@article{MammadovaBachBraun2019, author = {Mammadova-Bach, Elmina and Braun, Attila}, title = {Zinc homeostasis in platelet-related diseases}, series = {International Journal of Molecular Sciences}, volume = {20}, journal = {International Journal of Molecular Sciences}, number = {21}, issn = {1422-0067}, doi = {10.3390/ijms20215258}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285554}, year = {2019}, abstract = {Zn\(^{2+}\) deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn\(^{2+}\)-deficient diets, accounting for 1-4\% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn\(^{2+}\) deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn\(^{2+}\) status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn\(^{2+}\) uptake in the gut using different nutritional supplementation of Zn\(^{2+}\) could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn\(^{2+}\) diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn\(^{2+}\) in hemostasis. Storage protein metallothionein maintains or releases Zn\(^{2+}\) in the cytoplasm, and the dynamic change of this cytoplasmic Zn\(^{2+}\) pool is regulated by the redox status of the cell. An increase of labile Zn\(^{2+}\) pool can be toxic for the cells, and therefore cytoplasmic Zn\(^{2+}\) levels are tightly regulated by several Zn\(^{2+}\) transporters located on the cell surface and also on the intracellular membrane of Zn\(^{2+}\) storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn\(^{2+}\) is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn\(^{2+}\) transport and the physiological role of Zn\(^{2+}\) store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn\(^{2+}\) to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn\(^{2+}\) homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.}, language = {en} } @article{KraftDrechslerGunrebenetal.2014, author = {Kraft, Peter and Drechsler, Christiane and Gunreben, Ignaz and Nieswandt, Bernhard and Stoll, Guido and Heuschmann, Peter Ulrich and Kleinschnitz, Christoph}, title = {Von Willebrand Factor Regulation in Patients with Acute and Chronic Cerebrovascular Disease: A Pilot, Case-Control Study}, series = {PLoS ONE}, volume = {9}, journal = {PLoS ONE}, number = {6}, issn = {1932-6203}, doi = {10.1371/journal.pone.0099851}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119588}, pages = {e99851}, year = {2014}, abstract = {Background and Purpose In animal models, von Willebrand factor (VWF) is involved in thrombus formation and propagation of ischemic stroke. However, the pathophysiological relevance of this molecule in humans, and its potential use as a biomarker for the risk and severity of ischemic stroke remains unclear. This study had two aims: to identify predictors of altered VWF levels and to examine whether VWF levels differ between acute cerebrovascular events and chronic cerebrovascular disease (CCD). Methods A case-control study was undertaken between 2010 and 2013 at our University clinic. In total, 116 patients with acute ischemic stroke (AIS) or transitory ischemic attack (TIA), 117 patients with CCD, and 104 healthy volunteers (HV) were included. Blood was taken at days 0, 1, and 3 in patients with AIS or TIA, and once in CCD patients and HV. VWF serum levels were measured and correlated with demographic and clinical parameters by multivariate linear regression and ANOVA. Results Patients with CCD (158±46\%) had significantly higher VWF levels than HV (113±36\%, P<0.001), but lower levels than AIS/TIA patients (200±95\%, P<0.001). Age, sex, and stroke severity influenced VWF levels (P<0.05). Conclusions VWF levels differed across disease subtypes and patient characteristics. Our study confirms increased VWF levels as a risk factor for cerebrovascular disease and, moreover, suggests that it may represent a potential biomarker for stroke severity, warranting further investigation.}, language = {en} } @phdthesis{Gromova2007, author = {Gromova, Kira V.}, title = {Visualization of the Smad direct signaling response to Bone Morphogenetic Protein 4 activation with FRET-based biosensors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-25855}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {The Transforming Growth Factor (TGF) superfamily of cytokines and their serine/threonine kinase receptors play an important role in the regulation of cell division, differentiation, adhesion, migration, organization, and death. Smad proteins are the major intracellular signal transducers for the TGF receptor superfamily that mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 (BMP-4) is a representative of the TGF superfamily, which regulates the formation of teeth, limbs and bone, and also plays a role in fracture repair. Binding of BMP-4 to its receptor stimulates phosphorylation of Smad1, which subsequently recruits Smad4. A hetero-oligomeric complex consisting of Smad1 and Smad4 then translocates into the nucleus and regulates transcription of target genes by interacting with transcription factors. Although the individual steps of the signaling cascade from the receptor to the nucleus have been identified, the exact kinetics and the rate limiting step(s) have remained elusive. Standard biochemical techniques are not suitable for resolving these issues, as they do not offer sufficiently high sensitivity and temporal resolution. In this study, advanced optical techniques were used for direct visualization of Smad signaling in live mammalian cells. Novel fluorescent biosensors were developed by fusing cyan and yellow fluorescent proteins to the signaling molecules Smad1 and Smad4. By measuring Fluorescence Resonance Energy Transfer (FRET) between the two fluorescent proteins, the kinetics of BMP/Smad signaling was unraveled. A rate-limiting delay of 2 - 5 minutes occurred between BMP receptor stimulation and Smad1 activation. A similar delay was observed in the complex formation between Smad1 and Smad4. Further experimentation indicated that the delay is dependent on the Mad homology 1 (MH1) domain of Smad1. These results give new insights into the dynamics of the BMP receptor - Smad1/4 signaling process and provide a new tool for studying Smads and for testing inhibitory drugs.}, subject = {FRET}, language = {en} } @phdthesis{Blachutzik2012, author = {Blachutzik, J{\"o}rg O.}, title = {Visualisierung von Plasmamembran-Dom{\"a}nen in Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71925}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Unter Verwendung fluoreszenzmarkierter Remorine der taxonomischen Gruppe 1b wurden Nanodom{\"a}nen in Arabidopsis Plasmamembranen (PM) unter Verwendung hoch aufl{\"o}sender Laser Scanning-Systeme sichtbar gemacht. In diesen kompartimentierten Membranbereichen lagerten sich Sterol-abh{\"a}ngige Remorine aus verschiedenen Pflanzen-familien zusammen und zeigten dort Kolokalisation. Dies wurde statistisch belegt durch hohe Pearson und Spearman Korrelationskoeffizienten. Remorine konnten schließlich als pflanzliche Markerproteine f{\"u}r kompartimentierte Membranbereiche etabliert werden. Die Nanodom{\"a}nen zeigten zu keinem Zeitpunkt laterale Bewegungen in der PM und scheinen sowohl von zytoskelett{\"a}ren Strukturen als auch von Komponenten der Zellwand stabilisiert zu werden. M{\"o}glicherweise spielen transmembrane Tetraspanine sowie GPI-verankerte SKU5-Proteine eine Rolle bei der stabilen Verankerung. F{\"u}r zwei native Arabidopsis Remorine wurden posttranslationale Modifikationsstellen aufgedeckt, die der Anheftung dieser hydrophilen Proteine an die PM dienen. Weiterhin scheinen gleichartige Remorine miteinander zu interagieren. Beispielsweise waren im Zytosol lokalisierte Remorin-Mutanten bei einer gleichzeitigen Expression der entsprechenden Voll{\"a}ngenproteine erneut an der PM zu finden. F{\"u}r die Remorine wurde postuliert, dass sie mit anderen Proteinen interagieren und dabei makromolekulare Strukturen ausbilden. Den Remorinen k{\"o}nnte daher eine Aufgabe bei der molekularen Organisation pflanzlicher Membrandom{\"a}nen zukommen, indem sie ein filamentartiges Netzwerk innerhalb distinkter Dom{\"a}nen ausbilden, das m{\"o}glicherweise zur Stabilit{\"a}t und Aufrechterhaltung dieser spezialisierten Bereiche beitr{\"a}gt. Unter Einbeziehung der STED-Mikroskopie wurde eine empirische Gr{\"o}ßenverteilung von 97±4nm Durchmesser f{\"u}r PM-st{\"a}ndige Dom{\"a}nen in Arabidopsis ermittelt. Hinsichtlich der physiologischen Relevanz konnte gezeigt werden, dass die Dom{\"a}nen eine Rolle bei der ABA-vermittelten, kalziumabh{\"a}ngigen Regulation des Anionenkanals SLAH3 einnehmen. SLAH3 wird durch kalziumabh{\"a}ngige Kinasen aus der CDPK-Familie aktiviert, im Speziellen durch CPK21 und CPK23. Beide Kinasen werden durch die ABA-sensitiven Phosphatasen ABI1 und ABI2 reguliert. Die spezifisch stattfindenden Interaktionen zwischen SLAH3 und CPK21, sowie zwischen CPK21 und ABI1 waren auf Nanodom{\"a}nen beschr{\"a}nkt und wurden durch die Methodik der bimolekularen Fluoreszenzkomplementation erstmals in planta nachgewiesen, mit Remorinen der taxonomischen Gruppe 1b als etablierte Markerproteine f{\"u}r Membrandom{\"a}nen.}, subject = {Plasmamembran}, language = {de} } @article{KoenigWolfHeisenberg2016, author = {Koenig, Sebastian and Wolf, Reinhard and Heisenberg, Martin}, title = {Visual Attention in Flies-Dopamine in the Mushroom Bodies Mediates the After-Effect of Cueing}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {8}, doi = {10.1371/journal.pone.0161412}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179564}, year = {2016}, abstract = {Visual environments may simultaneously comprise stimuli of different significance. Often such stimuli require incompatible responses. Selective visual attention allows an animal to respond exclusively to the stimuli at a certain location in the visual field. In the process of establishing its focus of attention the animal can be influenced by external cues. Here we characterize the behavioral properties and neural mechanism of cueing in the fly Drosophila melanogaster. A cue can be attractive, repulsive or ineffective depending upon (e.g.) its visual properties and location in the visual field. Dopamine signaling in the brain is required to maintain the effect of cueing once the cue has disappeared. Raising or lowering dopamine at the synapse abolishes this after-effect. Specifically, dopamine is necessary and sufficient in the αβ-lobes of the mushroom bodies. Evidence is provided for an involvement of the αβ\(_{posterior}\) Kenyon cells.}, language = {en} } @phdthesis{Sareen2011, author = {Sareen, Preeti}, title = {Visual attention in Drosophila melanogaster}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69616}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {There is such vast amount of visual information in our surroundings at any time that filtering out the important information for further processing is a basic requirement for any visual system. This is accomplished by deploying attention to focus on one source of sensory inputs to the exclusion of others (Luck and Mangun 2009). Attention has been studied extensively in humans and non human primates (NHPs). In Drosophila, visual attention was first demonstrated in 1980 (Wolf and Heisenberg 1980) but this field remained largely unexplored until recently. Lately, however, studies have emerged that hypothesize the role of attention in several behaviors but do not specify the characteristic properties of attention. So, the aim of this research was to characterize the phenomenon of visual attention in wild-type Drosophila, including both externally cued and covert attention using tethered flight at a torque meter. Development of systematic quantifiable behavioral tests was a key aspect for this which was not only important for analyzing the behavior of a population of wild-type flies but also for comparing the wild-type flies with mutant flies. The latter would help understand the molecular, genetic, and neuronal bases of attention. Since Drosophila provides handy genetic tools, a model of attention in Drosophila will serve to the greater questions about the neuronal circuitry and mechanisms involved which might be analogous to those in primates. Such a model might later be used in research involving disorders of attention. Attention can be guided to a certain location in the visual field by the use of external cues. Here, using visual cues the attention of the fly was directed to one or the other of the two visual half-fields. A simple yet robust paradigm was designed with which the results were easily quantifiable. This paradigm helped discover several interesting properties of the cued attention, the most substantial one being that this kind of external guidance of attention is restricted to the lower part of the fly's visual field. The guiding cue had an after-effect, i.e. it could occur at least up to 2 seconds before the test and still bias it. The cue could also be spatially separated from the test by at least 20° and yet attract the attention although the extent of the focus of attention (FoA) was smaller than one lower visual half-field. These observations excluded the possibility of any kind of interference between the test and the cue stimuli. Another interesting observation was the essentiality of continuous visibility of the test stimulus but not the cue for effective cuing. When the contrast of the visual scene was inverted, differences in response frequencies and cuing effects were observed. Syndirectional yaw torque responses became more frequent than the antidirectional responses and cuing was no longer effective in the lower visual field with inverted contrast. Interestingly, the test stimulus with simultaneous displacement of two stripes not only effectuated a phasic yaw torque response but also a landing response. A 50 landing response was produced in more than half of the cases whenever a yaw torque response was produced. Elucidation of the neuronal correlates of the cued attention was commenced. Pilot experiments with hydroxyurea (HU) treated flies showed that mushroom bodies were not required for the kind of guidance of attention tested in this study. Dopamine mutants were also tested for the guidance of attention in the lower visual field. Surprisingly, TH-Gal4/UAS-shits1 flies flew like wild-type flies and also showed normal optomotor response during the initial calibration phase of the experiment but did not show any phasic yaw torque or landing response at 18 °C, 25 °C or 30 °C. dumb2 flies that have almost no D1 dopamine receptor dDA1 expression in the mushroom bodies and the central complex (Kim et al. 2007) were also tested and like THGal4/ UAS-shits1 flies did not show any phasic yaw torque or landing response. Since the dopamine mutants did not show the basic yaw torque response for the test the role of dopamine in attention could not be deduced. A different paradigm would be needed to test these mutants. Not only can attention be guided through external cues, it can also be shifted endogenously (covert attention). Experiments with the windows having oscillating stripes nicely demonstrated the phenomenon of covert attention due to the production of a characteristic yaw torque pattern by the flies. However, the results were not easily quantifiable and reproducible thereby calling for a more systematic approach. Experiments with simultaneous opposing displacements of two stripes provide a promising avenue as the results from these experiments showed that the flies had a higher tendency to deliver one type of response than when the responses would be produced stochastically suggesting that attention increased this tendency. Further experiments and analysis of such experiments could shed more light on the mechanisms of covert attention in flies.}, subject = {Visuelle Aufmerksamkeit}, language = {en} } @article{KoenigWolfHeisenberg2016, author = {Koenig, Sebastian and Wolf, Reinhard and Heisenberg, Martin}, title = {Vision in Flies: Measuring the Attention Span}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {2}, doi = {10.1371/journal.pone.0148208}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179947}, year = {2016}, abstract = {A visual stimulus at a particular location of the visual field may elicit a behavior while at the same time equally salient stimuli in other parts do not. This property of visual systems is known as selective visual attention (SVA). The animal is said to have a focus of attention (FoA) which it has shifted to a particular location. Visual attention normally involves an attention span at the location to which the FoA has been shifted. Here the attention span is measured in Drosophila. The fly is tethered and hence has its eyes fixed in space. It can shift its FoA internally. This shift is revealed using two simultaneous test stimuli with characteristic responses at their particular locations. In tethered flight a wild type fly keeps its FoA at a certain location for up to 4s. Flies with a mutation in the radish gene, that has been suggested to be involved in attention-like mechanisms, display a reduced attention span of only 1s.}, language = {en} } @article{PatilGentschevAdelfingeretal.2012, author = {Patil, Sandeep S. and Gentschev, Ivaylo and Adelfinger, Marion and Donat, Ulrike and Hess, Michael and Weibel, Stephanie and Nolte, Ingo and Frentzen, Alexa and Szalay, Aladar A.}, title = {Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {10}, doi = {10.1371/journal.pone.0047472}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130039}, pages = {e47472}, year = {2012}, abstract = {Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.}, language = {en} } @article{GholamiChenBelinetal.2013, author = {Gholami, Sepideh and Chen, Chun-Hao and Belin, Laurence J. and Lou, Emil and Fujisawa, Sho and Antonacci, Caroline and Carew, Amanda and Chen, Nanhai G. and De Brot, Marina and Zanzonico, Pat B. and Szalay, Aladar A. and Fong, Yuman}, title = {Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer}, series = {Breast Cancer Research}, volume = {15}, journal = {Breast Cancer Research}, number = {R26}, doi = {10.1186/bcr3404}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122140}, year = {2013}, abstract = {Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model. Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90\% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide. Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10\% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05). Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.}, language = {en} } @article{BarcenaUribarriTheinMaieretal.2013, author = {B{\´a}rcena-Uribarri, Iv{\´a}n and Thein, Marcus and Maier, Elke and Bonde, Mari and Bergstr{\"o}m, Sven and Benz, Roland}, title = {Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {11}, doi = {10.1371/journal.pone.0078272}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129965}, pages = {e78272}, year = {2013}, abstract = {In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than 'normal' Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be \(\leq\)1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80-90\% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.}, language = {en} } @phdthesis{Karus2022, author = {Karus, Christine}, title = {Untersuchung der Architektur von Proteinstrukturen des Ranvier-Schn{\"u}rrings mittels der super-hochaufl{\"o}senden Mikroskopiemethode dSTORM}, doi = {10.25972/OPUS-27456}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-274568}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Ranvier-Schn{\"u}rringe spielen eine entscheidende Rolle bei der schnellen Weiterleitung von elektrischen Impulsen in Nervenzellen. Bei bestimmten neurologischen Erkrankungen, den Neuropathien, kann es zu St{\"o}rungen in der ultrastrukturellen Organisation verschiedener Schn{\"u}rring-Proteine kommen (Doppler et al., 2018, Doppler et al., 2016). Eine detailliertere Kenntnis der genauen Anordnung dieser Schn{\"u}rring-Proteine und eventueller Abweichungen von dieser Anordnung im Krankheitsfall, k{\"o}nnte der Schl{\"u}ssel zu einer vereinfachten Diagnostik von bestimmten Neuropathie- Formen sein. Ziel meiner Arbeit war es daher, die Untersuchung der ultrastrukturellen Architektur der (para-)nodalen Adh{\"a}sionsproteine Neurofascin-155 und Caspr1 unter Verwendung der super-hochaufl{\"o}senden Mikroskopiemethode dSTORM (direct Stochastic Optical Reconstruction Microscopy) an murinen Zupfnervenpr{\"a}paraten zu etablieren. Nach erster Optimierung der Probenpr{\"a}paration f{\"u}r die 2-Farben-dSTORM sowie der korrelationsbasierten Bildanalyse, konnte ich mittels modellbasierter Simulation die zugrundeliegende Molek{\"u}lorganisation identifizieren und mit Hilfe der Ergebnisse aus fr{\"u}heren Untersuchungen validieren. In einem translationalen Ansatz habe ich anschließend humane Zupfnervenpr{\"a}parate von 14 Probanden mit unterschiedlichen Formen einer Neuropathie mikroskopiert und ausgewertet, um die Anwendbarkeit dieses Ansatzes in der Diagnostik zu testen. Obgleich keine signifikanten Unterschiede zwischen physiologischem und pathologischem neurologischem Gewebe hinsichtlich Neurofascin-155 und Caspr1 festgestellt werden konnten, scheint der Ansatz grunds{\"a}tzlich dennoch vielversprechend zu sein, bedarf jedoch noch weiteren Anstrengungen hinsichtlich Probenpr{\"a}paration, Auswertungs- und Versuchsprotokollen und einer gr{\"o}ßeren Anzahl an humanen Biopsien mit homogenerem Krankheitsbild.}, language = {de} } @article{BalakrishnanHemmenChoudhuryetal.2022, author = {Balakrishnan, Ashwin and Hemmen, Katherina and Choudhury, Susobhan and Krohn, Jan-Hagen and Jansen, Kerstin and Friedrich, Mike and Beliu, Gerti and Sauer, Markus and Lohse, Martin J. and Heinze, Katrin G.}, title = {Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence}, series = {Communications Biology}, volume = {5}, journal = {Communications Biology}, number = {1}, doi = {10.1038/s42003-022-03106-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301140}, year = {2022}, abstract = {G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β\(_{2}\)-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm\(^{2}\)/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbr{\"u}ck model.}, language = {en} } @article{WoelfelSaetteleZechmeisteretal.2020, author = {W{\"o}lfel, Angela and S{\"a}ttele, Mathias and Zechmeister, Christina and Nikolaev, Viacheslov O. and Lohse, Martin J. and Boege, Fritz and Jahns, Roland and Boivin-Jahns, Val{\´e}rie}, title = {Unmasking features of the auto-epitope essential for β\(_1\)-adrenoceptor activation by autoantibodies in chronic heart failure}, series = {ESC Heart Failure}, volume = {7}, journal = {ESC Heart Failure}, number = {4}, doi = {10.1002/ehf2.12747}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235974}, pages = {1830-1841}, year = {2020}, abstract = {Aims Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β\(_1\)-adrenoceptor (β1ECII) that is targeted by stimulating β\(_1\)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. Methods and results Non-conserved amino acids within the β\(_1\)EC\(_{II}\) loop (compared with the amino acids constituting the ECII loop of the β\(_2\)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine-serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β1ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β\(_1\)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β\(_1\)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β\(_1\)EC\(_{II}\) loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK\(^{211-214}\) motif and (ii) the intra-loop disulfide bond C\(^{209}\)↔C\(^{215}\). Of note, aberrant intra-loop disulfide bond C\(^{209}\)↔C\(^{216}\) almost fully disrupted the functional auto-epitope in cyclopeptides. Conclusions The conformational auto-epitope targeted by cardio-pathogenic β\(_1\)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β\(_1\)EC\(_{II}\) loop bearing the NDPK\(^{211-214}\) motif and the C\(^{209}\)↔C\(^{215}\) bridge while lacking cysteine C216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β\(_1\)-autoantibodypositive CHF.}, language = {en} } @phdthesis{LiessneeEller2021, author = {Liess [n{\´e}e Eller], Anna Katharina Luise}, title = {Understanding the regulation of the ubiquitin-conjugating enzyme UBE2S}, doi = {10.25972/OPUS-20419}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204190}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The ubiquitination of proteins serves as molecular signal to control an enormous number of physiological processes and its dysregulation is connected to human diseases like cancer. The versatility of this signal stems from the diverse ways by which ubiquitin can be attached to its targets. Thus, specificity and tight regulation of the ubiquitination are pivotal requirements of ubiquitin signaling. Ubiquitin-conjugating enzymes (E2s) act at the heart of the ubiquitination cascade, transferring ubiquitin from a ubiquitin-activating enzyme (E1) to a ubiquitin ligase (E3) or substrate. When cooperating with a RING-type E3, ubiquitin-conjugating enzymes can determine linkage specificity in ubiquitin chain formation. Our understanding of the regulation of E2 activities is still limited at a structural level. The work described here identifies two regulation mechanisms in UBE2S, a cognate E2 of the human RING-type E3 anaphase-promoting complex/cyclosome (APC/C). UBE2S elongates ubiquitin chains on APC/C substrates in a Lys11 linkage-specific manner, thereby targeting these substrates for degradation and driving mitotic progression. In addition, UBE2S was found to have a role in DNA repair by enhancing non-homologous end-joining (NHEJ) and causing transcriptional arrest at DNA damage sites in homologous recombination (HR). Furthermore, UBE2S overexpression is a characteristic feature of many cancer types and is connected to poor prognosis and diminished response to therapy. The first regulatory mechanism uncovered in this thesis involves the intramolecular auto-ubiquitination of a particular lysine residue (Lys+5) close to the active site cysteine, presumably through conformational flexibility of the active site region. The Lys+5-linked ubiquitin molecule adopts a donor-like, 'closed' orientation towards UBE2S, thereby conferring auto-inhibition. Notably, Lys+5 is a major physiological ubiquitination site in ~25\% of the human E2 enzymes, thus providing regulatory opportunities beyond UBE2S. Besides the active, monomeric state and the auto-inhibited state caused by auto-ubiquitination, I discovered that UBE2S can adopt a dimeric state. The latter also provides an auto-inhibited state, in which ubiquitin transfer is blocked via the obstruction of donor binding. UBE2S dimerization is promoted by its unique C-terminal extension, suppresses auto-ubiquitination and thereby the proteasomal degradation of UBE2S. Taken together, the data provided in this thesis illustrate the intricate ways by which UBE2S activity is fine-tuned and the notion that structurally diverse mechanisms have evolved to restrict the first step in the catalytic cycle of E2 enzymes.}, subject = {E2}, language = {en} } @phdthesis{Truongvan2023, author = {Truongvan, Ngoc}, title = {Understanding the dual specificity of UBA6}, doi = {10.25972/OPUS-24457}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244579}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Ubiquitylation is a protein post translational modification, in which ubiquitin is covalently attached to target protein substrates resulting in diverse cellular outcomes. Besides ubiquitin, various ubiquitin-like proteins including FAT10 exist, which are also conjugated to target proteins. The underlying modification mechanisms are conserved. In the initial step, ubiquitin or a ubiquitin-like protein is thioester-linked to a catalytic cysteine in the E1activating enzyme in an ATP-dependent manner. The respective protein modifier is then transferred to an E2 conjugating enzyme in a transthioesterification reaction. Finally, an E3 ubiquitin ligase E3 catalyzes the covalent attachment of the protein modifier to a substrate. In the case of ubiquitin, multiple ubiquitin molecules can be attached to a substrate in the form of either linear or branched polyubiquitin chains but also as single ubiquitin modifications. Depending on the nature of the ubiquitin chain, the substrates are destined to various cellular processes such as their targeted destruction by the proteasome but also non-degradative outcomes may occur. As stated above FAT10 is a ubiquitin-like protein modifier which typically targets proteins for proteasomal degradation. It consists of two ubiquitin-like domains and is mainly expressed in cells of the human immune system. The reported involvement of FAT10 modifications in cancers and other diseases has caught the attention of the scientific community as an inhibition of the FAT10ylation process may provide avenues for novel therapeutic approaches. UBA6 is the E1 activating enzyme that resides at the apex of the FAT10 proteasomal degradation pathway. UBA6 not only recognizes FAT10 but can also activate ubiquitin as efficiently as the ubiquitin specific E1 UBA1. The dual specificity of UBA6 may complicate the inhibition FAT10ylation since targeting the active site of UBA6 will also inhibit the UBA6-catalyzed ubiquitin activation. Therefore, it is important to understand the underlying principles for the dual specificity of UBA6 prior to the development of compounds interfering with FAT10ylation. In this thesis important novel insights into the structure and function of UBA6 were derived by X-ray crystallography and biochemical methods. The first crystal structure of UBA6 reveals the multidomain architecture of this enzyme in atomic detail. The enzyme is composed of a rigid core including its active and inactive adenylation domains as well as a 4 helix bundle. Overall, the molecule adopts a "Y" shape architecture with the core at the base and the first and second catalytic half domains forming one arm of the "Y" and the ubiquitin fold domain constituting the other arm. While UBA6 shares the same domain architecture as UBA1, substantial differences were revealed by the crystal structure. In particular, the first catalytic half domain undergoes a significant shift to a position more distal from the core. This rigid body movement is assumed to generate room to accommodate the second ubiquitin-like domain of FAT10. Differences are also observed in a hydrophobic platform between the core and the first catalytic half domain and the adenylation active site in the core, which together from the binding sites for ubiquitin and FAT10. Site directed mutagenesis of key residues in these areas altered the UBA6-catalyzed activation of ubiquitin and FAT10. UBA6 variants were generated with the goal of trying to block the activation of FAT10 while still maintaining that of ubiquitin activation, in order to fully explain the dual specificity of UBA6. However, none of these mutations could block the activation of FAT10, while some of these UBA6 variants blocked ubiquitin activation. Preliminary inhibition assays with a group of E1 inhibitors belonging to the adenosyl sulfamate family demonstrated potent inhibition of FAT10ylation for two compounds. The dual specificity of UBA6 hence needs to be further examined by biochemical and structural methods. In particular, the structure of a complex between UBA6 and ubiquitin or FAT10 would provide key insights for further biochemical studies, ultimately allowing the targeted inhibition of the FAT10ylation machinery.}, language = {en} } @article{GoosDejungWehmanetal.2019, author = {Goos, Carina and Dejung, Mario and Wehman, Ann M. and M-Natus, Elisabeth and Schmidt, Johannes and Sunter, Jack and Engstler, Markus and Butter, Falk and Kramer, Susanne}, title = {Trypanosomes can initiate nuclear export co-transcriptionally}, series = {Nucleic Acids Research}, volume = {47}, journal = {Nucleic Acids Research}, number = {1}, doi = {10.1093/nar/gky1136}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177709}, pages = {266-282}, year = {2019}, abstract = {The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.}, language = {en} } @article{SzalayWeibelHofmannetal.2013, author = {Szalay, Aladar A and Weibel, Stephanie and Hofmann, Elisabeth and Basse-Luesebrink, Thomas Christian and Donat, Ulrike and Seubert, Carolin and Adelfinger, Marion and Gnamlin, Prisca and Kober, Christina and Frentzen, Alexa and Gentschev, Ivaylo and Jakob, Peter Michael}, title = {Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer}, series = {Journal of Translational Medicine}, journal = {Journal of Translational Medicine}, doi = {doi:10.1186/1479-5876-11-106}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96016}, year = {2013}, abstract = {Background Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. Methods In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. Results We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. Conclusions Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.}, subject = {Lungenkrebs}, language = {en} } @phdthesis{Luckner2009, author = {Luckner, Sylvia}, title = {Towards the development of high affinity InhA and KasA inhibitors with activity against drug-resistant strains of Mycobacterium tuberculosis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-43621}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Mycobacterium tuberculosis is the causative agent of tuberculosis and responsible for more than eight million new infections and about two million deaths each year. Novel chemotherapeutics are urgently needed to treat the emerging threat of multi drug resistant and extensively drug resistant strains. Cell wall biosynthesis is a widely used target for chemotherapeutic intervention in bacterial infections. In mycobacteria, the cell wall is comprised of mycolic acids, very long chain fatty acids that provide protection and allow the bacteria to persist in the human macrophage. The type II fatty acid biosynthesis pathway in Mycobacterium tuberculosis synthesizes fatty acids with a length of up to 56 carbon atoms that are the precursors of the critical mycobacterial cell wall components mycolic acids. KasA, the mycobacterial ß-ketoacyl synthase and InhA, the mycobacterial enoyl reductase, are essential enzymes in the fatty acid biosynthesis pathway and validated drug targets. In this work, KasA was expressed in Mycobacterium smegmatis, purified and co-crystallized in complex with the natural thiolactone antibiotic thiolactomycin (TLM). High-resolution crystal structures of KasA and the C171Q KasA variant, which mimics the acyl enzyme intermediate of the enzyme, were solved in absence and presence of bound TLM. The crystal structures reveal how the inhibitor is coordinated by the enzyme and thus specifically pinpoint towards possible modifications to increase the affinity of the compound and develop potent new drugs against tuberculosis. Comparisons between the TLM bound crystal structures explain the preferential binding of TLM to the acylated form of KasA. Furthermore, long polyethylene glycol molecules are bound to KasA that mimic a fatty acid substrate of approximately 40 carbon atoms length. These structures thus provide the first insights into the molecular mechanism of substrate recognition and reveal how a wax-like substance can be accommodated in a cytosolic environment. InhA was purified and co-crystallized in complex with the slow, tight binding inhibitor 2-(o-tolyloxy)-5-hexylphenol (PT70). Two crystal structures of the ternary InhA-NAD+-PT70 were solved and reveal how the inhibitor is bound to the substrate binding pocket. Both structures display an ordered substrate binding loop and corroborate the hypothesis that slow onset inhibition is coupled to loop ordering. Upon loop ordering, the active site entrance is more restricted and the inhibitor is kept inside more tightly. These studies provide additional information on the mechanistic imperatives for slow onset inhibition of enoyl ACP reductases.}, subject = {Tuberkelbakterium}, language = {en} } @phdthesis{Chouhan2017, author = {Chouhan, Nitin Singh}, title = {Time-odor learning in \(Drosophila\) \(melanogaster\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145675}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Endogenous clocks help animals to anticipate the daily environmental changes. These internal clocks rely on environmental cues, called Zeitgeber, for synchronization. The molecular clock consists of transcription-translation feedback loops and is located in about 150 neurons (Helfrich-F{\"o}rster and Homberg, 1993; Helfrich-F{\"o}rster, 2005). The core clock has the proteins Clock (CLK) and Cycle (CYC) that together act as a transcription activator for period (per) and timeless (tim) which then, via PER and TIM block their own transcription by inhibiting CLK/CYC activity (Darlington et al., 1998; Hardin, 2005; Dubruille and Emery, 2008). Light signals trigger the degradation of TIM through a blue-light sensing protein Cryptochrome (CRY) and thus, allows CLK/CYC to resume per and tim transcription (Emery et al., 1998; Stanewsky et al., 1998). Therefore, light acts as an important Zeitgeber for the clock entrainment. The mammalian clock consists of similarly intertwined feedback loops. Endogenous clocks facilitate appropriate alterations in a variety of behaviors according to the time of day. Also, these clocks can provide the phase information to the memory centers of the brain to form the time of day related associations (TOD). TOD memories promote appropriate usage of resources and concurrently better the survival success of an animal. For instance, animals can form time-place associations related to the availability of a biologically significant stimulus like food or mate. Such memories will help the animal to obtain resources at different locations at the appropriate time of day. The significance of these memories is supported by the fact that many organisms including bees, ants, rats and mice demonstrate time-place learning (Biebach et al. 1991; Mistlberger et al. 1997; Van der Zee et al. 2008; Wenger et al. 1991). Previous studies have shown that TOD related memories rely on an internal clock, but the identity of the clock and the underlying mechanism remain less well understood. The present study demonstrates that flies can also form TOD associated odor memories and further seeks to identify the appropriate mechanism. Hungry flies were trained in the morning to associate odor A with the sucrose reward and subsequently were exposed to odor B without reward. The same flies were exposed in the afternoon to odor B with and odor A without reward. Two cycles of the 65 reversal training on two subsequent days resulted in the significant retrieval of specific odor memories in the morning and afternoon tests. Therefore, flies were able to modulate their odor preference according to the time of day. In contrast, flies trained in a non-reversal manner were unable to form TOD related memories. The study also demonstrates that flies are only able to form time-odor memories when the two reciprocal training cycles occur at a minimum 6 h interval. This work also highlights the role of the internal state of flies in establishing timeodor memories. Prolonged starvation motivates flies to appropriate their search for the food. It increases the cost associated with a wrong choice in the T-maze test as it precludes the food discovery. Accordingly, an extended starvation promotes the TOD related changes in the odor preference in flies already with a single cycle of reversal training. Intriguingly, prolonged starvation is required for the time-odor memory acquisition but is dispensable during the memory retrieval. Endogenous oscillators promote time-odor associations in flies. Flies in constant darkness have functional rhythms and can form time-odor memories. In contrast, flies kept in constant light become arrhythmic and demonstrated no change in their odor preference through the day. Also, clock mutant flies per01 and clkAR, show compromised performance compared to CS flies when trained in the time-odor conditioning assay. These results suggest that flies need a per and clk dependent oscillator for establishing TOD related memories. Also, the clock governed rhythms are necessary for the timeodor memory acquisition but not for the retrieval. Pigment-Dispersing Factor (PDF) neuropeptide is a clock output factor (Park and Hall, 1998; Park et al., 2000; Helfrich-F{\"o}rster, 2009). pdf01 mutant flies are unable to form significant time-odor memories. PDF is released by 8 neurons per hemisphere in the fly brain. This cluster includes the small (s-LNvs) and large (l-LNvs) ventral lateral neurons. Restoring PDF in these 16 neurons in the pdf01 mutant background rescues the time-odor learning defect. The PDF neuropeptide activates a seven transmembrane G-protein coupled receptor (PDFR) which is broadly expressed in the fly brain (Hyun et al., 2005). The present study shows that the expression of PDFR in about 10 dorsal neurons (DN1p) is sufficient for robust time-odor associations in flies. 66 In conclusion, flies use distinct endogenous oscillators to acquire and retrieve time-odor memories. The first oscillator is light dependent and likely signals through the PDF neuropeptide to promote the usage of the time as an associative cue during appetitive conditioning. In contrast, the second clock is light independent and specifically signals the time information for the memory retrieval. The identity of this clock and the underlying mechanism are open to investigation.}, subject = {Taufliege}, language = {en} } @phdthesis{WeigelverhHoffmann2024, author = {Weigel [verh. Hoffmann], Mathis Leonard}, title = {Thrombozytenfunktionsanalyse als potenzielles Instrument zur Fr{\"u}herkennung von Sepsis}, doi = {10.25972/OPUS-35819}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358193}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Sepsis ist ein h{\"a}ufiges und akut lebensbedrohliches Syndrom, das eine Organfunktionsst{\"o}rung in Folge einer dysregulierten Immunantwort auf eine Infektion beschreibt. Eine fr{\"u}hzeitige Diagnosestellung und Therapieeinleitung sind von zentraler Bedeutung f{\"u}r das {\"U}berleben der Patient:innen. In einer Pilotstudie konnte unsere Forschungsgruppe mittels Durchflusszytometrie eine ausgepr{\"a}gte Hyporeaktivit{\"a}t der Thrombozyten bei Sepsis nachweisen, die einen potenziell neuen Biomarker zur Sepsis-Fr{\"u}herkennung darstellt. Zur Evaluation des Ausmaßes und Entstehungszeitpunktes der detektierten Thrombozytenfunktionsst{\"o}rung wurden im Rahmen der vorliegenden Arbeit zus{\"a}tzlich zu Patient:innen mit Sepsis (SOFA-Score ≥ 2; n=13) auch hospitalisierte Patient:innen mit einer Infektion ohne Sepsis (SOFA-Score < 2; n=12) rekrutiert. Beide Kohorten wurden zu zwei Zeitpunkten (t1: <24h; t2: Tag 5-7) im Krankheitsverlauf mittels Durchflusszytometrie und PFA-200 untersucht und mit einer gesunden Kontrollgruppe (n=28) verglichen. Ph{\"a}notypische Auff{\"a}lligkeiten der Thrombozyten bei Sepsis umfassten: (i) eine ver{\"a}nderte Expression verschiedener Untereinheiten des GPIb-IX-V-Rezeptorkomplexes, die auf ein verst{\"a}rktes Rezeptor-Shedding hindeutet; (ii) ein ausgepr{\"a}gtes Mepacrin-Beladungsdefizit, das auf eine zunehmend reduzierte Anzahl von δ-Granula entlang des Infektion-Sepsis Kontinuums hinweist; (iii) eine Reduktion endst{\"a}ndig gebundener Sialins{\"a}ure im Sinne einer verst{\"a}rkten Desialylierung. Die funktionelle Analyse der Thrombozyten bei Sepsis ergab bei durchflusszytometrischer Messung der Integrin αIIbβ3-Aktivierung (PAC-1-Bindung) eine ausgepr{\"a}gte generalisierte Hyporeaktivit{\"a}t gegen{\"u}ber multiplen Agonisten, die abgeschw{\"a}cht bereits bei Infektion nachweisbar war und gem{\"a}ß ROC-Analysen gut zwischen Infektion und Sepsis diskriminierte (AUC >0.80 f{\"u}r alle Agonisten). Im Gegensatz dazu zeigten Thrombozyten bei Sepsis und Analyse mittels PFA-200 unter Einfluss physiologischer Scherkr{\"a}fte eine normale bis gar beschleunigte Aggregation. Die Reaktivit{\"a}tsmessung von Thrombozyten mittels Durchflusszytometrie stellt weiterhin einen vielversprechenden Biomarker f{\"u}r die Sepsis-Fr{\"u}herkennung dar. F{\"u}r weitere Schlussfolgerungen ist jedoch eine gr{\"o}ßere Kohorte erforderlich. In nachfolgenden Untersuchungen sollten zudem mechanistische Ursachen der beschriebenen ph{\"a}notypischen und funktionellen Auff{\"a}lligkeiten von Thrombozyten bei Infektion und Sepsis z.B. mittels Koinkubationsexperimenten untersucht werden.}, subject = {Sepsis}, language = {de} } @article{StegnervanEeuwijkAngayetal.2017, author = {Stegner, David and van Eeuwijk, Judith M.M. and Angay, Oğuzhan and Gorelashvili, Maximilian G. and Semeniak, Daniela and Pinnecker, J{\"u}rgen and Schmithausen, Patrick and Meyer, Imke and Friedrich, Mike and D{\"u}tting, Sebastian and Brede, Christian and Beilhack, Andreas and Schulze, Harald and Nieswandt, Bernhard and Heinze, Katrin G.}, title = {Thrombopoiesis is spatially regulated by the bone marrow vasculature}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, number = {127}, doi = {10.1038/s41467-017-00201-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170591}, year = {2017}, abstract = {In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.}, language = {en} } @phdthesis{Schmithausen2019, author = {Schmithausen, Patrick Alexander Gerhard}, title = {Three-dimensional fluorescence image analysis of megakaryocytes and vascular structures in intact bone}, doi = {10.25972/OPUS-17854}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178541}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The thesis provides insights in reconstruction and analysis pipelines for processing of three-dimensional cell and vessel images of megakaryopoiesis in intact murine bone. The images were captured in a Light Sheet Fluorescence Microscope. The work presented here is part of Collaborative Research Centre (CRC) 688 (project B07) of the University of W{\"u}rzburg, performed at the Rudolf-Virchow Center. Despite ongoing research within the field of megakaryopoiesis, its spatio-temporal pattern of megakaryopoiesis is largely unknown. Deeper insight to this field is highly desirable to promote development of new therapeutic strategies for conditions related to thrombocytopathy as well as thrombocytopenia. The current concept of megakaryopoiesis is largely based on data from cryosectioning or in vitro studies indicating the existence of spatial niches within the bone marrow where specific stages of megakaryopoiesis take place. Since classic imaging of bone sections is typically limited to selective two-dimensional views and prone to cutting artefacts, imaging of intact murine bone is highly desired. However, this has its own challenges to meet, particularly in image reconstruction. Here, I worked on processing pipelines to account for irregular specimen staining or attenuation as well as the extreme heterogeneity of megakaryocyte morphology. Specific challenges for imaging and image reconstruction are tackled and solution strategies as well as remaining limitations are presented and discussed. Fortunately, modern image processing and segmentation strongly benefits from continuous advances in hardware as well as software-development. This thesis exemplifies how a combined effort in biomedicine, computer vision, data processing and image technology leads to deeper understanding of megakaryopoiesis. Tailored imaging pipelines significantly helped elucidating that the large megakaryocytes are broadly distributed throughout the bone marrow facing a surprisingly dense vessel network. No evidence was found for spatial niches in the bone marrow, eventually resulting in a revised model of megakaryopoiesis.}, subject = {Megakaryozytopoese}, language = {en} } @article{MeinertJessenHufnageletal.2024, author = {Meinert, Madlen and Jessen, Christina and Hufnagel, Anita and Kreß, Julia Katharina Charlotte and Burnworth, Mychal and D{\"a}ubler, Theo and Gallasch, Till and Da Xavier Silva, Thamara Nishida and Dos Santos, Anc{\´e}ly Ferreira and Ade, Carsten Patrick and Schmitz, Werner and Kneitz, Susanne and Friedmann Angeli, Jos{\´e} Pedro and Meierjohann, Svenja}, title = {Thiol starvation triggers melanoma state switching in an ATF4 and NRF2-dependent manner}, series = {Redox Biology}, volume = {70}, journal = {Redox Biology}, doi = {10.1016/j.redox.2023.103011}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350328}, year = {2024}, abstract = {The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; Braf\(^{CA}\); Pten\(^{lox/+}\) melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.}, language = {en} } @article{JessenKressBaluapurietal.2020, author = {Jessen, Christina and Kreß, Julia K. C. and Baluapuri, Apoorva and Hufnagel, Anita and Schmitz, Werner and Kneitz, Susanne and Roth, Sabine and Marquardt, Andr{\´e} and Appenzeller, Silke and Ade, Casten P. and Glutsch, Valerie and Wobser, Marion and Friedmann-Angeli, Jos{\´e} Pedro and Mosteo, Laura and Goding, Colin R. and Schilling, Bastian and Geissinger, Eva and Wolf, Elmar and Meierjohann, Svenja}, title = {The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression}, series = {Oncogene}, volume = {39}, journal = {Oncogene}, issn = {0950-9232}, doi = {10.1038/s41388-020-01477-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235064}, pages = {6841-6855}, year = {2020}, abstract = {The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H\(_2\)O\(_2\) or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.}, language = {en} } @article{KaiserSauerKisker2017, author = {Kaiser, Sebastian and Sauer, Florian and Kisker, Caroline}, title = {The structural and functional characterization of human RecQ4 reveals insights into its helicase mechanism}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, number = {15907}, doi = {10.1038/ncomms15907}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170769}, year = {2017}, abstract = {RecQ4 is a member of the RecQ helicase family, an evolutionarily conserved class of enzymes, dedicated to preserving genomic integrity by operating in telomere maintenance, DNA repair and replication. While reduced RecQ4 activity is associated with cancer predisposition and premature aging, RecQ4 upregulation is related to carcinogenesis and metastasis. Within the RecQ family, RecQ4 assumes an exceptional position, lacking several characteristic RecQ domains. Here we present the crystal structure of human RecQ4, encompassing the conserved ATPase core and a novel C-terminal domain that lacks resemblance to the RQC domain observed in other RecQ helicases. The new domain features a zinc-binding site and two distinct types of winged-helix domains, which are not involved in canonical DNA binding or helicase activity. Based on our structural and functional analysis, we propose that RecQ4 exerts a helicase mechanism, which may be more closely related to bacterial RecQ helicases than to its human family members.}, language = {en} } @phdthesis{Pleines2009, author = {Pleines, Irina}, title = {The role of the Rho GTPases Rac1 and Cdc42 for platelet function and formation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48572}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Platelet activation induces cytoskeletal rearrangements involving a change from discoid to spheric shape, secretion, and eventually adhesion and spreading on immobilized ligands. Small GTPases of the Rho family, such as Rac1 and Cdc42, are known to be involved in these processes by facilitating the formation of lamellipodia and filopodia, respectively. This thesis focuses on the role Rac1 and Cdc42 for platelet function and formation from their precursor cells, the megakaryocytes (MKs), using conditional knock-out mice. In the first part of the work, the involvement of Rac1 in the activation of the enzyme phospholipase (PL) C2 in the signaling pathway of the major platelet collagen receptor glycoprotein (GP) VI was investigated. It was found that Rac1 is essential for PLC2 activation independently of tyrosine phosphorylation of the enzyme, resulting in a specific platelet activation defect downstream of GPVI, whereas signaling of other activating receptors remains unaffected. Since Rac1-deficient mice were protected from arterial thrombosis in two different in vivo models, the GTPase might serve as a potential target for the development of new drugs for the treatment and prophylaxis of cardio- and cerebrovascular diseases. The second part of the thesis deals with the first characterization of MK- and platelet-specific Cdc42 knock-out mice. Cdc42-deficient mice displayed mild thrombo-cytopenia and platelet production from mutant MKs was markedly reduced. Unexpectedly, Cdc42-deficient platelets showed increased granule content and release upon activation, leading to accelerated thrombus formation in vitro and in vivo. Furthermore, Cdc42 was not generally required for filopodia formation upon platelet activation. Thus, these results indicate that Cdc42, unlike Rac1, is involved in multiple signaling pathways essential for proper platelet formation and function. Finally, the outcome of combined deletion of Rac1 and Cdc42 was studied. In contrast to single deficiency of either GTPase, platelet production from double-deficient MKs was virtually abrogated, resulting in dramatic macrothrombocytopenia in the animals. Formed platelets were largely non-functional leading to a severe hemostatic defect and defective thrombus formation in double-deficient mice in vivo. These results demonstrate for the first time a functional redundancy of Rac1 and Cdc42 in the hematopoietic system.}, subject = {Thrombose}, language = {en} } @phdthesis{Stritt2017, author = {Stritt, Simon}, title = {The role of the cytoskeleton in platelet production and the pathogenesis of platelet disorders in humans and mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122662}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Platelets are continuously produced from megakaryocytes (MK) in the bone marrow by a cytoskeleton-driven process of which the molecular regulation is not fully understood. As revealed in this thesis, MK/ platelet-specific Profilin1 (Pfn1) deficiency results in micro- thrombocytopenia, a hallmark of the Wiskott-Aldrich syndrome (WAS) in humans, due to accelerated platelet turnover and premature platelet release into the bone marrow. Both Pfn1-deficient mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyper-stable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. In contrast, Twinfilin2a (Twf2a) was established as a central regulator of platelet reactivity and turnover. Twf2a-deficient mice revealed an age-dependent macrothrombocytopenia that could be explained by a markedly decreased platelet half-life, likely due to the pronounced hyper-reactivity of \(Twf2a^{-/-}\) platelets. The latter was characterized by sustained integrin acti- vation and thrombin generation in vitro that translated into accelerated thrombus formation in vivo. To further elucidate mechanisms of integrin activation, Rap1-GTP-interacting adaptor molecule (RIAM)-null mice were generated. Despite the proposed critical role of RIAM for platelet integrin activation, no alterations in this process could be found and it was concluded that RIAM is dispensable for the activation of β1 and β3 integrins, at least in platelets. These findings change the current mechanistic understanding of platelet integrin activation. Outside-in signaling by integrins and other surface receptors was supposed to regulate MK migration, but also the temporal and spatial formation of proplatelet protrusions. In this the- sis, phospholipase D (PLD) was revealed as critical regulator of actin dynamics and podo- some formation in MKs. Hence, the unaltered platelet counts and production in \(Pld1/2^{-/-}\) mice and the absence of a premature platelet release in the bone marrow of \(Itga2^{-/-}\) mice question the role of podosomes in platelet production and raise the need to reconsider the proposed inhibitory signaling by α2β1 integrins on proplatelet formation. Non-muscle myosin IIA (NMMIIA) has been implicated as a downstream effector of the in- hibitory signals transmitted via α2β1 integrins. Besides Rho-GTPase signaling, also \(Mg^{2+}\) and transient receptor potential melastatin-like 7 (TRPM7) channel α-kinase are known regulators of NMMIIA activity. In this thesis, TRPM7 was identified as major regulator of \(Mg^{2+}\) homeostasis in MKs and platelets. Furthermore, decreased \([Mg^{2+}]_i\) led to deregulated NMMIIA activity and altered cytoskeletal dynamics that impaired thrombopoiesis and resulted in macrothrombocytopenia in humans and mice.}, subject = {Thrombozytopoese}, language = {en} } @phdthesis{May2011, author = {May, Frauke}, title = {The role of the (hem)ITAM-coupled receptors C-type lectin-like receptor 2 (CLEC-2) and Glycoprotein (GP) VI for platelet function: in vitro and in vivo studies in mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65383}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Die Thrombozytenaktivierung und -adh{\"a}sion sowie die nachfolgende Thrombusbildung ist ein essentieller Prozess in der prim{\"a}ren H{\"a}mostase, der aber auch irreversible Gef{\"a}ßverschl{\"u}sse und damit Herzinfarkt oder Schlaganfall verursachen kann. Erst k{\"u}rzlich wurde beschrieben, dass der C-type lectin-like receptor 2 (CLEC-2) auf der Thrombozytenoberfl{\"a}che exprimiert wird, jedoch wurde f{\"u}r diesen Rezeptor noch keine Funktion in den Prozessen der H{\"a}mostase und Thrombose gezeigt. In der vorliegenden Arbeit wurde die Rolle von CLEC-2 in der Thrombozytenfunktion und Thrombusbildung im Mausmodel untersucht. In dem ersten Teil dieser Arbeit konnte gezeigt werden, dass die Behandlung von M{\"a}usen mit dem neu generierten monoklonalen Antik{\"o}rper INU1, der gegen murines CLEC-2 gerichtet ist, zu dem vollst{\"a}ndigen und hochspezifischen Verlust des Rezeptors in zirkulierenden Thrombozyten f{\"u}hrte, ein Prozess, der als „Immundepletion" bezeichnet wird. Die CLEC-2-defizienten Thrombozyten waren nicht mehr durch den CLEC-2-spezifischen Agonisten Rhodozytin aktivierbar, w{\"a}hrend die Aktivierung durch alle anderen getesteten Agonisten nicht beeintr{\"a}chtigt war. Dieser selektive Defekt f{\"u}hrte unter Flussbedingungen ex vivo zu stark verminderter Aggregatbildung der Thrombozyten. Außerdem zeigten in vivo-Thrombosestudien, dass die gebildeten Thromben instabil waren und vermehrt embolisierten. Infolgedessen war die CLEC-2 Defizienz mit einem deutlichen Schutz vor arterieller Thrombose verbunden. Außerdem ließ die in INU1-behandelten M{\"a}usen beobachtete variable Verl{\"a}ngerung der Blutungszeit auf einen moderaten h{\"a}mostatischen Defekt schließen. Diese Ergebnisse zeigen zum ersten Mal, dass CLEC-2 in vitro und in vivo signifikant zur Thrombusstabilit{\"a}t beitr{\"a}gt und eine essentielle Rolle in der H{\"a}mostase und arteriellen Thrombose spielt. Daher stellt CLEC-2 eine potentiell neue antithrombotische Zielstruktur dar, die in vivo inaktiviert werden kann. Diese in vivo-Herabregulierung von Thrombozytenoberfl{\"a}chenrezeptoren k{\"o}nnte einen vielversprechenden Ansatz f{\"u}r zuk{\"u}nftige antithrombotische Therapien darstellen. Der zweite Teil dieser Arbeit behandelte den Effekt einer Doppelimmundepletion der immunoreceptor tyrosine-based activation motiv (ITAM)- und hemITAM-gekoppelten Rezeptoren Glykoprotein (GP) VI und CLEC-2 auf H{\"a}mostase und Thrombose mittels einer Kombination der GPVI- beziehungsweise CLEC-2-spezifischen Antik{\"o}rper JAQ1 und INU1. Eine Einzeldepletion von GPVI oder CLEC-2 in vivo beeintr{\"a}chtigte nicht die Expression und Funktion des jeweils anderen Rezeptors. Eine gleichzeitige Behandlung mit beiden Antik{\"o}rpern f{\"u}hrte jedoch zu dem nachhaltigen Verlust der GPVI- und CLEC-2-vermittelten Signale in Thrombozyten, w{\"a}hrend andere Signalwege nicht betroffen waren. Im Gegensatz zu den Einzeldefizienzen, wiesen die GPVI/CLEC-2 doppeldefizienten M{\"a}use einen schwerwiegenden Blutungsph{\"a}notyp auf. Außerdem f{\"u}hrte die Behandlung zu einer starken Beeintr{\"a}chtigung der arteriellen Thrombusbildung, die die Effekte der Einzeldefizienzen weit {\"u}bertraf. Von Bedeutung ist auch, dass gleiche Ergebnisse in Gp6-/- M{\"a}usen gefunden wurden, die mittels INU1-Behandlung CLEC-2-depletiert wurden. Dies veranschaulicht, dass der Blutungsph{\"a}notyp nicht durch Sekund{\"a}reffekte der kombinierten Antik{\"o}rperbehandlung hervorgerufen wurde. Diese Daten deuten darauf hin, dass GPVI und CLEC-2 sowohl unabh{\"a}ngig voneinander als auch gleichzeitig in vivo von der Thrombozytenoberfl{\"a}che herabreguliert werden k{\"o}nnen und lassen unerwartete redundante Funktionen der beiden Rezeptoren in H{\"a}mostase und Thrombose erkennen. Da beide Rezeptoren, GPVI und CLEC-2, als neue antithrombotische Zielstrukturen diskutiert werden, k{\"o}nnten diese Ergebnisse wichtige Auswirkungen auf die Entwicklung von anti-GPVI oder anti-CLEC-2-basierenden Antithrombotika haben.}, subject = {Thrombozyt}, language = {en} } @phdthesis{Slotta2019, author = {Slotta, Anja Maria}, title = {The Role of Protein Kinase D 1 in the regulation of murine adipose tissue function under physiological and pathophysiological conditions}, doi = {10.25972/OPUS-17911}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179112}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Adipocytes are specialized cells found in vertebrates to ensure survival in terms of adaption to food deficit and abundance. However, their dysfunction accounts for the pathophysiology of metabolic diseases such as T2DM. Preliminary data generated by Mona L{\"o}ffler suggested that PKD1 is involved in adipocyte function. Here, I show that PKD1 expression and activity is linked to lipid metabolism of murine adipocytes. PKD1 gene expression and activity was reduced in murine white adipose tissue upon fasting, a physiological condition which induces lipolysis. Isoproterenol-stimulated lipolysis in adipose tissue and 3T3-L1 adipocytes reduced PKD1 gene expression. Silencing ATGL in adipocytes inhibited isoproterenol-stimulated lipolysis, however, the β-adrenergic stimulation of ATGL-silenced adipocytes lowered PKD1 expression levels as well. Adipose tissue of obese mice exhibited high PKD1 RNA levels but paradoxically lower protein levels of phosphorylated PKD1-Ser916. However, HFD generated a second PKD1 protein product of low molecular weight in mouse adipose tissue. Furthermore, constitutively active PKD1 predominantly displayed nuclear localization in 3T3-L1 adipocytes containing many fat vacuoles. However, adipocytes overexpressing non-functional PKD1 contained fewer lipid droplets and PKD1-KD was distributed in cytoplasm. Most importantly, deficiency of PKD1 in mouse adipose tissue caused expression of genes involved in adaptive thermogenesis such as UCP-1 and thus generated brown-like phenotype adipocytes. Thus, PKD1 is implicated in adipose tissue function and presents an interesting target for therapeutic approaches in the prevention of obesity and associated diseases.}, subject = {adipocyte}, language = {en} } @phdthesis{Cam2006, author = {Cam, Hakan}, title = {The role of p53 family members in myogenic differentiation and rhabdomyosarcoma development}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-20240}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Krebserkrankungen zeichnen sich h{\"a}ufig durch St{\"o}rungen zellul{\"a}rer Differenzierungsprozesse aus. So weisen Rhabdomyosarkome, die aus Muskelvorl{\"a}uferzellen hervorgehen, Differenzierungsdefekte auf, die zur unkontrollierten Proliferation der Tumorzellen f{\"u}hren. Bislang ist ungekl{\"a}rt, ob die Differenzierungsdefekte auf der verst{\"a}rkten Expression von Inhibitoren, der defekten Funktion von Aktivatoren oder einer Kombination von beidem beruht. In dieser Arbeit wird gezeigt, dass im Unterschied zu normalen Muskelzellen RMS-Zellen verst{\"a}rkt DeltaNp73, einen Pan-Inhibitor der p53-Tumorsuppressorfamilie, exprimieren. Die experimentelle {\"U}berexpression von DeltaNp73 in normalen Myoblasten blockierte die Muskeldifferenzierung und f{\"o}rderte in Kombination mit klassischen RMS-Onkogenen wie IGF2 oder PAX3/FKHR die maligne Transformation. Umgekehrt f{\"u}hrte die Hemmung von DeltaNp73 durch RNAi zur Reduktion der Tumorigenit{\"a}t von RMS-Tumorzellen. Da DeltaNp73 als dominant-negativer Inhibitor der p53-Familie wirkt, lies die Hemmung von Differenzierungsprozessen durch DeltaNp73 vermuten, dass die p53-Familienmitglieder (p53, p63, und p73) an der Regulation der Muskeldifferenzierung beteiligt sind. Tats{\"a}chlich konnte in dieser Arbeit gezeigt werden, dass die drei p53-Familienmitglieder bei der Induktion sp{\"a}ter Differenzierungsstadien kooperieren, indem sie die Aktivit{\"a}t des Retinoblastoma-Proteins RB regulieren. Die Funktion von RB ist bekanntermassen sowohl f{\"u}r den permanenten Zellzyklusarrest als auch f{\"u}r die Aktivierung Muskel-spezifischer Gene notwendig. W{\"a}hrend p53 die Proteinspiegel von RB reguliert, kontrollieren p63 und p73 den Aktivierungsgrad von RB, indem sie dessen Phoshphorylierungszustand {\"u}ber den Zyklin-abh{\"a}ngigen Kinaseinhibitor p57KIP2 modifizieren. Eine Hemmung dieser Funktionen blockiert das Differenzierungsprogramm und f{\"o}rdert die Tumorentstehung. Die Aktivierung zellul{\"a}rer Differenzierungsprozesse stellt somit einen entscheidenden Bestandteil der Tumorsuppressoraktivit{\"a}t der p53-Familie dar und liefert eine Erkl{\"a}rung f{\"u}r die H{\"a}ufigkeit von Mutationen im p53-Signalweg bei Rhabdomyosarkom-Patienten.}, subject = {Rhabdomyosarkom}, language = {de} } @phdthesis{Becker2021, author = {Becker, Isabelle Carlotta}, title = {The role of megakaryocytes and platelets in vascular and osteogenic development}, doi = {10.25972/OPUS-21024}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-210241}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Platelets, small anucleate cell fragments in the blood stream, derive from large precursor cells, so-called megakaryocytes (MK) residing in the bone marrow (BM). In addition to their role in wound healing, platelets have been shown to play a significant role during inflammatory bleeding. Above all, the immunoreceptor tyrosine-based activation motif (ITAM) receptors GPVI as well as CLEC-2 have been identified as main regulators of vascular integrity. In addition to ITAM-bearing receptors, our group identified GPV as another potent regulator of hemostasis and thrombosis. Surprisingly, concomitant lack of GPV and CLEC-2 deteriorated blood-lymphatic misconnections observed in Clec2-/- mice resulting in severe edema formation and intestinal inflammation. Analysis of lymphatic and vascular development in embryonic mesenteries revealed severely defective blood-lymph-vessel separation, which translated into thrombocytopenia and increased vascular permeability due to reduced tight junction density in mesenteric blood vessels and consequent leakage of blood into the peritoneal cavity. Recently, platelet granule release has been proposed to ameliorate the progression of retinopathy of prematurity (ROP), a fatal disease in newborns leading to retinal degradation. The mechanisms governing platelet activation in this process remained elusive nonetheless, which prompted us to investigate a possible role of ITAM signaling. In the second part of this thesis, granule release during ROP was shown to be GPVI- and partly CLEC-2-triggered since blockade or loss of these receptors markedly deteriorated ROP progression. Proplatelet formation from MKs is highly dependent on a functional microtubule and actin cytoskeleton, the latter of which is regulated by several actin-monomer binding proteins including Cofilin1 and Twinfilin1 that have been associated with actin-severing at pointed ends. In the present study, a redundancy between both proteins especially important for the guided release of proplatelets into the bloodstream was identified, since deficiency in both proteins markedly impaired MK functionality mainly due to altered actin-microtubule crosstalk. Besides ITAM-triggered activation, platelets and MKs are dependent on inhibitory receptors, which prevent overshooting activation. We here identified macrothrombocytopenic mice with a mutation within Mpig6b encoding the ITIM-bearing receptor G6b-B. G6b-B-mutant mice developed a severe myelofibrosis associated with sex-specific bone remodeling defects resulting in osteosclerosis and -porosis in female mice. Moreover, G6b-B was shown to be indispensable for MK maturation as verified by a significant reduction in MK-specific gene expression in G6b-B-mutant MKs due to reduced GATA-1 activity.}, subject = {Megakaryozyt}, language = {en} } @article{Rasmussen2023, author = {Rasmussen, Tim}, title = {The potassium efflux system Kef: bacterial protection against toxic electrophilic compounds}, series = {Membranes}, volume = {13}, journal = {Membranes}, number = {5}, issn = {2077-0375}, doi = {10.3390/membranes13050465}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313686}, year = {2023}, abstract = {Kef couples the potassium efflux with proton influx in gram-negative bacteria. The resulting acidification of the cytosol efficiently prevents the killing of the bacteria by reactive electrophilic compounds. While other degradation pathways for electrophiles exist, Kef is a short-term response that is crucial for survival. It requires tight regulation since its activation comes with the burden of disturbed homeostasis. Electrophiles, entering the cell, react spontaneously or catalytically with glutathione, which is present at high concentrations in the cytosol. The resulting glutathione conjugates bind to the cytosolic regulatory domain of Kef and trigger activation while the binding of glutathione keeps the system closed. Furthermore, nucleotides can bind to this domain for stabilization or inhibition. The binding of an additional ancillary subunit, called KefF or KefG, to the cytosolic domain is required for full activation. The regulatory domain is termed K+ transport-nucleotide binding (KTN) or regulator of potassium conductance (RCK) domain, and it is also found in potassium uptake systems or channels in other oligomeric arrangements. Bacterial RosB-like transporters and K+ efflux antiporters (KEA) of plants are homologs of Kef but fulfill different functions. In summary, Kef provides an interesting and well-studied example of a highly regulated bacterial transport system.}, language = {en} } @article{KooMatthewsHarrisonetal.2022, author = {Koo, Chek Ziu and Matthews, Alexandra L. and Harrison, Neale and Szyroka, Justyna and Nieswandt, Bernhard and Gardiner, Elizabeth E. and Poulter, Natalie S. and Tomlinson, Michael G.}, title = {The platelet collagen receptor GPVI is cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 molecular scissors}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {5}, issn = {1422-0067}, doi = {10.3390/ijms23052440}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284468}, year = {2022}, abstract = {The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a 'molecular scissor' that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.}, language = {en} } @article{KitzenmaierSchaeferKasaragodetal.2019, author = {Kitzenmaier, Alexandra and Schaefer, Natascha and Kasaragod, Vikram Babu and Polster, Tilman and Hantschmann, Ralph and Schindelin, Hermann and Villmann, Carmen}, title = {The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia}, series = {European Journal of Neuroscience}, volume = {50}, journal = {European Journal of Neuroscience}, number = {12}, doi = {10.1111/ejn.14533}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206158}, pages = {3906-3920}, year = {2019}, abstract = {Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α-helix 5, presumably disrupting the integrity of this α-helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild-type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non-symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild-type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α-helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium-binding site of the transporter.}, language = {en} } @article{MeirMaurusKuperetal.2021, author = {Meir, Michael and Maurus, Katja and Kuper, Jochen and Hankir, Mohammed and Wardelmann, Eva and Rosenwald, Andreas and Germer, Christoph-Thomas and Wiegering, Armin}, title = {The novel KIT exon 11 germline mutation K558N is associated with gastrointestinal stromal tumor, mastocytosis, and seminoma development}, series = {Genes, Chromosomes \& Cancer}, volume = {60}, journal = {Genes, Chromosomes \& Cancer}, number = {12}, doi = {10.1002/gcc.22988}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-257476}, pages = {827-832}, year = {2021}, abstract = {Familial gastrointestinal stromal tumors (GIST) are dominant genetic disorders that are caused by germline mutations of the type III receptor tyrosine kinase KIT. While sporadic mutations are frequently found in mastocytosis and GISTs, germline mutations of KIT have only been described in 39 families until now. We detected a novel germline mutation of KIT in exon 11 (p.Lys-558-Asn; K558N) in a patient from a kindred with several GISTs harboring different secondary somatic KIT mutations. Structural analysis suggests that the primary germline mutation alone is not sufficient to release the autoinhibitory region of KIT located in the transmembrane domain. Instead, the KIT kinase module becomes constitutively activated when K558N combines with different secondary somatic mutations. The identical germline mutation in combination with an additional somatic KIT mutation was detected in a second patient of the kindred with seminoma while a third patient within the family had a cutaneous mastocytosis. These findings suggest that the K558N mutation interferes with the juxtamembranous part of KIT, since seminoma and mastocystosis are usually not associated with exon 11 mutations.}, language = {en} } @article{EisenbergAlbertTeuffeletal.2022, author = {Eisenberg, Philip and Albert, Leon and Teuffel, Jonathan and Zitzow, Eric and Michaelis, Claudia and Jarick, Jane and Sehlke, Clemens and Große, Lisa and Bader, Nicole and Nunes-Alves, Ariane and Kreikemeyer, Bernd and Schindelin, Hermann and Wade, Rebecca C. and Fiedler, Tomas}, title = {The Non-phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase GapN Is a Potential New Drug Target in Streptococcus pyogenes}, series = {Frontiers in Microbiology}, volume = {13}, journal = {Frontiers in Microbiology}, issn = {1664-302X}, doi = {10.3389/fmicb.2022.802427}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-262869}, year = {2022}, abstract = {The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.}, language = {en} } @phdthesis{Jurak2006, author = {Jurak, Igor}, title = {The molecular mechanism of the Cytomegalovirus species specificity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-19233}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Viruses have undergone a coevolution with their hosts, resulting in a specific adaptation to them. Consequently, many viruses have a limited host range. Occasionally, viruses acquire an adaptive mutation, which allows infection and replication in a different species as shown recently for the human immunodeficiency virus and influenza virus. Cross-species infections are responsible for the majority of emerging and re-emerging viral diseases. However, little is known about the mechanisms that restrict viruses to a certain host species, and the factors viruses need to cross the species barrier and replicate in a different host. Cytomegaloviruses are prototypes of the beta-herpesvirus subfamily and are highly species specific. They replicate only in cells of their own or a closely related species. The molecular mechanism underlying their species specificity is poorly understood and was investigated in this study. An initial observation showed that murine cytomegalovirus (MCMV) can replicate in human 293 and 911 cells, but not in any other human cells tested. Both cell lines are transformed with adenoviral E1 genes that encode a transcriptional transactivator (E1A) and two suppressors of apoptosis (E1B-55k and E1B-19k). This has led to the hypothesis that these functions are required for MCMV replication in human cells. Further analysis revealed that normal human cells died rapidly after infection of caspase-9-mediated apoptosis. Apoptosis induced by MCMV can be suppressed by broad-spectrum caspase inhibitors, and virus replication can be rescued, indicating a major role of caspases in this process. Furthermore, over-expression of a mitochondria-localized inhibitor of apoptosis, a Bcl-2-like protein, prevented apoptosis induced by this virus. Human cells resistant to apoptosis allowed also an efficient MCMV replication. The important role of Bcl-2-like proteins for cytomegalovirus cross-species infections was subsequently confirmed by inserting the corresponding genes, and other inhibitors of apoptosis and control genes into the MCMV genome. Only recombinant viruses expressing a Bcl-2-like protein were able to replicate in human cells. A single gene of human cytomegalovirus encoding a mitochondrial inhibitor of apoptosis was sufficient to allow MCMV replication in human cells. Moreover, the same principle facilitated replication of the rat cytomegalovirus in human cells. Thus, induction of apoptosis limits rodent cytomegalovirus cross-species infection.}, subject = {Cytomegalie-Virus}, language = {en} } @article{MayerLoefflerLozaValdesetal.2019, author = {Mayer, Alexander E. and L{\"o}ffler, Mona C. and Loza Vald{\´e}s, Angel E. and Schmitz, Werner and El-Merahbi, Rabih and Trujillo-Viera, Jonathan and Erk, Manuela and Zhang, Thianzhou and Braun, Ursula and Heikenwalder, Mathias and Leitges, Michael and Schulze, Almut and Sumara, Grzegorz}, title = {The kinase PKD3 provides negative feedback on cholesterol and triglyceride synthesis by suppressing insulin signaling}, series = {Science Signaling}, journal = {Science Signaling}, edition = {accepted manuscript}, doi = {10.1126/scisignal.aav9150}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250025}, year = {2019}, abstract = {Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity.}, language = {en} } @article{RaduSchoenwetterBraunetal.2017, author = {Radu, Laura and Schoenwetter, Elisabeth and Braun, Cathy and Marcoux, Julien and Koelmel, Wolfgang and Schmitt, Dominik R. and Kuper, Jochen and Cianf{\´e}rani, Sarah and Egly, Jean M. and Poterszman, Arnaud and Kisker, Caroline}, title = {The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH}, series = {Nucleic Acids Research}, volume = {45}, journal = {Nucleic Acids Research}, number = {18}, doi = {10.1093/nar/gkx743}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170173}, pages = {10872-10883}, year = {2017}, abstract = {The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.}, language = {en} } @article{PetrusevaNaumenkoKuperetal.2021, author = {Petruseva, Irina and Naumenko, Natalia and Kuper, Jochen and Anarbaev, Rashid and Kappenberger, Jeannette and Kisker, Caroline and Lavrik, Olga}, title = {The Interaction Efficiency of XPD-p44 With Bulky DNA Damages Depends on the Structure of the Damage}, series = {Frontiers in Cell and Developmental Biology}, volume = {9}, journal = {Frontiers in Cell and Developmental Biology}, issn = {2296-634X}, doi = {10.3389/fcell.2021.617160}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231806}, year = {2021}, abstract = {The successful elimination of bulky DNA damages via the nucleotide excision repair (NER) system is largely determined by the damage recognition step. This step consists of primary recognition and verification of the damage. The TFIIH helicase XPD plays a key role in the verification step during NER. To date, the mechanism of damage verification is not sufficiently understood and requires further detailed research. This study is a systematic investigation of the interaction of ctXPD (Chaetomium thermophilum) as well as ctXPD-ctp44 with model DNAs, which contain structurally different bulky lesions with previously estimated NER repair efficiencies. We have used ATPase and DNA binding studies to assess the interaction of ctXPD with damaged DNA. The result of the analysis of ctXPD-ctp44 binding to DNA containing fluorescent and photoactivatable lesions demonstrates the relationship between the affinity of XPD for DNAs containing bulky damages and the ability of the NER system to eliminate the damage. Photo-cross-linking of ctXPD with DNA probes containing repairable and unrepairable photoactivatable damages reveals differences in the DNA interaction efficiency in the presence and absence of ctp44. In general, the results obtained indicate the ability of ctXPD-ctp44 to interact with a damage and suggest a significant role for ctp44 subunit in the verification process.}, language = {en} } @article{KressJessenHufnageletal.2023, author = {Kreß, Julia Katharina Charlotte and Jessen, Christina and Hufnagel, Anita and Schmitz, Werner and Da Xavier Silva, Thamara Nishida and Ferreira Dos Santos, Anc{\´e}ly and Mosteo, Laura and Goding, Colin R. and Friedmann Angeli, Jos{\´e} Pedro and Meierjohann, Svenja}, title = {The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2}, series = {Cell Reports}, volume = {42}, journal = {Cell Reports}, number = {7}, doi = {10.1016/j.celrep.2023.112724}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350312}, year = {2023}, abstract = {Highlights • The integrated stress response leads to a general ATF4-dependent activation of NRF2 • ATF4 causes a CHAC1-dependent GSH depletion, resulting in NRF2 stabilization • An elevation of NRF2 transcript levels fosters this effect • NRF2 supports the ISR/ATF4 pathway by improving cystine and antioxidant supply Summary The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.}, language = {en} } @article{YoungClementsLangetal.2014, author = {Young, Joanna C. and Clements, Abigail and Lang, Alexander E. and Garnett, James A. and Munera, Diana and Arbeloa, Ana and Pearson, Jaclyn and Hartland, Elizabeth L. and Matthews, Stephen J. and Mousnier, Aurelie and Barry, David J. and Way, Michael and Schlosser, Andreas and Aktories, Klaus and Frankel, Gad}, title = {The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation}, series = {Nature Communications}, volume = {5}, journal = {Nature Communications}, number = {5887}, doi = {10.1038/ncomms6887}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121157}, year = {2014}, abstract = {The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck-WIP-N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.}, language = {en} } @article{TessmerMargison2023, author = {Tessmer, Ingrid and Margison, Geoffrey P.}, title = {The DNA alkyltransferase family of DNA repair proteins: common mechanisms, diverse functions}, series = {International Journal of Molecular Sciences}, volume = {25}, journal = {International Journal of Molecular Sciences}, number = {1}, issn = {1422-0067}, doi = {10.3390/ijms25010463}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-355790}, year = {2023}, abstract = {DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O\(^6\)-alkylguanine and O\(^4\)-alkylthymine bases in DNA. Their mechanism involves binding to the damaged DNA and flipping the base out of the DNA helix into the active site pocket in the protein. Alkyltransferases then directly and irreversibly transfer the alkyl group from the base to the active site cysteine residue. In contrast, alkyltransferase-like proteins recruit nucleotide excision repair components for O\(^6\)-alkylguanine elimination. One or more of these proteins are found in all kingdoms of life, and where this has been determined, their overall DNA repair mechanism is strictly conserved between organisms. Nevertheless, between species, subtle as well as more extensive differences that affect target lesion preferences and/or introduce additional protein functions have evolved. Examining these differences and their functional consequences is intricately entwined with understanding the details of their DNA repair mechanism(s) and their biological roles. In this review, we will present and discuss various aspects of the current status of knowledge on this intriguing protein family.}, language = {en} } @article{NieswandtMorowskiBrachsetal.2014, author = {Nieswandt, Bernhard and Morowski, Martina and Brachs, Sebastian and Mielenz, Dirk and D{\"u}tting, Sebastian}, title = {The Adaptor Protein Swiprosin-1/EFhd2 Is Dispensable for Platelet Function in Mice}, doi = {10.1371/journal.pone.0107139}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113316}, year = {2014}, abstract = {Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown. Objective Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements. Methods and Results We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation. Conclusion These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss.}, language = {en} } @article{NavarroStegnerNieswandtetal.2021, author = {Navarro, Stefano and Stegner, David and Nieswandt, Bernhard and Heemskerk, Johan W. M. and Kuijpers, Marijke J. E.}, title = {Temporal roles of platelet and coagulation pathways in collagen- and tissue factor-induced thrombus formation}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {1}, issn = {1422-0067}, doi = {10.3390/ijms23010358}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284219}, year = {2021}, abstract = {In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.}, language = {en} } @phdthesis{Eltschkner2020, author = {Eltschkner, Sandra}, title = {Targeting the Bacterial Fatty-Acid Synthesis Pathway: Towards the Development of Slow-Onset Inhibitors and the Characterisation of Protein-Protein Interactions}, doi = {10.25972/OPUS-15664}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156643}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {A continuous arms race between the development of novel antibiotics and the evolution of corresponding resistance mechanisms in bacteria has been observed, since antibiotic agents like arsphenamines (e.g. Salvarsan, developed by Paul Ehrlich [1]), sulphonamides (e.g. Prontosil, Gerhard Domagk [2]) and penicillin (Alexander Fleming [3]) were first applied to effectively cure bacterial infections in the early 20th century. The rapid emergence of resistances in contrast to the currently lagging discovery of antibiotics displays a severe threat to human health. Some serious infectious diseases, such as tuberculosis or melioidosis, which were either thought to be an issue only in Third-World countries in case of tuberculosis, or regionally restricted with respect to melioidosis, are now on the rise to expand to other areas. In contrast, methicillin-resistant Staphylococcus aureus (MRSA) is already present in clinical setups all over the world and causes severe infections in immunocompromised patients. Thus, there is an urgent need for new and effective antimicrobial agents, which impair vital functions of the pathogen's metabolism. One central metabolic pathway is represented by the bacterial fatty-acid synthesis pathway (FAS II), which is essential for the synthesis of long and branched-chain fatty acids, as well as mycolic acids. These substances play a major role as modulating components of the properties of the most important protective barrier - the cell envelope. The integrity of the bacterial cell wall and the associated membrane(s) is crucial for cell growth and for protection against physical strain, intrusion of antibiotic agents and regulation of uptake of ions and other small molecules. Thus, this central pathway represents a promising target for antibiotic action against pathogens to combat infectious diseases. The last and rate-limiting step is catalysed by the trans-2-enoyl-ACP reductase (ENR) FabI or InhA (in mycobacteria), which has been demonstrated to be a valuable target for drug design and can be addressed, amongst others, by diphenyl ether (DPE) compounds, derived from triclosan (TCL) - the first one of this class which was discovered to bind to ENR enzymes [4, 5]. Based on this scaffold, inhibitors containing different combinations of substituents at crucial positions, as well as a novel type of substituent at position five were investigated regarding their binding behaviour towards the Burkholderia pseudomallei and Mycobacterium tuberculosis ENR enzymes bpFabI and InhA, respectively, by structural, kinetic and in-vivo experiments. Generally, substitution patterns modulate the association and dissociation velocities of the different ENR inhibitors in the context of the two-step slow-onset binding mechanism, which is observed for both enzymes. These alterations in the rapidity of complex formation and decomposition have a crucial impact on the residence time of a compound and hence, on the pharmacokinetic properties of potential drug candidates. For example, the substituents at the 2'-position of the DPE scaffold influence the ground- and transition state stability during the binding process to bpFabI, whereas 4'-substituents primarily alter the transition state [6]. The novel triazole group attached to the 5-position of the scaffold, targeting the hydrophobic part of the substrate-binding pocket in InhA, significantly enhances the energy barrier of the transition state of inhibitor binding [7] and decelerates the association- as well as the dissociation processes. Combinations with different substituents at the 2'-position can enhance or diminish this effect, e.g. by ground-state stabilisation, which will result in an increased residence time of the respective inhibitor on InhA. Further structural investigations carried out in this work, confirm the proposed binding mode of a customised saFabI inhibitor [8], carrying a pyridone moiety on the DPE scaffold to expand interactions with the protein environment. Structural and preliminary kinetic data confirm the binding of the same inhibitor to InhA in a related fashion. Comparisons with structures of the ENR inhibitor AFN-1252 [9] bound to ENR enzymes from other organisms, addressing a similar region as the pyridone-moiety of the DPE inhibitor, suggest that also the DPE inhibitor bears the potential to display binding to homologues of saFabI and InhA and may be optimised accordingly. Both of the newly investigated substituents, the pyridone moiety at the 4'-position as well as the 5-triazole substituent, provide a good starting point to modify the DPE scaffold also towards improved kinetic properties against ENR enzymes other than the herein studied and combining both groups on the DPE scaffold may have beneficial effects. The understanding of the underlying binding mechanism is a crucial factor to promote the dedicated design of inhibitors with superior pharmacokinetic characteristics. A second target for a structure-based drug-design approach is the interaction surface between ENR enzymes and the acyl-carrier protein (ACP), which delivers the growing acyl chain to each distinct enzyme of the dissociated FAS-II system and presumably recognises its respective interaction partner via electrostatic contacts. The interface between saACP and saFabI was investigated using different approaches including crosslinking experiments and the design of fusion constructs connecting the ACP and the FabI subunits via a flexible linker region of varying lengths and compositions. The crosslinking studies confirmed a set of residues to be part of the contact interface of a previously proposed complex model [10] and displayed high crosslinking efficiency of saACP to saFabI when mutated to cysteine residues. However, crystals of the complex obtained from either the single components, or of the fusion constructs usually displayed weak diffraction, which supports the assumption that complex formation is highly transient. To obtain ordered crystals for structural characterisation of the complex it is necessary to trap the complex in a fixed state, e.g. by a high-affinity substrate attached to ACP [11], which abolishes rapid complex dissociation. For this purpose, acyl-coupled long-residence time inhibitors might be a valuable tool to elucidate the detailed architecture of the ACP-FabI interface. This may provide a novel basis for the development of inhibitors that specifically target the FAS-II biosynthesis pathway.}, subject = {Fetts{\"a}urestoffwechsel}, language = {en} } @article{SchuhmannKraftBieberetal.2019, author = {Schuhmann, Michael K. and Kraft, Peter and Bieber, Michael and Kollikowski, Alexander M. and Schulze, Harald and Nieswandt, Bernhard and Pham, Mirko and Stegner, David and Stoll, Guido}, title = {Targeting platelet GPVI plus rt-PA administration but not α2β1-mediated collagen binding protects against ischemic brain damage in mice}, series = {International Journal of Molecular Science}, volume = {20}, journal = {International Journal of Molecular Science}, number = {8}, issn = {1422-0067}, doi = {10.3390/ijms20082019}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201700}, year = {2019}, abstract = {Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{-/-}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.}, language = {en} } @article{NavarroStarkeHeemskerketal.2022, author = {Navarro, Stefano and Starke, Andreas and Heemskerk, Johan W. M. and Kuijpers, Marijke J. E. and Stegner, David and Nieswandt, Bernhard}, title = {Targeting of a conserved epitope in mouse and human GPVI differently affects receptor function}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {15}, issn = {1422-0067}, doi = {10.3390/ijms23158610}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286227}, year = {2022}, abstract = {Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6\(^{tg/tg\)). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.}, language = {en} } @article{KhayenkoMaric2019, author = {Khayenko, Vladimir and Maric, Hans Michael}, title = {Targeting GABA\(_A\)R-associated proteins: new modulators, labels and concepts}, series = {Frontiers in Molecular Neuroscience}, volume = {12}, journal = {Frontiers in Molecular Neuroscience}, number = {162}, doi = {10.3389/fnmol.2019.00162}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201876}, year = {2019}, abstract = {γ-aminobutyric acid type A receptors (GABA\(_A\)Rs) are the major mediators of synaptic inhibition in the brain. Aberrant GABA\(_A\)R activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer's and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review article, we introduce a new strategy to modulate GABAeric signaling: targeting the intracellular protein interactors of GABA\(_A\)Rs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABA\(_A\)R subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABA\(_A\)R-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABA\(_A\)R numbers and clustering, modifying neuronal transmission. Interference with GABA\(_A\)R trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABA\(_A\)R to AP2 increase the surface concentration of GABA\(_A\)R clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with submicron resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABA\(_A\)R accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABA\(_A\)R modulators and following in vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.}, language = {en} } @article{HoppAlbertWeissenbergerMencletal.2016, author = {Hopp, Sarah and Albert-Weissenberger, Christiane and Mencl, Stine and Bieber, Michael and Schuhmann, Michael K. and Stetter, Christian and Nieswandt, Bernhard and Schmidt, Peter M. and Monoranu, Camelia-Maria and Alafuzoff, Irina and Marklund, Niklas and Nolte, Marc W. and Sir{\´e}n, Anna-Leena and Kleinschnitz, Christoph}, title = {Targeting coagulation factor XII as a novel therapeutic option in brain trauma}, series = {Annals of Neurology}, volume = {79}, journal = {Annals of Neurology}, number = {6}, doi = {10.1002/ana.24655}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-188800}, pages = {970-982}, year = {2016}, abstract = {Objective: Traumatic brain injury is a major global public health problem for which specific therapeutic interventions are lacking. There is, therefore, a pressing need to identify innovative pathomechanism-based effective therapies for this condition. Thrombus formation in the cerebral microcirculation has been proposed to contribute to secondary brain damage by causing pericontusional ischemia, but previous studies have failed to harness this finding for therapeutic use. The aim of this study was to obtain preclinical evidence supporting the hypothesis that targeting factor XII prevents thrombus formation and has a beneficial effect on outcome after traumatic brain injury. Methods: We investigated the impact of genetic deficiency of factor XII and acute inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused infestin-4 (rHA-Infestin-4) on trauma-induced microvascular thrombus formation and the subsequent outcome in 2 mouse models of traumatic brain injury. Results: Our study showed that both genetic deficiency of factor XII and an inhibition of activated factor XII in mice minimize trauma-induced microvascular thrombus formation and improve outcome, as reflected by better motor function, reduced brain lesion volume, and diminished neurodegeneration. Administration of human factor XII in factor XII-deficient mice fully restored injury-induced microvascular thrombus formation and brain damage. Interpretation: The robust protective effect of rHA-Infestin-4 points to a novel treatment option that can decrease ischemic injury after traumatic brain injury without increasing bleeding tendencies.}, language = {en} }