligasen einen vergleichbaren Mechanismus bei der Donor-Ubiquitin-Erkennung verwenden, unabhängig von ihrer Bindung und Substratspezifität. Mit Hilfe biochemischer Analysen validierte ich auch spezifische Merkmale der Struktur, insbesondere die Positionierung des C-terminal tail der Ligase, der entscheidend für die Aktivität ist. In der neu bestimmten Struktur, die einen "L-förmigen", aktiven Zustand der HECT-Domäne widerspiegelt, ist der C-terminal tail an der Grenzfläche von N-lobe und C-lobe vollständig aufgelöst und koordiniert. Ich konnte Seitenketten festmachen, die für diese Koordination kritisch sind, und habe gezeigt, dass sie auch für die Aktivität von HUWE1 wesentlich sind, einschließlich der Auto-Ubiquitinierung, der freien Ubiquitin-Kettenbildung und der Substrat-Ubiquitinierung. Darüber hinaus entdeckte ich, dass der N-lobe von HUWE1 eine Ubiquitin-bindende exosite aufweist, ähnlich wie für die Klasse der NEDD4-Ligasen und E6AP. Meine in vitro Aktivitäts- und Bindungstests ergaben, dass HUWE1 die exosite für die Bildung von Isopeptidbindungen verwendet, diese aber für die Bildung von Thioesterbindungen entbehrlich ist. Die Bindungstests zeigten ferner, dass die Donor-Ubiquitin-beladene HECT-Domäne ein zusätzliches Ubiquitin-Molekül an der exosite stärker bindet als die Apo-HECT-Domäne, was möglicherweise auf eine allosterische Kommunikation zwischen den beiden Ubiquitin-Bindestellen hindeutet. Schließlich zeigte ich, dass die ´ubiquitin activity-based probe´ (Ubiquitin-Propargylamin) das katalytische Cystein von HUWE1 und NEDD4 mit nahezu quantitativem Umsatz markieren kann, während es nicht mit der HECT-Domäne des evolutionär stärker divergierenden E6AP reagiert. Die Faktoren, die diesen unterschiedlichen Reaktivitäten zugrunde liegen, müssen noch erforscht werden. Zusammengenommen verbessern meine Ergebnisse unser mechanistisches Verständnis der katalytischen Domäne von HUWE1 und geben uns die Dreh- und Angelpunkte für therapeutische Interventionen mit der Aktivität dieses krankheitsrelevanten Enzyms an die Hand. KW - HUWE1 KW - Ubiquitin-PA Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221030 ER - TY - JOUR A1 - Truongvan, Ngoc A1 - Li, Shurong A1 - Misra, Mohit A1 - Kuhn, Monika A1 - Schindelin, Hermann T1 - Structures of UBA6 explain its dual specificity for ubiquitin and FAT10 JF - Nature Communications N2 - 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. KW - enzyme mechanisms KW - post-translational modifications KW - X-ray crystallography Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301161 VL - 13 ER - TY - JOUR A1 - Pauls, Dennis A1 - Hamarat, Yasmin A1 - Trufasu, Luisa A1 - Schendzielorz, Tim M. A1 - Gramlich, Gertrud A1 - Kahnt, Jörg A1 - Vanselow, Jens A1 - Schlosser, Andreas A1 - Wegener, Christian T1 - Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate JF - European Journal of Neuroscience N2 - 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. KW - direct muss spectrometric profiling KW - friut fly behaviour KW - M14 carboxypeptidasses KW - peptidomoics KW - protein processing Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204863 VL - 50 IS - 9 ER - TY - JOUR A1 - Balakrishnan, Ashwin A1 - Hemmen, Katherina A1 - Choudhury, Susobhan A1 - Krohn, Jan-Hagen A1 - Jansen, Kerstin A1 - Friedrich, Mike A1 - Beliu, Gerti A1 - Sauer, Markus A1 - Lohse, Martin J. A1 - Heinze, Katrin G. T1 - Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence JF - Communications Biology N2 - 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-Delbrück model. KW - G-protein-coupled receptors KW - molecular mobility KW - temporal range Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301140 VL - 5 IS - 1 ER - TY - THES A1 - Balakrishnan, Ashwin T1 - Fast molecular mobility of β\(_2\)-adrenergic receptor revealed by time-resolved fluorescence spectroscopy T1 - Schnelle molekulare Beweglichkeit des β\(_2\)-adrenergen Rezeptors durch zeitaufgelöste Fluoreszenzspektroskopie N2 - 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-Delbrück 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. N2 - G¬Protein¬gekoppelte Rezeptoren (GPCRs) sind die größte Familie der Membran¬Rezeptoren und durch Bindung von Liganden leiten sie extrazlluläre Signal in das Innere der Zelle weiter. Fast 40% der Medikamente auf der Welt zielen aufgrund ihrer Funktion auf GPCRs ab, obwohl man relative wenig über ihre Aktivierung weiß. Das Verständnis ihres dynamischen Verhaltens im basalen und aktivierten Zustand könnte sich in Zukunft als Schlüssel zur Medikamentenentwicklung erweisen. GPCRs sind dafür bekannt, dass sie komplexe molekulare Bewegungsmuster aufweisen. Eine Fülle von Studien wurden und werden durchgeführt, um die Beweglichkeit von GPCRs zu verstehen. Aufgrund der Einschränkungen der gängigen bildgebenden und spektroskopischen Techniken sind die relevanten Zeitskalen nur schwer messbar. Die am häufigsten verwendeten Techniken sind die paramagnetische Elektronenresonanz oder die Doppel¬Elektron¬Elektron¬Resonanz, die magnetische Kernresonanz, die zeitaufgelöste Fluoreszenz, die Einzelpartikelverfolgung und die Fluoreszenzwiederherstellung nach Photobleichung. Unter diesen Techniken haben nur die Fluoreszenz¬basierten Techniken das Potential, lebende Zellen zu untersuchen. In dieser Arbeit werden verschiedene zeitaufgelöste fluoreszenzspektroskopische Techniken zur Quantifizierung der Diffusionsdynamik oder molekularen Mobilität des β2¬adrenergen Rezeptors (β2¬AR) in lebenden Zellen verwendet. Diese Arbeit zeigt, dass β2-AR eine Beweglichkeit über einen außergewöhnlich breiten, zeitlichen Bereich (Nanosekunde bis Sekunde) aufweist, der mit dem jeweiligen physiologischen Szenario verknüpft werden kann. Es wird gezeigt, wie β2¬AR eine überraschend schnelle, laterale Bewegung (~10 μm²/s) besitzt, welche mit vesikulärem Transport in Verbindung gebracht werden kann. Im Gegensatz zu früheren Berichten, wonach die beobachtete Komponente von der Photophysik der Fluorophore und freien Fluorophoren im Zytosol abstammt. Zusätzlich weist β2¬AR eine Rotationsbeweglichkeit (~100 μs) auf, welche es ¬ im Gegensatz zu früheren Studien ¬ dem Saffman¬Delbrück¬Modell der Membrandiffusion zuordnen lässt. Dieser Unterschied ist auf die Beschränkungen der verwendeten Techniken zurückzuführen. Die Einschränkungen werden in dieser Arbeit durch die Verwendung verschiedener zeitaufgelöster Fluoreszenztechniken überwunden, z. B. der Fluoreszenzkorrelationsspektroskopie (FCS) im Bereich von mehreren hundert Nanosekunden bis Sekunden, der zeitaufgelösten Anisotropie (TRA) im Nanosekundenbereich und der polarisationsaufgelösten FullFCS (FullFCS), die die zeitlich Lücke zwischen FCS und TRA schließt. Zuletzt wird eine Abnahme der lateralen Beweglichkeit durch Ligandenstimulation gezeigt, was ein Hinweis auf Clusterbildung aufgrund von Internalisierung sein könnte, und dass β2¬AR eine basale Oligomerisierung aufweist, die sich bei Aktivierung nicht ändert. Zusammenfassend kann man sagen, dass verschiedene komplementäre Fluoreszenztechniken notwendig sind, um die Einschränkungen der einzelnen Techniken zu überwinden und dadurch die funktionelle Dynamik der GPCR¬Aktivierung und deren Bedeutung für die nachgeschaltete Signalübertragung aufzuklären. KW - Fluorescence correlation spectroscopy KW - GPCR KW - time-resolved anisotropy KW - adrenergic receptor KW - homoFRET Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250856 ER - TY - JOUR A1 - Klenk, Christoph A1 - Hommers, Leif A1 - Lohse, Martin J. T1 - Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor JF - Frontiers in Endocrinology N2 - 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. KW - GPCRs KW - parathyroid hormone 1 receptor KW - matrix metalloproteinase KW - ectodomain cleavage KW - biased signaling Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262055 SN - 1664-2392 VL - 13 ER - TY - THES A1 - Stetter, Maurice T1 - LC3-associated phagocytosis seals the fate of the second polar body in \(Caenorhabditis\) \(elegans\) T1 - LC3-assoziierte Phagozytose besiegelt das Schicksal des zweiten Polkörpers in \(Caenorhabditis\) \(elegans\) N2 - 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. N2 - Um besser zu verstehen, wie undifferenzierte pluripotente Zellen mit abgestorbenen Zellen umgehen, wird in dieser Dissertation die Phagozytose und der Abbau des 2. Polkörpers der weiblichen Meiose im Fadenwurm C. elegans untersucht. Mithilfe von fluorenzenzmikroskopischen Aufnahmen wird in dieser Arbeit gezeigt, dass beide Polkörper schon früh ihre Membranintegrität verlieren. Der 2. Polkörper, welcher direkten Kontakt zu embryonischen Zellen hat, wird daraufhin mithilfe des Rac1-Orthologs CED-10 phagozytiert. Es wird gezeigt, dass es sich bei dem Abbauprozess um LC3-assoziierte Phagozytose handelt. Die RAB-7 GTPase ist notwendig für die Rekrutierung von Lysosomen, während LC3 darauf keinen Einfluss hat, aber trotzdem den Abbau des Polkörpers beschleunigt. Mit dieser Arbeit konnte ein genetisches Modell für die Erforschung von Zelltod und der LC3-assoziierten Phagozytose entwickelt werden und weitere Aspekte der Phagosomreifung und des -abbaus aufgedeckt werden. KW - Polkörper KW - Phagozytose KW - Autophagie KW - Zelltod KW - Caenorhabditis elegans KW - LC3-assoziierte Phagozytose KW - phagosome maturation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231981 ER - TY - JOUR A1 - Kaiser, Sebastian A1 - Sauer, Florian A1 - Kisker, Caroline T1 - The structural and functional characterization of human RecQ4 reveals insights into its helicase mechanism JF - Nature Communications N2 - 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. KW - x-ray crystallography KW - enzymes KW - RecQ4 KW - humans Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170769 VL - 8 IS - 15907 ER - TY - JOUR A1 - Kronhardt, Angelika A1 - Beitzinger, Christoph A1 - Barth, Holger A1 - Benz, Roland T1 - Chloroquine Analog Interaction with C2-and Iota-Toxin in Vitro and in Living Cells JF - Toxins N2 - 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. KW - C2-toxin KW - iota-toxin KW - binding components KW - chloroquine KW - black lipid bilayer KW - aminoquinolinium salts Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168286 VL - 8 IS - 8 ER - TY - THES A1 - Beer, Katharina Beate T1 - Identification and characterization of TAT-5 interactors that regulate extracellular vesicle budding T1 - Identifizierung und Charakterisierung von TAT-5 Interaktoren, welche die Ausschüttung von Extrazellulären Vesikeln regulieren N2 - 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. N2 - Zellen von Bakterien bis zum Menschen produzieren Extrazelluläre Vesikel (EV) wie zum Beispiel Mikrovesikel (MV). MV können Signal Moleküle wie Morphogene und miRNA transportieren, welche die normale oder krankheitsbedingte interzelluläre Kommunikation kontrollieren. Bei der Produktion von MVs werden Membranen verformt, wie auch für die Reparatur von beschädigten Membranen um den Zelltod zu verhindern. Außerdem knospen HIV-Virus Partikel von der Plasma Membrane durch eine ähnliche Art und Weise. Um zu verstehen welche in vivo Funktion MV haben, müssen wir die Mechanismen der MV Knospung von der Plasma Membran (Ektozytose) verstehen. Die konservierte Phospholipid Flippase TAT-5 hält die asymmetrische Verteilung von Phosphatidylethanolamine (PE) in der Plasma Membrane aufrecht und war der einzig bekannte Inhibitor der von ESCRT Proteinen durchgeführten Ektozytose in C. elegans. Wenn die Lipid-flippende Funktion von TAT-5 verloren geht, wird PE externalisiert und MV sammeln sich außerhalb der Zelle an. Allerdings ist es unklar mit welchen Mechanismen die Aktivität von TAT-5 reguliert wird um die richtige Menge an MV zur richtigen Zeit zu produzieren, da die vorgeschalteten Regulatoren unbekannt sind. Um konservierte TAT-5 Regulatoren zu identifizieren suchten wir nach neuen Proteinen, die die Produktion von MV inhibieren. Dazu entwickelten wir eine Degradations-Technik um MV spezifisch zu kennzeichnen. Wir markierten einen fluoreszierenden Plasma Membran Marker mit dem endogenen ZF1 Degradations-Kennzeichen (Degron) und exprimierten es im C. elegans Embryo. Der Marker wird vor der Degradation geschützt, wenn er in einem MV von der Zelle ausgesondert wurde. Dadurch bleibt die Fluoreszenz speziell in MV erhalten, während sie innerhalb der Zelle abgebaut wird. Dadurch wurde die Sichtbarkeit von ausgeschütteten MV erhöht. Wir fanden vier Proteine, welche mit Protein Recycling in Verbindung gebracht werden, die die Ausschüttung von MV verhindern: Class III PI3Kiase VPS-34, Beclin1 Homolog BEC-1, DnaJ Protein RME-8 und das nicht näher charakterisierte Dopey Homolog PAD-1. Wir benutzten dieses Set an Proteinen, um zu testen ob und wie diese TAT-5 regulieren können. Wir fanden, dass Class III PI3Kinase, RME-8 und redundante Sorting Nexins für die Plasma Membran Lokalisierung von TAT-5 verantwortlich sind, was wichtig ist um die PE Asymmetrie aufrecht zu erhalten und die MV Produktion zu verhindern. Wenn auch PAD-1 und das GEF-ähnliche MON-2 für endosomales Recycling verantwortlich sind, regulieren sie die Lokalisation von TAT-5 nur in Abwesenheit von Sorting Nexins-reguliertem Transport. Zudem scheint PAD-1 direkt für die Lipid Translokations-Aktivität von TAT-5 verantwortlich zu sein. Demnach konnten wir zeigen, dass TAT-5 und PE Schlüsselregulatoren für MV Produktion sind, was weiterhin die Ansicht unterstützt, dass PE Externalisierung für die Ektozytose verantwortlich ist. Außerdem fanden wir, dass redundante intrazelluläre Transportwege für die Größe von Organellen verantwortlich sind und deckten neue TAT-5 Aktivitäts-Regulatoren auf. Diese neu aufgedeckten Ektozytose Inhibitoren könnten Werkzeuge sein um die in vivo Funktionen von MV zu testen. Längerfristig kann unsere Forschung dazu beitragen die Mechanismen der MV Produktion zu identifizieren und die Regulation während der krankheitsbedingten EV Produktion, der Membrane Reparatur und der Virus Knospung besser zu verstehen. KW - C. elegans KW - Extracellular Vesicles KW - Microvesicle KW - Flippase KW - P4-ATPase KW - Caenorhabditis elegans KW - Vesikelbildung Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-206724 ER - TY - JOUR A1 - Grabarczyk, Daniel B. A1 - Berks, Ben C. T1 - Intermediates in the Sox sulfur oxidation pathway are bound to a sulfane conjugate of the carrier protein SoxYZ JF - PLoS ONE N2 - 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. KW - thiosulfates KW - oxidation KW - sulfur KW - cysteine KW - sulfides KW - thermodynamics KW - sulfates KW - sulfites Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171147 VL - 12 IS - 3 ER - TY - THES A1 - Imam, Nasir T1 - Molecular basis of collybistin conformational activation T1 - Molekulare Prinzipien der konformellen Aktivierung von Collybistin N2 - 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 (Förster 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. N2 - Das Nervensystem ist eine komplexe Ansammlung zellulärer Einheiten, darunter sind die Neuronen, die speziell für die Verarbeitung und Übertragung von Informationen zuständig sind. Die Kommunikation zwischen Neuronen erfolgt über Synapsen, spezialisierte Membran-Membran-Kontakte, die eine effiziente Signalübertragung gewährleisten. Die synaptische Neurotransmission umfasst die präsynaptische Freisetzung von Neurotransmitters und deren Empfang durch entsprechende Rezeptoren in den Postsynapsen. Die inhibitorische Neurotransmission wird in erster Linie durch die Freisetzung der Neurotransmitter GABA (γ-Aminobuttersäure-Typ) und Glycin vermittelt, die von GABA-Typ-A-Rezeptor (GABAAR) bzw. Glycinrezeptoren (GlyR) präzise wahrgenommen werden. Der Aufbau und die Aufrechterhaltung von GABAAR Clustern wird durch verschiedene postsynaptische neuronale Faktoren koordiniert, darunter das Gerüstprotein Gephyrin, das neuronale Adaptorprotein Collybistin (CB) und Zelladhäsionsproteine der Neuroligin (NL)-Familie, insbesondere NL2 und NL4. Es wird angenommen, dass Gephyrin an hemmenden postsynaptischen Spezialisierungen ausgedehnte Strukturen unterhalb der Plasmamembran bildet, und durch Interaktion mit den Rezeptoren deren laterale Diffusion verhindert. Gephyrin-Mutationen wurden mit verschiedenen Hirnkrankheiten in Verbindung gebracht, darunter Autismus, Schizophrenie, Alzheimer und Epilepsie. Der Verlust von Gephyrin ist tödlich und führt dazu, dass Mäuse innerhalb des ersten postnatalen Tages sterben. Die Rekrutierung von Gephyrin aus intrazellulären Ablagerungen zu postsynaptischen Membranen hängt in erster Linie von CB ab. Als Moonlighting-Protein katalysiert CB, ein Guanin-Nukleotid-Austauschfaktor (GEF), auch den Nukleotidaustausch und somit die Reaktivierung der kleinen GTPase Cdc42 . Das CB-Gen wird durch alternatives Spleißen modifiziert; die meisten CB-Spleißvarianten weisen eine N-terminale SH3-Domäne auf, gefolgt von Tandem aus einer Dbl-Homologie (DH)- und einer Pleckstrin-Homologie (PH)-Domäne. Frühere Studien zeigten, dass die am häufigsten exprimierte Spleißvariante, die eine SH3-Domäne enthält (CB2SH3+), vorzugsweise eine geschlossene Konformation annimmt, bei der die N-terminal gelegene SH3-Domäne eine intra-molekulare Interaktion mit dem DH-PH- Tandem eingeht. Zellbasierte Studien zeigten, dass CB-Varianten, die für die SH3-Domäne kodieren, sich innerhalb der Zelle nicht an spezifischen Orten anreichern und stattdessen mit intrazellulären Gephyrin-Ablagerungen kolokalisieren. Zusätzliche Faktoren werden benötigt, die mit der SH3-Domäne interagieren und so eine offene oder aktive Konformation hervorrufen. Die SH3-Domänen-defiziente CB-Isoform (CB2SH3-) hingegen nimmt eine offene Konformation an, die eine verstärkte postsynaptische Gephyrin-Anhäufung aufweist und die GTP-gebundene kleine GTPase Cdc42 aus ihrem GDP-gebundenen Zustand effektiv wieder regeneriert. Trotz der grundlegenden Rolle von CB als neuronales Adaptorprotein, das die ordnungsgemäße Funktion hemmender GABAerger Synapsen aufrechterhält, ist seine Interaktion mit dem neuronalen Gerüstprotein Gephyrin und anderen post-synaptischen neuronalen Faktoren nach wie vor unzureichend verstanden. Darüber hinaus sind die Interaktionsstudien von CB mit der kleinen GTPase Cdc42 und TC10, einem eng verwandten Mitglied der Cdc42-Unterfamilie, noch immer unzureichend charakterisiert. Somit war die Frage, ob diese neuronalen Faktoren sowie die kleinen GTPasen an der CB-Konformationsaktivierung beteiligt sind. Diese Dissertation konzentriert sich in erster Linie auf die Beschreibung der molekularen Grundlagen der Interaktion von CB mit postsynaptischen neuronalen Faktoren. Im Rahmen meiner Dissertation habe ich eine Reihe von CB-FRET-Sensoren (Förster-Resonanz-Energie-Transfer) entwickelt, um die CB-Interaktion mit seinen Bindungspartnern zu charakterisieren und ihre Rolle bei der CB-Konformationsaktivierung zu beschreiben. Mit Hilfe der CB-FRET-Sensoren entschlüsselte ich das langjährige Rätsel der Gephyrin-CB-Interaktion, das aufgrund technischer Beschränkungen mehr als zwei Jahrzehnte lang ungelöst blieb (siehe Kapitel 2 für weitere Einzelheiten). In der Folge habe ich auch die molekularen Grundlagen der CB-Wechselwirkung und damit ihre konformelle Aktivierung durch den neuronalen Zelladhäsionsfaktor Neuroligin 2 (siehe Kapitel 2) und die kleinen GTPasen Cdc42 und TC10 (siehe Kapitel 3) analysiertt. Zusammengefasst liefert diese Dissertation starke Beweise für ein engmaschiges CB-Kommunikationsnetzwerk mit Gephyrin, Neuroligin und der kleinen GTPase TC10, in dem der CB-Konformationswechsel vom geschlossenen/inaktiven zum offenen/aktiven Zustand effektiv durch die Liganden ausgelöst wird. KW - Guanine nucleotide exchange factor (GEF) KW - Rho GTPasw KW - inhibitory postsynapse KW - Autoinhibition KW - conformational activation KW - collybistin KW - fluorescence resonance energy transfer (FRET) KW - gephyrin KW - neurologin-2 KW - time-correlated single photon counting (TCSPC) Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311458 ER - TY - JOUR A1 - Kuhlemann, Alexander A1 - Beliu, Gerti A1 - Janzen, Dieter A1 - Petrini, Enrica Maria A1 - Taban, Danush A1 - Helmerich, Dominic A. A1 - Doose, Sören A1 - Bruno, Martina A1 - Barberis, Andrea A1 - Villmann, Carmen A1 - Sauer, Markus A1 - Werner, Christian T1 - Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy JF - Frontiers in Synaptic Neuroscience N2 - 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. KW - super-resolution microscopy (SRM) KW - click-chemistry KW - dSTORM KW - GABA-A receptor KW - genetic code expansion Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251035 SN - 1663-3563 VL - 13 ER - TY - JOUR A1 - Jeanclos, Elisabeth A1 - Knobloch, Gunnar A1 - Hoffmann, Axel A1 - Fedorchenko, Oleg A1 - Odersky, Andrea A1 - Lamprecht, Anna‐Karina A1 - Schindelin, Hermann A1 - Gohla, Antje T1 - Ca\(^{2+}\) functions as a molecular switch that controls the mutually exclusive complex formation of pyridoxal phosphatase with CIB1 or calmodulin JF - FEBS Letters N2 - 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. KW - calmodulin KW - chronophin KW - CIB1 KW - haloacid dehalogenase KW - pyridoxal phosphatase KW - vitamin B6 Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-217963 VL - 594 IS - 13 SP - 2099 EP - 2115 ER - TY - JOUR A1 - Schuhmann, Michael K. A1 - Kraft, Peter A1 - Bieber, Michael A1 - Kollikowski, Alexander M. A1 - Schulze, Harald A1 - Nieswandt, Bernhard A1 - Pham, Mirko A1 - Stegner, David A1 - Stoll, Guido T1 - Targeting platelet GPVI plus rt-PA administration but not α2β1-mediated collagen binding protects against ischemic brain damage in mice JF - International Journal of Molecular Science N2 - 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. KW - ischemic stroke KW - integrin α2 KW - glycoprotein VI KW - recombinant tissue-type plasminogen activator KW - intracranial bleeding KW - transient middle cerebral artery occlusion Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201700 SN - 1422-0067 VL - 20 IS - 8 ER - TY - JOUR A1 - Makbul, Cihan A1 - Khayenko, Vladimir A1 - Maric, Hans Michael A1 - Böttcher, Bettina T1 - Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype JF - Microorganisms N2 - 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. KW - hepatitis B core protein KW - hepatitis B virus KW - peptide inhibitor of envelopment KW - isothermal titration calorimetry KW - electron cryo microscopy KW - low-secretion phenotype mutants KW - peptide microarray Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236720 SN - 2076-2607 VL - 9 IS - 5 ER - TY - JOUR A1 - Kitzenmaier, Alexandra A1 - Schaefer, Natascha A1 - Kasaragod, Vikram Babu A1 - Polster, Tilman A1 - Hantschmann, Ralph A1 - Schindelin, Hermann A1 - Villmann, Carmen T1 - The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia JF - European Journal of Neuroscience N2 - 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. KW - glycine transporter 2 KW - glyvine uptake KW - loss of function KW - presynaptic hyperekplexia KW - protein transport KW - structural disruption Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-206158 VL - 50 IS - 12 ER - TY - JOUR A1 - Kalleda, Natarajaswamy A1 - Amich, Jorge A1 - Arslan, Berkan A1 - Poreddy, Spoorthi A1 - Mattenheimer, Katharina A1 - Mokhtari, Zeinab A1 - Einsele, Hermann A1 - Brock, Matthias A1 - Heinze, Katrin Gertrud A1 - Beilhack, Andreas T1 - Dynamic Immune Cell Recruitment After Murine Pulmonary Aspergillus fumigatus Infection under Different Immunosuppressive Regimens JF - Frontiers in Microbiology N2 - 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. KW - corticosteroids and cyclophosphamide KW - aspergillus fumigatus KW - CD11b+ myeloid cells KW - immune cell recruitment Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165368 VL - 7 IS - 1107 ER - TY - JOUR A1 - Koo, Chek Ziu A1 - Matthews, Alexandra L. A1 - Harrison, Neale A1 - Szyroka, Justyna A1 - Nieswandt, Bernhard A1 - Gardiner, Elizabeth E. A1 - Poulter, Natalie S. A1 - Tomlinson, Michael G. T1 - The platelet collagen receptor GPVI is cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 molecular scissors JF - International Journal of Molecular Sciences N2 - 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. KW - ADAM10 KW - GPVI KW - tetraspanin KW - platelet KW - shedding KW - TspanC8 KW - metalloproteinase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284468 SN - 1422-0067 VL - 23 IS - 5 ER - TY - JOUR A1 - Stegner, David A1 - Klaus, Vanessa A1 - Nieswandt, Bernhard T1 - Platelets as modulators of cerebral ischemia/reperfusion injury JF - Frontiers in Immunology N2 - 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. KW - thrombo-inflammation KW - ischemic stroke KW - platelet KW - glycoprotein Ibα KW - platelet degranulation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195748 SN - 1664-3224 VL - 10 IS - 2505 ER - TY - JOUR A1 - Dütting, Sebastian A1 - Gaits-Iacovoni, Frederique A1 - Stegner, David A1 - Popp, Michael A1 - Antkowiak, Adrien A1 - van Eeuwijk, Judith M.M. A1 - Nurden, Paquita A1 - Stritt, Simon A1 - Heib, Tobias A1 - Aurbach, Katja A1 - Angay, Oguzhan A1 - Cherpokova, Deya A1 - Heinz, Niels A1 - Baig, Ayesha A. A1 - Gorelashvili, Maximilian G. A1 - Gerner, Frank A1 - Heinze, Katrin G. A1 - Ware, Jerry A1 - Krohne, Georg A1 - Ruggeri, Zaverio M. A1 - Nurden, Alan T. A1 - Schulze, Harald A1 - Modlich, Ute A1 - Pleines, Irina A1 - Brakebusch, Cord A1 - Nieswandt, Bernhard T1 - A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis JF - Nature Communications N2 - 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. KW - megakaryocytes KW - blood platelets KW - regulatory circuit downstream KW - glycoprotein Ib Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170797 VL - 8 IS - 15838 ER - TY - JOUR A1 - Stegner, David A1 - van Eeuwijk, Judith M.M. A1 - Angay, Oğuzhan A1 - Gorelashvili, Maximilian G. A1 - Semeniak, Daniela A1 - Pinnecker, Jürgen A1 - Schmithausen, Patrick A1 - Meyer, Imke A1 - Friedrich, Mike A1 - Dütting, Sebastian A1 - Brede, Christian A1 - Beilhack, Andreas A1 - Schulze, Harald A1 - Nieswandt, Bernhard A1 - Heinze, Katrin G. T1 - Thrombopoiesis is spatially regulated by the bone marrow vasculature JF - Nature Communications N2 - 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. KW - bone marrow KW - megakaryocytes KW - thrombopoiesis Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170591 VL - 8 IS - 127 ER - TY - JOUR A1 - Kollikowski, Alexander M. A1 - Schuhmann, Michael K. A1 - Nieswandt, Bernhard A1 - Müllges, Wolfgang A1 - Stoll, Guido A1 - Pham, Mirko T1 - Local Leukocyte Invasion during Hyperacute Human Ischemic Stroke JF - Annals of Neurology N2 - 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 KW - neurology Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212168 VL - 87 IS - 3 ER - TY - JOUR A1 - Göb, Vanessa A1 - Voll, Maximilian G. A1 - Zimmermann, Lena A1 - Hemmen, Katharina A1 - Stoll, Guido A1 - Nieswandt, Bernhard A1 - Schuhmann, Michael K. A1 - Heinze, Katrin G. A1 - Stegner, David T1 - Infarct growth precedes cerebral thrombosis following experimental stroke in mice JF - Scientific Reports N2 - 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. KW - cerebrovascular disorders KW - thrombosis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265791 VL - 11 IS - 1 ER - TY - JOUR A1 - Beck, Sarah A1 - Stegner, David A1 - Loroch, Stefan A1 - Baig, Ayesha A. A1 - Göb, Vanessa A1 - Schumbutzki, Cornelia A1 - Eilers, Eva A1 - Sickmann, Albert A1 - May, Frauke A1 - Nolte, Marc W. A1 - Panousis, Con A1 - Nieswandt, Bernhard T1 - Generation of a humanized FXII knock-in mouse-A powerful model system to test novel anti-thrombotic agents JF - Journal of Thrombosis and Haemostasis N2 - 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. KW - hemostasis, KW - blood coagulation KW - factor XII KW - animal models KW - thrombosis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259567 VL - 19 IS - 11 ER - TY - THES A1 - Aigner, Max T1 - Establishing successful protocols and imaging pipelines for Expansion Microscopy in murine blood platelets T1 - Etablierung erfolgreicher Protokolle zur Probenpräparation und Bildgebung für die ‚Expansion Microscopy‘ in murinen Thrombozyten N2 - 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. N2 - Thrombozyten spielen eine wichtige Rolle im Körper, denn als Teil des Gerinnungssystems, sind sie daran beteiligt Blutverlust vorzubeugen und zu stoppen. Gleichwohl können sie bei Störungen des Gerinnungssystems zu unkontrollierbaren Blutungen und auch durch Aggregation zu kardiovaskulären Ereignissen, wie Herzinfarkt und Schlaganfällen führen. Für ein besseres Verständnis von Hämostase und Gerinnungsstörungen ist es deshalb nötig die Funktion und Aktivierung von Thrombozyten zu verstehen, welche durch eine Vielzahl von Membranrezeptoren und anderen Faktoren gesteuert wird. Eine Methode, um weitere Einblicke in diese Prozesse zu bekommen ist die mikroskopische Darstellung von Rezeptorverteilungen auf der Zellmembran und deren Interaktionen. Dies zu realisieren ist aus verschiedenen Gründen anspruchsvoll. Der mikroskopisch kleine Durchmesser der Thrombozyten macht es konventioneller Lichtmikroskopie schwer, einzelne Rezeptoren auf der Membran darzustellen. Außerdem befinden sich sehr viele Rezeptoren dicht gepackt auf der Membran, sodass sogar superhochauflösende Mikroskope Schwierigkeiten haben, die Rezeptoren quantitativ zu beurteilen. Mit ‚Expansion microscopy‘ (ExM) wurde eine relativ junge superhochaufösende Technik auf Thrombozyten angewendet. Diese Technik erreicht Auflösungen vergleichbar mit sogenannten ‚super-resolution‘ Mikroskopen, ohne die Benutzung selbiger, sondern durch die Kombination von konfokaler Mikroskopie mit einer physikalisch expandierten Probe. In dieser Arbeit evaluierte ich das Potential dieser Technik für superhochauflösende Bilder von Thrombozytenrezeptoren und optimierte die Probenvorbereitung, sodass zweifarbige 3D Bilder von verschiedenen Membranrezeptoren möglich waren. Die Ergebnisse der Kolokationsanalyse zeigten einen deutlich vergrößerten Dynamikumfang durch ExM. Außerdem katalogisierte ich fluoreszenzmarkierte Antikörper gegen verschiedene Thrombozyten Rezeptoren bezüglich ihrer Tauglichkeit mit ExM und zeigte, dass es möglich ist Membranrezeptoren und Bestandteile des Zytoskeletts gleichzeitig zu färben. KW - Expansion Microscopy KW - platelets KW - Mikroskopie KW - Microscopy Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-309003 ER - TY - JOUR A1 - Mammadova-Bach, Elmina A1 - Braun, Attila T1 - Zinc homeostasis in platelet-related diseases JF - International Journal of Molecular Sciences N2 - 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. KW - Zinc KW - platelets KW - hemostasis KW - thrombosis KW - ischemic stroke KW - storage-pool diseases Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285554 SN - 1422-0067 VL - 20 IS - 21 ER - TY - JOUR A1 - Ries, Lena K. A1 - Liess, Anna K. L. A1 - Feiler, Christian G. A1 - Spratt, Donald E. A1 - Lowe, Edward D. A1 - Lorenz, Sonja T1 - Crystal structure of the catalytic C‐lobe of the HECT‐type ubiquitin ligase E6AP JF - Protein Science N2 - 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. KW - dimerization KW - domain swapping KW - E3 enzyme KW - UBE3A KW - X‐ray crystallography Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214812 VL - 29 IS - 6 SP - 1550 EP - 1554 ER - TY - JOUR A1 - Schuhmann, Michael K. A1 - Bieber, Michael A1 - Franke, Maximilian A1 - Kollikowski, Alexander M. A1 - Stegner, David A1 - Heinze, Katrin G. A1 - Nieswandt, Bernhard A1 - Pham, Mirko A1 - Stoll, Guido T1 - Platelets and lymphocytes drive progressive penumbral tissue loss during middle cerebral artery occlusion in mice JF - Journal of Neuroinflammation N2 - 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. KW - ischemic penumbra KW - glycoprotein receptor Ib KW - T-cells KW - ischemic stroke KW - thrombo-inflammation KW - middle cerebral artery occlusion Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259172 VL - 18 IS - 1 ER - TY - THES A1 - Mietrach, Nicole Aline T1 - Structural and functional elucidation of the Type VIIb secretion system from Staphylococcus aureus T1 - Strukturelle und funktionelle Analyse des Typ VIIb Sekretionssystems aus Staphylococcus aureus N2 - 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 Å 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 Å. 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. N2 - Das Typ VII Sekretionssystem (T7SS) ist wichtig für Virulenz und Langzeit- Pathogenität von Gram-positiven Bakterien. Zu diesen gehört auch Staphylococcus aureus, bekannt als Kommensal und Pathogen im Menschen. Das Typ VIIb Sekretionssystem (T7SSb) exportiert kleine, toxische Proteine, die antibakterielle Immunantworten auslösen und für bakterielle Persistenz verantwortlich sind. Außerdem ist es an dem Konkurrenzkampf zwischen Bakterien beteiligt. Das System benötigt verschiedene Komponenten, um eine Sekretion zu ermöglichen. Diese Doktorarbeit konzentriert sich auf zwei dieser Proteine, die ATPase EssC und das Membranprotein EsaA. Beide Komponenten sind unentbehrlich für eine vollständige Funktionalität. EssC gehört zu der Familie der FtsK/SpoIIIE ATPasen und ist evolutionär in allen T7SSs erhalten. EssC besitzt drei C-terminale, zytosolische ATPase Domänen, bezeichnet als EssC-D1, -D2 und D3, wobei EssC-D3 C-terminal gelegen ist. In dieser Arbeit präsentiere ich die Kristallstruktur der ATPase Domäne EssC-D3, aufgelöst bis zu 1.7 Å. Die Domäne ist unabdingbar für die Sekretion. Durch die Strukturauflösung wurde eine hydrophobe, Oberflächen-exponierte Substrat- Bindetasche bestimmt, die eine essenzielle Rolle für den Export der toxischen Substrate einnimmt. Durch dieses Projekt konnten zwei Aminosäuren in dieser Tasche bestimmt werden, die für den Prozess der Substratsekretion wichtig sind. Weiterhin wurde bewiesen, dass nicht nur EssC-D3, sondern auch die ATPase Domäne EssC-D2 und/oder EssC-D1 mit den Substraten interagieren kann. Im Gegensatz zu dem verwandten T7SSb in Bacillus subtilis, verfügt EssC-D3 über ATPase Aktivität und ermöglicht dadurch den Substratexport. Das Membranprotein EsaA besitzt einen extrazellulären Abschnitt, der sich durch die Zellwand von S. aureus erstreckt. Dieser extrazelluläre Part besteht aus einer stabilen Domäne, welche kristallisiert werden konnte und bis zu 4 Å diffraktiert. Aufgrund von fehlenden homologen Strukturen konnte die Struktur der Domäne noch nicht bestimmt werden. Für die Phasenbestimmung, die wichtig für die Strukturauflösung ist, wurden Kristalle mit Selenomethionyl-Substituten hergestellt. Die Strukturauflösung ist noch nicht beendet. Erste Experimente bezüglich der extrazellulären Domäne zeigen, dass diese ebenfalls wichtig für die Substratsekretion und zusätzlich am Konkurrenzkampf zwischen Bakterien beteiligt ist. KW - Secretion KW - Gram-positive bacteria KW - Type VIIb secretion system KW - Staphylococcus aureus Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214824 ER - TY - JOUR A1 - Petruseva, Irina A1 - Naumenko, Natalia A1 - Kuper, Jochen A1 - Anarbaev, Rashid A1 - Kappenberger, Jeannette A1 - Kisker, Caroline A1 - Lavrik, Olga T1 - The Interaction Efficiency of XPD-p44 With Bulky DNA Damages Depends on the Structure of the Damage JF - Frontiers in Cell and Developmental Biology N2 - 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. KW - nucleotide excision repair KW - XPD helicase KW - DNA damage KW - protein-DNA interaction KW - bulky damages recognition KW - photo-cross-linking Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231806 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Peissert, Stefan A1 - Sauer, Florian A1 - Grabarczyk, Daniel B. A1 - Braun, Cathy A1 - Sander, Gudrun A1 - Poterszman, Arnaud A1 - Egly, Jean-Marc A1 - Kuper, Jochen A1 - Kisker, Caroline T1 - In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair JF - Nature Communications N2 - 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. KW - nucleotide excision repair KW - nuclear receptors KW - helicase KW - transactivation KW - fluorescence KW - recognition KW - subunit KW - binding KW - sulfur KW - kinease Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229857 VL - 11 IS - 1 ER - TY - JOUR A1 - Wagner, Nicole A1 - Mott, Kristina A1 - Upcin, Berin A1 - Stegner, David A1 - Schulze, Harald A1 - Ergün, Süleyman T1 - CXCL12-abundant reticular (CAR) cells direct megakaryocyte protrusions across the bone marrow sinusoid wall JF - Cells N2 - 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. KW - megakaryocytes KW - microvasculature KW - CXCL12-abundant reticular (CAR)-cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234180 SN - 2073-4409 VL - 10 IS - 4 ER - TY - JOUR A1 - Harnoš, Jakub A1 - Cañizal, Maria Consuelo Alonso A1 - Jurásek, Miroslav A1 - Kumar, Jitender A1 - Holler, Cornelia A1 - Schambony, Alexandra A1 - Hanáková, Kateřina A1 - Bernatík, Ondřej A1 - Zdráhal, Zbynêk A1 - Gömöryová, Kristína A1 - Gybeľ, Tomáš A1 - Radaszkiewicz, Tomasz Witold A1 - Kravec, Marek A1 - Trantírek, Lukáš A1 - Ryneš, Jan A1 - Dave, Zankruti A1 - Fernández-Llamazares, Ana Iris A1 - Vácha, Robert A1 - Tripsianes, Konstantinos A1 - Hoffmann, Carsten A1 - Bryja, Vítězslav T1 - Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1 JF - Nature Communications N2 - 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. KW - biological techniques KW - cell signalling KW - phosphorylation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227837 VL - 10 ER - TY - JOUR A1 - Heil, Hannah S. A1 - Schreiber, Benjamin A1 - Götz, Ralph A1 - Emmerling, Monika A1 - Dabauvalle, Marie-Christine A1 - Krohne, Georg A1 - Höfling, Sven A1 - Kamp, Martin A1 - Sauer, Markus A1 - Heinze, Katrin G. T1 - Sharpening emitter localization in front of a tuned mirror JF - Light: Science & Applications N2 - 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. KW - imaging and sensing KW - super-resolution microscopy Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228080 VL - 7 ER - TY - THES A1 - Weigel [verh. Hoffmann], Mathis Leonard T1 - Thrombozytenfunktionsanalyse als potenzielles Instrument zur Früherkennung von Sepsis T1 - Platelet function analysis as a potential tool for early sepsis diagnosis N2 - Sepsis ist ein häufiges und akut lebensbedrohliches Syndrom, das eine Organfunktionsstörung in Folge einer dysregulierten Immunantwort auf eine Infektion beschreibt. Eine frühzeitige Diagnosestellung und Therapieeinleitung sind von zentraler Bedeutung für das Überleben der Patient:innen. In einer Pilotstudie konnte unsere Forschungsgruppe mittels Durchflusszytometrie eine ausgeprägte Hyporeaktivität der Thrombozyten bei Sepsis nachweisen, die einen potenziell neuen Biomarker zur Sepsis-Früherkennung darstellt. Zur Evaluation des Ausmaßes und Entstehungszeitpunktes der detektierten Thrombozytenfunktionsstörung wurden im Rahmen der vorliegenden Arbeit zusätzlich 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. Phänotypische Auffälligkeiten der Thrombozyten bei Sepsis umfassten: (i) eine veränderte Expression verschiedener Untereinheiten des GPIb-IX-V-Rezeptorkomplexes, die auf ein verstärktes Rezeptor-Shedding hindeutet; (ii) ein ausgeprägtes Mepacrin-Beladungsdefizit, das auf eine zunehmend reduzierte Anzahl von δ-Granula entlang des Infektion-Sepsis Kontinuums hinweist; (iii) eine Reduktion endständig gebundener Sialinsäure im Sinne einer verstärkten Desialylierung. Die funktionelle Analyse der Thrombozyten bei Sepsis ergab bei durchflusszytometrischer Messung der Integrin αIIbβ3-Aktivierung (PAC-1-Bindung) eine ausgeprägte generalisierte Hyporeaktivität gegenüber multiplen Agonisten, die abgeschwächt bereits bei Infektion nachweisbar war und gemäß ROC-Analysen gut zwischen Infektion und Sepsis diskriminierte (AUC >0.80 für alle Agonisten). Im Gegensatz dazu zeigten Thrombozyten bei Sepsis und Analyse mittels PFA-200 unter Einfluss physiologischer Scherkräfte eine normale bis gar beschleunigte Aggregation. Die Reaktivitätsmessung von Thrombozyten mittels Durchflusszytometrie stellt weiterhin einen vielversprechenden Biomarker für die Sepsis-Früherkennung dar. Für weitere Schlussfolgerungen ist jedoch eine größere Kohorte erforderlich. In nachfolgenden Untersuchungen sollten zudem mechanistische Ursachen der beschriebenen phänotypischen und funktionellen Auffälligkeiten von Thrombozyten bei Infektion und Sepsis z.B. mittels Koinkubationsexperimenten untersucht werden. N2 - Sepsis is a frequent and life-threatening condition that describes organ dysfunction resulting from a dysregulated host immune response to infection. Early diagnosis and treatment are essential to improve patient survival. In a previous pilot study with sepsis patients, our research identified a severe platelet hyporeactivity using flow cytometry which could become a potential new biomarker for early sepsis diagnosis. To evaluate onset and extend of the detected platelet dysfunction in this study, we extended our patient cohort in addition to sepsis (SOFA-score ≥2; n=13) also to hospitalized patients with infection without sepsis (SOFA-score <2; n=12). Both cohorts were assessed at two time points during the disease (t1: <24h; t2: day 5-7) by flow cytometry and PFA-200 and compared with a healthy control group (n=28). Platelet phenotypic abnormalities during sepsis included: (i) altered expression of subunits of the GPIb-IX-V receptor complex, pointing to increased receptor shedding; (ii) a severe mepacrine loading deficit, indicating an increasingly reduced number of δ-granules along the infection-sepsis continuum; (iii) a reduction of terminally bound sialic acid, suggesting increased desialylation. Functional analysis of platelets in sepsis revealed a marked and generalized hyporeactivity toward multiple agonists when integrin αIIbβ3 activation (PAC-1 binding) was measured by flow cytometry, which was already to a lesser extend present in patients with infection and discriminated well between infection and sepsis according to ROC analysis (AUC >0.80 for all agonists). In contrast, platelets from septic patients showed normal to even accelerated aggregation when measured under flow condition and physiological shear forces by PFA-200. Analysis of platelet reactivity by flow cytometry remains a promising biomarker for early sepsis detection, but a larger cohort is needed for further conclusions. In subsequent studies, mechanistic causes of the described alterations in platelet phenotype and function during infection and sepsis should be investigated, e.g. by means of co-incubation experiments. KW - Sepsis KW - Thrombozyt KW - Biomarker KW - Frühdiagnostik KW - Durchflusscytometrie KW - Thrombozytenfunktionsanalyse Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-358193 ER - TY - JOUR A1 - Gotru, Sanjeev Kiran A1 - van Geffen, Johanna P. A1 - Nagy, Magdolna A1 - Mammadova-Bach, Elmina A1 - Eilenberger, Julia A1 - Volz, Julia A1 - Manukjan, Georgi A1 - Schulze, Harald A1 - Wagner, Leonard A1 - Eber, Stefan A1 - Schambeck, Christian A1 - Deppermann, Carsten A1 - Brouns, Sanne A1 - Nurden, Paquita A1 - Greinacher, Andreas A1 - Sachs, Ulrich A1 - Nieswandt, Bernhard A1 - Hermanns, Heike M. A1 - Heemskerk, Johan W. M. A1 - Braun, Attila T1 - Defective Zn2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases JF - Scientific Reports N2 - 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. KW - coagulation system KW - metals Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227455 VL - 9 ER - TY - JOUR A1 - Hines, Rochelle M. A1 - Maric, Hans Michael A1 - Hines, Dustin J. A1 - Modgil, Amit A1 - Panzanelli, Patrizia A1 - Nakamura, Yasuko A1 - Nathanson, Anna J. A1 - Cross, Alan A1 - Deeb, Tarek A1 - Brandon, Nicholas J. A1 - Davies, Paul A1 - Fritschy, Jean-Marc A1 - Schindelin, Hermann A1 - Moss, Stephen J. T1 - Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin JF - Nature Communications N2 - 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. KW - cellular neuroscience KW - ion channels in the nervous system KW - neurotransmitters KW - synaptic development Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320719 VL - 9 ER - TY - THES A1 - Karwen, Till T1 - Platelets promote insulin secretion of pancreatic β-cells T1 - Thrombozyten fördern die Insulinsekretion von pankreatischen β-Zellen N2 - 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. N2 - Der Pankreas ist das Schlüsselorgan für die Aufrechterhaltung der Glukosehomöostase. Diese wird insbesondere durch das von α-Zellen stammende Glukagon und von β-Zellen stammende Insulin reguliert, die als Reaktion auf Nährstoffmangel beziehungsweise erhöhte Glukosespiegel freigesetzt werden. Obwohl Glukose der Hauptregulator der Insulinsekretion ist, wird sie durch verschiedene Potentiatoren erheblich gesteigert. Thrombozyten sind kernlose Zellfragmente im Blutkreislauf, die für die Hämostase unerlässlich sind. Neben ihrer klassischen Funktion sind sie auch an anderen physiologischen und pathophysiologischen Prozessen beteiligt, etwa an der Tumorentwicklung, der Immunabwehr und der Angiogenese. Thrombozyten von Diabetikern weisen häufig eine erhöhte Reaktivität und basale Aktivierung auf. Außerdem speichern und sekretieren sie Substanzen, von denen bekannt ist, dass sie die Insulinsekretion durch β-Zellen verstärken. Aus diesen Gründen wurde in dieser Arbeit der Einfluss von Thrombozyten auf die Funktion von β-Zellen untersucht. Es konnte gezeigt werden, dass sowohl Glukose als auch eine aus β-Zellen stammende Substanz/en die Thrombozytenaktivierung und die Bindung an Kollagen fördern. Darüber hinaus wurde eine spezifische Thrombozytenadhäsion an der Mikrovaskulatur der pankreatischen Inseln festgestellt, was die Hypothese ihres Einflusses auf die Glukosehomöostase unterstützt. Eine genetische oder pharmakologische Ablation der Thrombozytenfunktion sowie eine Depletion von Thrombozyten führten zu einer verminderten Insulinsekretion und einer damit verbundenen Glukoseintoleranz. Hierbei erwies sich die Lipidfraktion von Thrombozyten als essentieller Potentiator für die glukosestimulierte Insulinsekretion, wobei 20-Hydroxyeicosatetraensäure (20-HETE) und die Lyso-Vorstufe des Plättchen-Aktivierenden Faktors (LysoPAF) als entscheidende Faktoren identifiziert werden konnten. Weiterhin wurde festgestellt, dass sowohl der direkte stimulierende Effekt von Thrombozyten auf die Insulinsekretion, als auch deren 20-HETE Sekretion mit zunehmendem Alter abnimmt. Thrombozyten beeinflussten außerdem die Inselvaskularisierung, welche mutmaßlich zusätzlich zu Glukoseintoleranz führt. Insgesamt deuten die Ergebnisse dieser Arbeit auf einen direkten und indirekten Mechanismus der Thrombozyten bei der Regulierung der Insulinsekretion hin, der die Glukosehomöostase insbesondere bei jungen Menschen gewährleistet. KW - platelet KW - β cell KW - insulin KW - pancreas KW - diabetes KW - Thrombozyt KW - Insulinsekretion Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313933 ER - TY - THES A1 - Nair, Radhika Karal T1 - Structural and biochemical characterization of USP28 inhibition by small molecule inhibitors T1 - Strukturelle und biochemische Charakterisierung der Hemmung von USP28 durch niedermolekulare Inhibitoren N2 - 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. N2 - Ubiquitinierung ist eine wichtige posttranslationale Modifikation, die die zelluläre Homöostase aufrechterhält, indem sie verschiedene biologische Prozesse reguliert. Deubiquitinasen (DUBs) sind Enzyme, die den Ubiquitinierungsprozess umkehren, indem sie die Entfernung von Ubiquitin von einem Substrat katalysieren. Eine abnorme Expression oder Funktion von DUBs wird häufig mit dem Auftreten und Fortschreiten verschiedener Krankheiten, einschließlich Krebs, in Verbindung gebracht. Ubiquitin-spezifische Proteasen (USPs), die im Menschen die größte Familie der DUBs bilden, sind als potenzielle Ziele in der Krebstherapie von besonderem Interesse, da viele von ihnen in bösartigen Tumoren oder deren Mikroumgebung abnormal aktiv oder überexprimiert sind. Die zwei eng verwandten Mitglieder der USP-Familie, USP28 und USP25, weisen eine hohe Sequenzidentität auf, sind aber an unterschiedlichen biologischen Prozessen beteiligt. USP28 reguliert die Zellproliferation, die Onkogenese, die Reparatur von DNA-Schäden und die Apoptose, während USP25 eine Rolle bei der antiviralen Reaktion, der angeborenen Immunität, dem ER-assoziierten Abbau und der Carcinogenese spielt. USP28 und USP25 weisen auch unterschiedliche oligomere Zustände auf. Während USP28 ein konstitutiv aktives Dimer bildet, tritt USP25 als auto-inhibiertes Tetramer auf. Strukturell bestehen die katalytischen Domänen sowohl von USP28 als auch von USP25 aus der kanonischen globulären USP-Domäne enthalten jedoch eine zusätzliche Insertion, die als „USP25/28 catalytic domain inserted domain (UCID)“ bezeichnet wird und die Oligomerisierung der Proteine vermittelt. Die Dissoziation des USP25 Tetramers in Dimere führt zu einem aktivierten USP25-Protein. Es ist jedoch immer noch nicht klar, was seine Aktivierung auslöst. Aufgrund ihrer Rolle bei der Aufrechterhaltung und Stabilisierung zahlreicher Onkoproteine haben sich USP28 und USP25 als interessante Kandidaten für die Entwicklung von Medikamenten in der Krebstherapie erwiesen. Jüngste Fortschritte in der Entwicklung von niedermolekularen Inhibitoren haben zur Entdeckung von relativ potenten Inhibitoren von USP28 und USP25 geführt. Diese Arbeit konzentriert sich auf die Strukturaufklärung von USP28 und die biochemische Charakterisierung von USP28/USP25, beide im Komplex mit Vertretern von drei der acht Verbindungsklassen, die als USP28/USP25-spezifische Inhibitoren bekannt sind. Die Kristallstrukturen von USP28 im Komplex mit den AZ-Verbindungen, Vismodegib und FT206 zeigen, dass alle Inhibitoren in einer ähnlichen Region an USP28 binden - einer allosterischen Tasche, die in der Nähe des katalytischen Zentrums liegt und sich zwischen der Handflächen- und der Daumen-Subdomäne befindet. Diese Bindungstasche ist identisch mit der Position, an der der „UCID-tip“ im USP25-Tetramer bindet und das Protein in eine verschränkte, inaktive Konformation versetzt. Die Bildung der Bindungstasche in USP28 erfordert eine Verschiebung der α5-Helix, die zu Konformationsänderungen und einer lokalen Verzerrung des Bindungskanalsführt, der normalerweise den C-terminus des Ubiquitin-Moleküls bindet und so die Katalyse verhindert und die Aktivität von USP28 hemmt. Die Schlüsselreste der USP28-Inhibitor-Bindungstasche sind in USP25 hoch konserviert. Mutagenese-Studien dieser Aminosäuren, begleitet von biochemischen und biophysikalischen Analysen, bestätigen den vorgeschlagenen Mechanismus der Hemmung und eine ähnliche Bindung der Inhibitoren an USP25. Diese Arbeit liefert wertvolle Einblicke in den Hemmungsmechanismus der Kleinmolekülverbindungen, die spezifisch für die DUBs USP28 und USP25 entwickelt worden sind. Die Strukturen der USP28-Inhibitor-Komplexe bieten eine Grundlage für die zukünftige Entwicklung spezifischerer und wirksamerer Inhibitoren. KW - USP KW - Inhibition KW - enzyme KW - crystallography KW - Unique Selling Proposition KW - Inhibition KW - Enzym KW - Kristallographie Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-281742 ER -