137 results on '"Günter Fritz"'
Search Results
2. Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL amyloidosis
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Lynn Radamaker, Yin-Hsi Lin, Karthikeyan Annamalai, Stefanie Huhn, Ute Hegenbart, Stefan O. Schönland, Günter Fritz, Matthias Schmidt, and Marcus Fändrich
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Science - Abstract
Systemic AL amyloidosis is caused by misfolding of immunoglobulin light chains and is one of the most frequently occurring forms of systemic amyloidosis. Here the authors present the 3.3 Å cryo-EM structure of a λ1 AL amyloid fibril that was isolated from an explanted human heart.
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- 2019
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3. Central Carbon Metabolism, Sodium-Motive Electron Transfer, and Ammonium Formation by the Vaginal Pathogen Prevotella bivia
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Lena Schleicher, Sebastian Herdan, Günter Fritz, Andrej Trautmann, Jana Seifert, and Julia Steuber
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bacterial vaginosis ,Prevotella bivia ,Na+-translocating NADH:quinone oxidoreductase ,fumarate reductase ,amino acid degradation ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.
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- 2021
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4. Vibrio natriegens as Host for Expression of Multisubunit Membrane Protein Complexes
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Lena Schleicher, Valentin Muras, Björn Claussen, Jens Pfannstiel, Bastian Blombach, Pavel Dibrov, Günter Fritz, and Julia Steuber
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Vibrio natriegens ,membrane proteins ,multisubunit complexes ,expression ,Na+-translocating NADH:quinone oxidoreductase ,multiple resistance and pH related antiporter ,Microbiology ,QR1-502 - Abstract
Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens.
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- 2018
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5. The Mouse-Specific Splice Variant mRAGE_v4 Encodes a Membrane-Bound RAGE That Is Resistant to Shedding and Does Not Contribute to the Production of Soluble RAGE.
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Stefania Di Maggio, Elena Gatti, Jaron Liu, Matteo Bertolotti, Günter Fritz, Marco E Bianchi, and Angela Raucci
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Medicine ,Science - Abstract
The receptor for advanced glycation end-products (RAGE) is involved in the onset and progression of several inflammatory diseases. The RAGE primary transcript undergoes numerous alternative splicing (AS) events, some of which are species-specific. Here, we characterize the mouse-specific mRAGE_v4 splice variant, which is conserved in rodents and absent in primates. mRAGE_v4 derives from exon 9 skipping and encodes a receptor (M-RAGE) that lacks 9 amino acids between the transmembrane and the immunoglobulin (Ig) domains. RNA-Seq data confirm that in mouse lung mRAGE_v4 is the most abundant RAGE mRNA isoform after mRAGE, which codes for full-length RAGE (FL-RAGE), while in heart all RAGE variants are almost undetectable. The proteins M-RAGE and FL-RAGE are roughly equally abundant in mouse lung. Contrary to FL-RAGE, M-RAGE is extremely resistant to shedding because it lacks the peptide motif recognized by both ADAM10 and MMP9, and does not contribute significantly to soluble cRAGE formation. Thus, a cassette exon in RAGE corresponds to a specific function of the RAGE protein-the ability to be shed. Given the differences in RAGE AS variants between rodents and humans, caution is due in the interpretation of results obtained in mouse models of RAGE-dependent human pathologies.
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- 2016
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6. High Affinity Binding of Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) to Lu/BCAM Adhesion Glycoprotein
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Franziska Reppin, Sylvie Cochet, Wassim El Nemer, Günter Fritz, and Gudula Schmidt
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Lu/BCAM ,CNF ,laminin ,toxin ,receptor ,immunoglobulin-like domain ,sickle cell disease ,Medicine - Abstract
The protein toxin Cytotoxic Necrotizing Factor 1 (CNF1) is a major virulence factor of pathogenic Escherichia coli strains. It belongs to a family of single chain AB-toxins, which enter mammalian cells by receptor-mediated endocytosis. Recently, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as a cellular receptor for CNF1. Here, we identified the Ig-like domain 2 of Lu/BCAM as main interaction site of the toxin by direct protein-protein interaction and competition studies. Using surface plasmon resonance, we showed a high affinity CNF-Lu/BCAM interaction with a KD of 2.8 nM. Furthermore, we performed small-angle X-ray scattering to define the molecular envelope of the Lu/BCAM-CNF1 complex, suggesting a 6:1 ratio of Lu/BCAM to CNF1 in the receptor-toxin complex. This study leads to a deeper understanding of the interaction between CNF1 and Lu/BCAM, and presents novel opportunities for the development of future anti-toxin strategies.
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- 2017
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7. The receptor for advanced glycation end-products (RAGE) is only present in mammals, and belongs to a family of cell adhesion molecules (CAMs).
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Luca Sessa, Elena Gatti, Filippo Zeni, Antonella Antonelli, Alessandro Catucci, Michael Koch, Giulio Pompilio, Günter Fritz, Angela Raucci, and Marco E Bianchi
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Medicine ,Science - Abstract
The human receptor for advanced glycation endproducts (RAGE) is a multiligand cell surface protein belonging to the immunoglobulin superfamily, and is involved in inflammatory and immune responses. Most importantly, RAGE is considered a receptor for HMGB1 and several S100 proteins, which are Damage-Associated Molecular Pattern molecules (DAMPs) released during tissue damage. In this study we show that the Ager gene coding for RAGE first appeared in mammals, and is closely related to other genes coding for cell adhesion molecules (CAMs) such as ALCAM, BCAM and MCAM that appeared earlier during metazoan evolution. RAGE is expressed at very low levels in most cells, but when expressed at high levels, it mediates cell adhesion to extracellular matrix components and to other cells through homophilic interactions. Our results suggest that RAGE evolved from a family of CAMs, and might still act as an adhesion molecule, in particular in the lung where it is highly expressed or under pathological conditions characterized by an increase of its protein levels.
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- 2014
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8. Intrinsically disordered and aggregation prone regions underlie β-aggregation in S100 proteins.
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Sofia B Carvalho, Hugo M Botelho, Sónia S Leal, Isabel Cardoso, Günter Fritz, and Cláudio M Gomes
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Medicine ,Science - Abstract
S100 proteins are small dimeric calcium-binding proteins which control cell cycle, growth and differentiation via interactions with different target proteins. Intrinsic disorder is a hallmark among many signaling proteins and S100 proteins have been proposed to contain disorder-prone regions. Interestingly, some S100 proteins also form amyloids: S100A8/A9 forms fibrils in prostatic inclusions and S100A6 fibrillates in vitro and seeds SOD1 aggregation. Here we report a study designed to investigate whether β-aggregation is a feature extensive to more members of S100 family. In silico analysis of seven human S100 proteins revealed a direct correlation between aggregation and intrinsic disorder propensity scores, suggesting a relationship between these two independent properties. Averaged position-specific analysis and structural mapping showed that disorder-prone segments are contiguous to aggregation-prone regions and that whereas disorder is prominent on the hinge and target protein-interaction regions, segments with high aggregation propensity are found in ordered regions within the dimer interface. Acidic conditions likely destabilize the seven S100 studied by decreasing the shielding of aggregation-prone regions afforded by the quaternary structure. In agreement with the in silico analysis, hydrophobic moieties become accessible as indicated by strong ANS fluorescence. ATR-FTIR spectra support a structural inter-conversion from α-helices to intermolecular β-sheets, and prompt ThT-binding takes place with no noticeable lag phase. Dot blot analysis using amyloid conformational antibodies denotes a high diversity of conformers; subsequent analysis by TEM shows fibrils as dominant species. Altogether, our data suggests that β-aggregation and disorder-propensity are related properties in S100 proteins, and that the onset of aggregation is likely triggered by loss of protective tertiary and quaternary interactions.
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- 2013
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9. Assessment of the Genetic Spectrum of Uncombable Hair Syndrome in a Cohort of 107 Individuals
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F. Buket Basmanav, Nicole Cesarato, Sheetal Kumar, Oleg Borisov, Pavlos Kokordelis, Damian J. Ralser, Maria Wehner, Daisy Axt, Xing Xiong, Holger Thiele, Vadim Dolgin, Yasmina Gossmann, Nadine Fricker, Malin Katharina Dewenter, Karsten Weller, Mohnish Suri, Herbert Reichenbach, Vinzenz Oji, Marie-Claude Addor, Karla Ramirez, Helen Stewart, Natalie Garcia Bartels, Lisa Weibel, Nicola Wagner, Susannah George, Arzu Kilic, Iliana Tantcheva-Poor, Alison Stewart, Nicola Dikow, Bettina Blaumeiser, Márta Medvecz, Ulrike Blume-Peytavi, Paul Farrant, Ramon Grimalt, Sara Bertok, Lisa Bradley, Marina Eskin-Schwartz, Ohad Samuel Birk, Anette Bygum, Michel Simon, Peter Krawitz, Christine Fischer, Henning Hamm, Günter Fritz, and Regina C. Betz
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Male ,Cohort Studies ,Transglutaminases ,Exome Sequencing ,Humans ,Female ,Human medicine ,Dermatology ,Hair Diseases ,Hair - Abstract
ImportanceUncombable hair syndrome (UHS) is a rare hair shaft anomaly that manifests during infancy and is characterized by dry, frizzy, and wiry hair that cannot be combed flat. Only about 100 known cases have been reported so far.ObjectiveTo elucidate the genetic spectrum of UHS.Design, Setting, and ParticipantsThis cohort study includes 107 unrelated index patients with a suspected diagnosis of UHS and family members who were recruited worldwide from January 2013 to December 2021. Participants of all ages, races, and ethnicities were recruited at referral centers or were enrolled on their own initiative following personal contact with the authors. Genetic analyses were conducted in Germany from January 2014 to December 2021.Main Outcomes and MeasuresClinical photographs, Sanger or whole-exome sequencing and array-based genotyping of DNA extracted from blood or saliva samples, and 3-dimensional protein modeling. Descriptive statistics, such as frequency counts, were used to describe the distribution of identified pathogenic variants and genotypes.ResultsThe genetic characteristics of patients with UHS were established in 80 of 107 (74.8%) index patients (82 [76.6%] female) who carried biallelic pathogenic variants in PADI3, TGM3, or TCHH (ie, genes that encode functionally related hair shaft proteins). Molecular genetic findings from 11 of these 80 individuals were previously published. In 76 (71.0%) individuals, the UHS phenotype were associated with pathogenic variants in PADI3. The 2 most commonly observed PADI3 variants account for 73 (48.0%) and 57 (37.5%) of the 152 variant PADI3 alleles in total, respectively. Two individuals carried pathogenic variants in TGM3, and 2 others carried pathogenic variants in TCHH. Haplotype analyses suggested a founder effect for the 4 most commonly observed pathogenic variants in the PADI3 gene.Conclusions and RelevanceThis cohort study extends and gives an overview of the genetic variant spectrum of UHS based on molecular genetic analyses of the largest worldwide collective of affected individuals, to our knowledge. Formerly, a diagnosis of UHS could only be made by physical examination of the patient and confirmed by microscopical examination of the hair shaft. The discovery of pathogenic variants in PADI3, TCHH, and TGM3 may open a new avenue for clinicians and affected individuals by introducing molecular diagnostics for UHS.
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- 2023
10. Concerning the photophysics of fluorophores towards tailored bioimaging compounds: a case study involving S100A9 inflammation markers
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Simon T. Steiner, Iván Maisuls, Anna Junker, Günter Fritz, Andreas Faust, and Cristian A. Strassert
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Fluorescent conjugates ,Fluorescence quantum yields ,S100A9 ,Fluorescence lifetimes - Abstract
A full understanding concerning the photophysical properties of a fluorescent label is crucial for a reliable and predictable performance in biolabelling applications. This holds true not only for the choice of a fluorophore in general, but also for the correct interpretation of data, considering the complexity of biological environments. In the frame of a case study involving inflammation imaging, we report the photophysical characterization of four fluorescent S100A9-targeting compounds in terms of UV–vis absorption and photoluminescence spectroscopy, fluorescence quantum yields (ΦF) and excited state lifetimes (τ) as well as the evaluation of the radiative and non-radiative rate constants (kr and knr, respectively). The probes were synthesized based on a 2-amino benzimidazole-based lead structure in combination with commercially available dyes, covering a broad color range from green (6-FAM) over orange (BODIPY-TMR) to red (BODIPY-TR) and near-infrared (Cy5.5) emission. The effect of conjugation with the targeting structure was addressed by comparison of the probes with their corresponding dye-azide precursors. Additionally, the 6-FAM and Cy5.5 probes were measured in the presence of murine S100A9 to determine whether protein binding influences their photophysical properties. An interesting rise in ΦF upon binding of 6-FAM-SST177 to murine S100A9 enabled the determination of its dissociation equilibrium constant, reaching up to KD = 324 nM. This result gives an outlook for potential applications of our compounds in S100A9 inflammation imaging and fluorescence assay developments. With respect to the other dyes, this study demonstrates how diverse microenvironmental factors can severely impair their performance while rendering them poor performers in biological media, showing that a preliminary photophysical screening is key to assess the suitability of a particular luminophore.
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- 2023
11. Fast IMAC purification of non-tagged S100A8/A9 (calprotectin) from Homo sapiens and Sus scrofa
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Jann-Louis Hau, Hannes Kremser, Sabrina Knogl-Tritschler, Volker Stefanski, Julia Steuber, and Günter Fritz
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Biotechnology - Published
- 2023
12. AgeR deletion decreases soluble fms-like tyrosine kinase 1 production and improves post-ischemic angiogenesis in uremic mice
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Günter Fritz, Rida Al-Rifai, Philippe Nguyen, Ann Marie Schmidt, Gael Poitevin, Jeremy Ortillon, Claire Tournois, Fatouma Touré, Vincent Dupont, Laura Jayyosi, Camile Boulagnon-Rombi, Philippe Rieu, Caroline Fichel, Christine Terryn, Caroline François, Hémostase et Remodelage Vasculaire Post-Ischémie (HERVI - EA 3801), Université de Reims Champagne-Ardenne (URCA), Department of Nephrology, Reims University Hospital, Reims, France, Matrice extracellulaire et dynamique cellulaire - UMR 7369 (MEDyC), SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Centre National de la Recherche Scientifique (CNRS), Plateforme en Imagerie Cellulaire et Tissulaire (PICT), Université de Reims Champagne-Ardenne (URCA)-SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV), Institute of Neuropathology, University of Freiburg [Freiburg], New York University Langone Medical Center (NYU Langone Medical Center), NYU System (NYU), Service de Néphrologie, Dialyse, Transplantations [CHU Limoges], and CHU Limoges
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Male ,0301 basic medicine ,Cancer Research ,medicine.medical_specialty ,Physiology ,Angiogenesis ,medicine.medical_treatment ,Receptor for Advanced Glycation End Products ,Clinical Biochemistry ,Neovascularization, Physiologic ,Ligands ,Revascularization ,Cell Line ,RAGE (receptor) ,03 medical and health sciences ,0302 clinical medicine ,Ischemia ,Glycation ,Internal medicine ,medicine ,Animals ,Humans ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,ComputingMilieux_MISCELLANEOUS ,Uremia ,Vascular Endothelial Growth Factor Receptor-1 ,business.industry ,medicine.disease ,Up-Regulation ,Mice, Inbred C57BL ,030104 developmental biology ,Endocrinology ,Solubility ,030220 oncology & carcinogenesis ,RNA ,Ligation ,business ,Tyrosine kinase ,Biomarkers ,Gene Deletion ,[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology ,Soluble fms-like tyrosine kinase-1 ,Kidney disease - Abstract
Peripheral arterial disease occurs more frequently and has a worse prognosis in patients with chronic kidney disease (CKD). The receptor for advanced glycation end products (RAGE) is involved in multiple aspects of uremia-associated vasculopathy. Previous data suggest that the RAGE pathway may promote soluble fms-like tyrosine kinase 1 (sFlt1) production, an anti-angiogenic molecule. Thus, we tested the hypothesis that the deletion of AgeR would decrease sFlt1 production and improve post-ischemic revascularization in uremic condition. We used a well-established CKD model (5/6 nephrectomy) in WT and AgeR−/− C57/Bl6 mice. Hindlimb ischemia was induced by femoral artery ligation. Revascularization was evaluated by complementary approaches: ischemic limb retraction, LASCA imagery, and capillary density. The production of sFlt1 was assessed at both RNA and protein levels. After hindlimb ischemia, uremic mice showed slower functional recovery (p
- Published
- 2020
13. Anoxic cell rupture of Prevotella bryantii by high-pressure homogenization protects the Na+-translocating NADH:quinone oxidoreductase from oxidative damage
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Lena Schleicher, Jana Seifert, Günter Fritz, and Julia Steuber
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chemistry.chemical_classification ,0303 health sciences ,biology ,030306 microbiology ,Superoxide ,General Medicine ,biology.organism_classification ,Quinone oxidoreductase ,Biochemistry ,Microbiology ,Anoxic waters ,Superoxide dismutase ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme ,chemistry ,Genetics ,Cell disruption ,biology.protein ,Molecular Biology ,Bacteria ,Prevotella bryantii ,030304 developmental biology - Abstract
Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L–1 culture and a NADH oxidation activity of 0.8 µmol min−1 mg−1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii.
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- 2020
14. Cryo-EM structure of a transthyretin-derived amyloid fibril from a patient with hereditary ATTR amyloidosis
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Per Westermark, Jonas Engler, Volkan Adak, Günter Fritz, Sebastian Wiese, Marcus Fändrich, Matthias Schmidt, Martin Zacharias, and Shubhangi Agarwal
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0301 basic medicine ,Male ,Models, Molecular ,Pathology ,medicine.medical_specialty ,Amyloid ,endocrine system ,Prions ,Science ,General Physics and Astronomy ,Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) ,macromolecular substances ,Biochemistry ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Prealbumin ,Protein folding ,Proteostasis Deficiencies ,lcsh:Science ,Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci) ,Aged ,Protein Unfolding ,Amyloid Neuropathies, Familial ,Multidisciplinary ,biology ,business.industry ,Cryoelectron Microscopy ,nutritional and metabolic diseases ,General Chemistry ,Amyloid fibril ,Systemic amyloidosis ,ddc ,Amyloid Neuropathy ,Transthyretin ,030104 developmental biology ,biology.protein ,lcsh:Q ,business ,030217 neurology & neurosurgery ,Attr amyloidosis - Abstract
ATTR amyloidosis is one of the worldwide most abundant forms of systemic amyloidosis. The disease is caused by the misfolding of transthyretin protein and the formation of amyloid deposits at different sites within the body. Here, we present a 2.97 Å cryo electron microscopy structure of a fibril purified from the tissue of a patient with hereditary Val30Met ATTR amyloidosis. The fibril consists of a single protofilament that is formed from an N-terminal and a C-terminal fragment of transthyretin. Our structure provides insights into the mechanism of misfolding and implies the formation of an early fibril state from unfolded transthyretin molecules, which upon proteolysis converts into mature ATTR amyloid fibrils., Systemic amyloidosis of the ATTR is one of the most abundant forms of systemic amyloidosis and caused by misfolding of the circulating blood protein transthyretin (TTR). Here the authors present the cryo-EM structure of patient-derived Val30Met ATTR amyloid fibrils which reveals that the protofilament consists of an N-terminal and a C-terminal TTR fragment and discuss implications for the mechanism of misfolding.
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- 2019
15. Structure and mechanism of sodium pumping NADH:quinone oxidoreductase
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Jann-Louis Hau, Susann Kaltwasser, Julia Steuber, Janet Vonck, and Günter Fritz
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Biophysics ,Cell Biology ,Biochemistry - Published
- 2022
16. Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL amyloidosis
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Stefan Schönland, Karthikeyan Annamalai, Yin-Hsi Lin, Lynn Radamaker, Matthias Schmidt, Stefanie Huhn, Günter Fritz, Marcus Fändrich, and Ute Hegenbart
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Models, Molecular ,0301 basic medicine ,Amyloid ,Protein Folding ,Protein Conformation ,Science ,General Physics and Astronomy ,02 engineering and technology ,macromolecular substances ,Protein aggregation ,Fibril ,Immunoglobulin light chain ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,DDC 570 / Life sciences ,Protein structure ,Cryoelectron microscopy ,ddc:570 ,AL amyloidosis ,medicine ,Humans ,Immunoglobulin Light-chain Amyloidosis ,Amino Acid Sequence ,lcsh:Science ,Peptide sequence ,Multidisciplinary ,biology ,Chemistry ,Myocardium ,Cryoelectron Microscopy ,General Chemistry ,Amyloidosis ,Amyloidose ,021001 nanoscience & nanotechnology ,medicine.disease ,3. Good health ,030104 developmental biology ,Mutation ,biology.protein ,Biophysics ,Immunoglobulin Light Chains ,Protein folding ,lcsh:Q ,Antibody ,0210 nano-technology - Abstract
Amyloid fibrils derived from antibody light chains are key pathogenic agents in systemic AL amyloidosis. They can be deposited in multiple organs but cardiac amyloid is the major risk factor of mortality. Here we report the structure of a λ1 AL amyloid fibril from an explanted human heart at a resolution of 3.3 Å which we determined using cryo-electron microscopy. The fibril core consists of a 91-residue segment presenting an all-beta fold with ten mutagenic changes compared to the germ line. The conformation differs substantially from natively folded light chains: a rotational switch around the intramolecular disulphide bond being the crucial structural rearrangement underlying fibril formation. Our structure provides insight into the mechanism of protein misfolding and the role of patient-specific mutations in pathogenicity., publishedVersion
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- 2019
17. A Sodium-Translocating Module Linking Succinate Production to Formation of Membrane Potential in Prevotella bryantii
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Michael Bott, Jochem Gätgens, Jörg Simon, Sascha Hein, Andrej Trautmann, Dennis P. Stegmann, Julia Steuber, Lena Schleicher, Jana Seifert, and Günter Fritz
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Anaerobic respiration ,Prevotella ,Quinone oxidoreductase ,Applied Microbiology and Biotechnology ,Cofactor ,Membrane Potentials ,Rumen ,anaerobic respiration ,Fumarates ,ddc:570 ,fumarate reductase ,Prevotella bryantii ,Environmental Microbiology ,Animals ,Electrochemical gradient ,rumen ,Sheep ,Ecology ,biology ,Chemistry ,Chemiosmosis ,Na+-translocating NADH:quinone oxidoreductase ,Sodium ,Succinates ,supercomplex ,Fumarate reductase ,NAD ,Electron transport chain ,Succinate Dehydrogenase ,Biochemistry ,biology.protein ,Cattle ,Food Science ,Biotechnology - Abstract
Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of the rumen microbiota belong to the family Prevotellaceae, which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na+-translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min−1 mg−1), quinone reduction (490 nmol min−1 mg−1), and fumarate reduction (1,200 nmol min−1 mg−1) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii. Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD+ and succinate. We propose that the regeneration of NAD+ in P. bryantii is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. IMPORTANCE Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella spp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na+-translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii. Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.
- Published
- 2021
18. Concise Synthesis of 1,4-Benzoquinone-Based Natural Products as Mitochondrial Complex I Substrates and Substrate-Based Inhibitors
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Hamid R. Nasiri, Karina Tuz, Iris Bischoff, Robert Fürst, Zhenyu Han, Oscar Juárez, Yihan Qin, Günter Fritz, Heike Angerer, Dana Lashley, and Dennis P. Stegmann
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Antineoplastic Agents ,Quinone oxidoreductase ,Biochemistry ,Substrate Specificity ,1,4-Benzoquinone ,chemistry.chemical_compound ,Mice ,Structure-Activity Relationship ,Cell Line, Tumor ,Drug Discovery ,Benzoquinones ,Molecule ,Animals ,Humans ,General Pharmacology, Toxicology and Pharmaceutics ,Enzyme Inhibitors ,Cell Proliferation ,Pharmacology ,chemistry.chemical_classification ,Biological Products ,Natural product ,Electron Transport Complex I ,biology ,Molecular Structure ,Succinate dehydrogenase ,Organic Chemistry ,Substrate (chemistry) ,Combinatorial chemistry ,Quinone ,Enzyme ,chemistry ,Drug Design ,biology.protein ,Molecular Medicine ,Female ,Drug Screening Assays, Antitumor - Abstract
A short, efficient one-step synthesis of 2-methyl-5-(3-methyl-2-butenyl)-1,4-benzoquinone, a natural product from Pyrola media is described. The synthesis is based on a direct late C-H functionalization of the quinone scaffold. The formation of the natural product was confirmed by means of 2D-NMR spectroscopy. Additional derivatives were synthesized and tested alongside the natural product as potential substrate and substrate-based inhibitors of mitochondrial complex I (MCI). The structure-activity relationship study led to the discovery of 3-methylbuteneoxide-1,4-anthraquinone (1 i), an inhibitor with an IC50 of 5 μM against MCI. The identified molecule showed high selectivity for MCI when tested against other quinone-converting enzymes, including succinate dehydrogenase, and the Na (+)-translocating NADH:quinone oxidoreductase. Moreover, the identified inhibitor was also active in cell-based proliferation assays. Therefore, 1 i can be considered as a novel chemical probe for MCI.
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- 2020
19. Impact of Na + -Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae
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Shubhangi Agarwal, Melanie Bernt, Alisha M. Block, Paul D’Alvise, Jens Pfannstiel, Julia Steuber, Hannes Kurz, Charlotte Toulouse, Martin Hasselmann, Claudia C. Häse, and Günter Fritz
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Operon ,Iron ,Antiporter ,Mutant ,Quinone oxidoreductase ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,Bacterial Proteins ,medicine ,Quinone Reductases ,Vibrio cholerae ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,ATP synthase ,biology ,030306 microbiology ,Structural gene ,Biological Transport ,Respiratory enzyme ,Biochemistry ,Mutation ,biology.protein ,Oxidation-Reduction ,Research Article - Abstract
The Na+ ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF, a flavin attachment gene, apbE, and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δnqr and ΔubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe2+ (Feo), was increased in V. choleraeΔnqr. In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. choleraeΔnqr with Fe2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. choleraeΔnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation. IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics.
- Published
- 2020
20. Functional implications of novel ADAM10 mutations in reticulate acropigmentation of Kitamura
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Regina C. Betz, Jorge Frank, Holger Thiele, A. Du-Thanh, P Kokordelis, Peter Nürnberg, Janine Altmüller, Günter Fritz, Gilles G. Lestringant, F.B.Ünalan Basmanav, J. Fischer, Damian J. Ralser, Vinzenz Oji, Sabrina Wolf, and Centre Hospitalier Régional Universitaire [Montpellier] ( CHRU Montpellier )
- Subjects
Male ,0301 basic medicine ,Skin Diseases, Papulosquamous ,Reticulate acropigmentation of Kitamura ,Mutation, Missense ,Haploinsufficiency ,Dermatology ,Biology ,ADAM10 Protein ,030207 dermatology & venereal diseases ,03 medical and health sciences ,0302 clinical medicine ,INDEL Mutation ,Hyperpigmentation ,Humans ,Frameshift Mutation ,ComputingMilieux_MISCELLANEOUS ,Genetics ,Membrane Proteins ,Skin Diseases, Genetic ,030104 developmental biology ,Codon, Nonsense ,Mutation ,Mutation (genetic algorithm) ,Female ,Amyloid Precursor Protein Secretases ,[ SDV.MHEP.DERM ] Life Sciences [q-bio]/Human health and pathology/Dermatology - Abstract
International audience
- Published
- 2017
21. A miniaturized assay for kinetic characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae
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Björn Claussen, Valentin Muras, Hamid R. Nasiri, Günter Fritz, and Julia Steuber
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0301 basic medicine ,chemistry.chemical_classification ,NADH-Ubiquinone Oxidoreductase ,High-throughput screening ,Mutagenesis ,Biophysics ,Cell Biology ,Sodium ion transport ,medicine.disease_cause ,Biochemistry ,Quinone ,03 medical and health sciences ,030104 developmental biology ,Enzyme ,chemistry ,Vibrio cholerae ,NADH quinone oxidoreductase ,medicine ,Molecular Biology - Abstract
We demonstrate the miniaturization of an enzymatic assay for the determination of NADH oxidation and quinone reduction by the Na+ -translocating NADH quinone oxidoreductase (NQR) in the 96-well plate format. The assay is based on the spectrophotometric detection of NADH consumption and quinol formation. We validated the new method with known inhibitors of the NQR and optimized conditions for high-throughput screening as demonstrated by excellent Z-factors well above the accepted threshold (≥0.5). Overall, the method allows the screening and identification of potential inhibitors of the NQR, and rapid characterization of NQR variants obtained by site-specific mutagenesis.
- Published
- 2017
22. Anoxic cell rupture of Prevotella bryantii by high-pressure homogenization protects the Na
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Lena, Schleicher, Günter, Fritz, Jana, Seifert, and Julia, Steuber
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Short Communication ,Na+-translocating NADH:quinone oxidoreductase (NQR) ,Prevotella ,Superoxide ,NAD ,Industrial Microbiology ,Oxidative Stress ,Bacterial Proteins ,Superoxides ,Cell rupture ,Prevotella bryantii ,Pressure ,EmulsiFlex-C3 homogenizer ,Quinone Reductases ,Oxidation-Reduction - Abstract
Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L–1 culture and a NADH oxidation activity of 0.8 µmol min−1 mg−1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii. Electronic supplementary material The online version of this article (10.1007/s00203-019-01805-x) contains supplementary material, which is available to authorized users.
- Published
- 2019
23. Respiratory Membrane Protein Complexes Convert Chemical Energy
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Valentin, Muras, Charlotte, Toulouse, Günter, Fritz, and Julia, Steuber
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Electron Transport ,Bacteria ,Cell Membrane ,Membrane Proteins ,Energy Metabolism ,Archaea - Abstract
The invention of a biological membrane which is used as energy storage system to drive the metabolism of a primordial, unicellular organism represents a key event in the evolution of life. The innovative, underlying principle of this key event is respiration. In respiration, a lipid bilayer with insulating properties is chosen as the site for catalysis of an exergonic redox reaction converting substrates offered from the environment, using the liberated Gibbs free energy (ΔG) for the build-up of an electrochemical H
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- 2019
24. Respiratory Membrane Protein Complexes Convert Chemical Energy
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Julia Steuber, Valentin Muras, Charlotte Toulouse, and Günter Fritz
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Exergonic reaction ,0303 health sciences ,biology ,030306 microbiology ,Chemiosmosis ,Chemistry ,Respiratory chain ,biology.organism_classification ,Unicellular organism ,Redox ,03 medical and health sciences ,Electron transfer ,Biophysics ,Lipid bilayer ,Electrochemical gradient ,030304 developmental biology - Abstract
The invention of a biological membrane which is used as energy storage system to drive the metabolism of a primordial, unicellular organism represents a key event in the evolution of life. The innovative, underlying principle of this key event is respiration. In respiration, a lipid bilayer with insulating properties is chosen as the site for catalysis of an exergonic redox reaction converting substrates offered from the environment, using the liberated Gibbs free energy (ΔG) for the build-up of an electrochemical H+ (proton motive force, PMF) or Na+ gradient (sodium motive force, SMF) across the lipid bilayer. Very frequently , several redox reactions are performed in a consecutive manner, with the first reaction delivering a product which is used as substrate for the second redox reaction, resulting in a respiratory chain. From today's perspective, the (mostly) unicellular bacteria and archaea seem to be much simpler and less evolved when compared to multicellular eukaryotes. However, they are overwhelmingly complex with regard to the various respiratory chains which permit survival in very different habitats of our planet, utilizing a plethora of substances to drive metabolism. This includes nitrogen, sulfur and carbon compounds which are oxidized or reduced by specialized, respiratory enzymes of bacteria and archaea which lie at the heart of the geochemical N, S and C-cycles. This chapter gives an overview of general principles of microbial respiration considering thermodynamic aspects, chemical reactions and kinetic restraints. The respiratory chains of Escherichia coli and Vibrio cholerae are discussed as models for PMF- versus SMF-generating processes, respectively. We introduce main redox cofactors of microbial respiratory enzymes, and the concept of intra-and interelectron transfer. Since oxygen is an electron acceptor used by many respiratory chains, the formation and removal of toxic oxygen radicals is described. Promising directions of future research are respiratory enzymes as novel bacterial targets, and biotechnological applications relying on respiratory complexes.
- Published
- 2019
25. Receptor for advanced glycation end products : a key molecule in the genesis of chronic kidney disease vascular calcification and a potential modulator of sodium phosphate co-transporter PIT-1 expression
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Günter Fritz, Manon Doué, Fatouma Touré, Karim Belmokhtar, Pascal Maurice, Philippe Rieu, Jeremy Ortillon, Philippe Gillery, Ziad A. Massy, Stéphane Jaisson, Camille Boulagnon Rombi, Ann Marie Schmidt, Matrice extracellulaire et dynamique cellulaire - UMR 7369 (MEDyC), Université de Reims Champagne-Ardenne (URCA)-SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en épidémiologie et santé des populations (CESP), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Laboratoire de Signalisation et Récepteurs Matriciels (SiRMa), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Centre National de la Recherche Scientifique (CNRS)-Université de Reims Champagne-Ardenne (URCA)-SFR CAP Santé (Champagne-Ardenne Picardie Santé), University of Fribourg, Université de Reims Champagne-Ardenne (URCA), New York University [New York] (NYU), NYU System (NYU), SFR CAP Santé (Champagne-Ardenne Picardie Santé), Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Université de Picardie Jules Verne (UPJV)-Université de Reims Champagne-Ardenne (URCA)-Centre National de la Recherche Scientifique (CNRS), Mécanismes physiologiques et conséquences des calcifications cardiovasculaires: rôle des remodelages cardiovasculaires et osseux, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Picardie Jules Verne (UPJV)
- Subjects
0301 basic medicine ,Apolipoprotein E ,Male ,medicine.medical_specialty ,Vascular smooth muscle ,mice ,Mice, Knockout, ApoE ,Receptor for Advanced Glycation End Products ,Core Binding Factor Alpha 1 Subunit ,030204 cardiovascular system & hematology ,urologic and male genital diseases ,[SDV.MHEP.UN]Life Sciences [q-bio]/Human health and pathology/Urology and Nephrology ,03 medical and health sciences ,0302 clinical medicine ,Glycation ,In vivo ,Internal medicine ,medicine ,Animals ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Renal Insufficiency, Chronic ,Receptor ,Cells, Cultured ,chemistry.chemical_classification ,Transplantation ,Reactive oxygen species ,Symporters ,business.industry ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,medicine.disease ,RAGE ,Mice, Inbred C57BL ,030104 developmental biology ,Endocrinology ,chemistry ,Nephrology ,vascular calcification ,business ,Reactive Oxygen Species ,Transcription Factor Pit-1 ,sodium–phosphate co-transporter ,Calcification ,Kidney disease ,uraemic toxins - Abstract
BackgroundChronic kidney disease (CKD) is associated with increased cardiovascular mortality, frequent vascular calcification (VC) and accumulation of uraemic toxins. Advanced glycation end products and S100 proteins interact with the receptor for advanced glycation end products (RAGE). In the present work, we aimed to investigate the role(s) of RAGE in the CKD–VC process.MethodsApoe−/− or Apoe−/−Ager (RAGE)−/− male mice were assigned to CKD or sham-operated groups. A high-phosphate diet was given to a subgroup of Apoe−/−and Apoe−/−Ager−/− CKD mice. Primary cultures of Ager+/+ and Ager−/− vascular smooth muscle cells (VSMCs) were established and stimulated with either vehicle, inorganic phosphate (Pi) or RAGE ligands (S100A12; 20 µM).ResultsAfter 12 weeks of CKD we observed a significant increase in RAGE ligand (AGE and S100 proteins) concentrations in the serum of CKD Apoe−/− mice. Ager messenger RNA (mRNA) levels were 4-fold higher in CKD vessels of Apoe−/− mice. CKD Apoe−/− but not CKD Apoe−/− or Ager−/− mice displayed a marked increase in the VC surface area. Similar trends were found in the high-phosphate diet condition. mRNA levels of Runx2 significantly increased in the Apoe−/− CKD group. In vitro, stimulation of Ager+/+VSMCs with Pi or S100A12 induced mineralization and osteoblast transformation, and this was inhibited by phosphonoformic acid (Pi co-transporters inhibitor) and Ager deletion. In vivo and in vitro RAGE was necessary for regulation of the expression of Pit-1, at least in part through production of reactive oxygen species.ConclusionRAGE, through the modulation of Pit-1 expression, is a key molecule in the genesis of VC.
- Published
- 2019
26. USP18 – a multifunctional component in the interferon response
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Klaus-Peter Knobeloch, Anja Basters, and Günter Fritz
- Subjects
0301 basic medicine ,ISG15 ,Proteases ,Protein Conformation ,medicine.medical_treatment ,Biophysics ,Review Article ,Biochemistry ,Deubiquitinating enzyme ,03 medical and health sciences ,Ubiquitin ,Interferon ,Endopeptidases ,medicine ,Humans ,Review Articles ,Ubiquitins ,Molecular Biology ,Gene ,chemistry.chemical_classification ,Isopeptide bond ,Protease ,biology ,protease ,ubiquitin like modifier proteins ,Cell Biology ,interferons ,USP18 ,030104 developmental biology ,chemistry ,Interferon Type I ,Proteolysis ,biology.protein ,Cytokines ,Ubiquitin Thiolesterase ,Peptide Hydrolases ,Signal Transduction ,medicine.drug - Abstract
Ubiquitin-specific proteases (USPs) represent the largest family of deubiquitinating enzymes (DUB). These proteases cleave the isopeptide bond between ubiquitin and a lysine residue of a ubiquitin-modified protein. USP18 is a special member of the USP family as it only deconjugates the ubiquitin-like protein ISG15 (interferon-stimulated gene (ISG) 15) from target proteins but is not active towards ubiquitin. Independent of its protease activity, USP18 functions as a major negative regulator of the type I interferon response showing that USP18 is – at least – a bifunctional protein. In this review, we summarise our current knowledge of protease-dependent and -independent functions of USP18 and discuss the structural basis of its dual activity.
- Published
- 2018
27. The neuronal S100B protein is a calcium-tuned suppressor of amyloid- aggregation
- Author
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Katrin Kierdorf, Bernd Reif, Vanessa K. Morris, Joana S. Cristóvão, Tobias Madl, Sónia S. Leal, Christoph Göbl, Mobina Alemi, Cláudio M. Gomes, Isabel Cardoso, Günter Fritz, Rodrigo David, Javier Martínez, Hugo M. Botelho, and Instituto de Investigação e Inovação em Saúde
- Subjects
0301 basic medicine ,Amyloid beta-Peptides / metabolismo ,Models, Molecular ,Amyloid ,Neurons / metabolismo ,S100 Calcium Binding Protein beta Subunit / metabolism ,Protein Conformation ,Plasma protein binding ,S100 Calcium Binding Protein beta Subunit ,Protein aggregation ,Fibril ,Protein Aggregation, Pathological ,Biochemistry ,Models, Biological ,S100 Calcium Binding Protein beta Subunit / chemistry ,03 medical and health sciences ,Protein Aggregates ,Structure-Activity Relationship ,0302 clinical medicine ,Protein structure ,medicine ,Humans ,Protein Interaction Domains and Motifs ,Senile plaques ,Neuroinflammation ,Research Articles ,Neurons ,Multidisciplinary ,Amyloid beta-Peptides ,Amyloid beta-Peptides / chemistry ,Chemistry ,Protein Aggregation, Pathological / metabolismo ,Neurodegeneration ,SciAdv r-articles ,medicine.disease ,3. Good health ,Transport protein ,Calcium / metabolismo ,Protein Transport ,030104 developmental biology ,Amyloid / metabolismo ,Biophysics ,Calcium ,Protein Multimerization ,030217 neurology & neurosurgery ,Research Article ,Neuroscience ,Protein Binding - Abstract
A novel role for S100B, a recognized brain distress marker, as a chaperone-like suppressor of Aβ42 aggregation and toxicity., Amyloid-β (Aβ) aggregation and neuroinflammation are consistent features in Alzheimer’s disease (AD) and strong candidates for the initiation of neurodegeneration. S100B is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is found associated with senile plaques. This recognized biomarker for brain distress may, thus, play roles in amyloid aggregation which remain to be determined. We report a novel role for the neuronal S100B protein as suppressor of Aβ42 aggregation and toxicity. We determined the structural details of the interaction between monomeric Aβ42 and S100B, which is favored by calcium binding to S100B, possibly involving conformational switching of disordered Aβ42 into an α-helical conformer, which locks aggregation. From nuclear magnetic resonance experiments, we show that this dynamic interaction occurs at a promiscuous peptide-binding region within the interfacial cleft of the S100B homodimer. This physical interaction is coupled to a functional role in the inhibition of Aβ42 aggregation and toxicity and is tuned by calcium binding to S100B. S100B delays the onset of Aβ42 aggregation by interacting with Aβ42 monomers inhibiting primary nucleation, and the calcium-bound state substantially affects secondary nucleation by inhibiting fibril surface–catalyzed reactions through S100B binding to growing Aβ42 oligomers and fibrils. S100B protects cells from Aβ42-mediated toxicity, rescuing cell viability and decreasing apoptosis induced by Aβ42 in cell cultures. Together, our findings suggest that molecular targeting of S100B could be translated into development of novel approaches to ameliorate AD neurodegeneration.
- Published
- 2018
28. High Affinity Binding of Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) to Lu/BCAM Adhesion Glycoprotein
- Author
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Gudula Schmidt, Sylvie Cochet, Wassim El Nemer, Franziska Reppin, and Günter Fritz
- Subjects
0301 basic medicine ,receptor ,Health, Toxicology and Mutagenesis ,Bacterial Toxins ,lcsh:Medicine ,Lu/BCAM ,CNF ,laminin ,toxin ,immunoglobulin-like domain ,sickle cell disease ,Toxicology ,medicine.disease_cause ,Endocytosis ,Article ,Virulence factor ,Cell Line ,03 medical and health sciences ,BCAM ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Receptor ,Escherichia coli ,chemistry.chemical_classification ,Membrane Glycoproteins ,030102 biochemistry & molecular biology ,Chemistry ,Toxin ,Escherichia coli Proteins ,lcsh:R ,Adhesion ,Lutheran Blood-Group System ,Molecular biology ,030104 developmental biology ,Glycoprotein ,Protein Binding - Abstract
The protein toxin Cytotoxic Necrotizing Factor 1 (CNF1) is a major virulence factor of pathogenic Escherichia coli strains. It belongs to a family of single chain AB-toxins, which enter mammalian cells by receptor-mediated endocytosis. Recently, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as a cellular receptor for CNF1. Here, we identified the Ig-like domain 2 of Lu/BCAM as main interaction site of the toxin by direct protein-protein interaction and competition studies. Using surface plasmon resonance, we showed a high affinity CNF-Lu/BCAM interaction with a KD of 2.8 nM. Furthermore, we performed small-angle X-ray scattering to define the molecular envelope of the Lu/BCAM-CNF1 complex, suggesting a 6:1 ratio of Lu/BCAM to CNF1 in the receptor-toxin complex. This study leads to a deeper understanding of the interaction between CNF1 and Lu/BCAM, and presents novel opportunities for the development of future anti-toxin strategies.
- Published
- 2017
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29. Identification of a novel mutation inRIPK4in a kindred with phenotypic features of Bartsocas-Papas and CHAND syndromes
- Author
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Sabrina Wolf, Benjamin Gollasch, Fitnat Buket Basmanav, Holger Thiele, Arti Nanda, Maria Wehner, Peter Nürnberg, Günter Fritz, Janine Altmüller, Hassnaa Mahmoudi, Jorge Frank, and Regina C. Betz
- Subjects
Male ,Models, Molecular ,Ectodermal dysplasia ,Cleft Lip ,DNA Mutational Analysis ,Molecular Sequence Data ,Nails, Malformed ,Protein Serine-Threonine Kinases ,Biology ,medicine.disease_cause ,Evolution, Molecular ,TP63 ,Genetics ,medicine ,Humans ,Missense mutation ,Exome ,Knee ,Amino Acid Sequence ,Eye Abnormalities ,Allele ,Conserved Sequence ,Genetics (clinical) ,Exome sequencing ,Mutation ,Base Sequence ,Tumor Suppressor Proteins ,Syndrome ,Disease gene identification ,medicine.disease ,Phenotype ,Pedigree ,Cleft Palate ,Structural Homology, Protein ,Eyelid Diseases ,Female ,Syndactyly ,Hair Diseases ,Transcription Factors - Abstract
Three children from an expanded consanguineous Kuwaiti kindred presented with ankyloblepharon, sparse and curly hair, and hypoplastic nails, suggestive of CHAND syndrome (OMIM 214350) that belongs to the heterogeneous spectrum of ectodermal dysplasias. After exclusion of pathogenic mutations in TP63 we performed homozygosity mapping, followed by exome sequencing of one affected individual. We initially identified three homozygous mutations in the linked region, located in PWP2, MX2 and RIPK4. Recently, mutations in RIPK4 have been reported in Bartsocas-Papas syndrome (OMIM 263650) that shows overlapping clinical symptoms with the phenotype observed in the affected individuals studied here. Subsequent analysis of affected and non-affected family members showed that mutation c.850G>A (p.Glu284Lys) in RIPK4 was in complete segregation with the disease phenotype, in accordance with an autosomal recessive inheritance pattern, thus supporting pathogenicity of this variant. Interestingly, however, our patients did not have cleft lip/palate, a common feature encountered in Bartsocas-Papas syndrome. Whereas in Bartsocas-Papas syndromes missense mutations are usually located within the serin/threonin kinase of RIPK4, the mutation detected in our family resides just outside of the kinase domain, which could explain the milder phenotype. Our data raise the question if CHAND syndrome indeed is a distinct entity. Alternatively, CHAND and Bartsocas-Papas syndrome might be allelic disorders or RIPK4 mutations could confer varying degrees of phenotypic severity, depending on their localization within or outside functionally important domains. Our findings indicate that making an accurate diagnosis based only on the prevailing clinical symptoms is challenging.
- Published
- 2015
30. Pathogenicity of POFUT1 in Dowling-Degos Disease: Additional Mutations and Clinical Overlap with Reticulate Acropigmentation of Kitamura
- Author
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Roland Kruse, Regina C. Betz, Jorge Frank, Sandra Hanneken, Arno Rütten, Susanne Pulimood, J. Fischer, Gilles G. Lestringant, Sabrina Wolf, Sumita Danda, Holger Thiele, Divya Pachat, Maria Wehner, Günter Fritz, F. Buket Basmanav, Janine Altmüller, Sabine Hoffjan, and Anette Bygum
- Subjects
Adult ,Dowling-Degos Disease ,Skin Diseases, Papulosquamous ,Reticulate acropigmentation of Kitamura ,Dermatology ,Biology ,Biochemistry ,030207 dermatology & venereal diseases ,03 medical and health sciences ,0302 clinical medicine ,Hyperpigmentation ,Humans ,Molecular Biology ,030304 developmental biology ,Genetics ,Skin Diseases, Papulosquamous/genetics ,0303 health sciences ,Skin Diseases, Genetic ,Fucosyltransferases/genetics ,Cell Biology ,Middle Aged ,Fucosyltransferases ,Pathogenicity ,3. Good health ,Hyperpigmentation/genetics ,Skin Diseases, Genetic/genetics ,Mutation ,Mutation (genetic algorithm) - Published
- 2015
31. A miniaturized assay for kinetic characterization of the Na
- Author
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Valentin, Muras, Björn, Claussen, Hamid, Nasiri, Günter, Fritz, and Julia, Steuber
- Subjects
Kinetics ,Electron Transport Complex I ,Miniaturization ,Bacterial Proteins ,Sodium ,Mutagenesis, Site-Directed ,Quinones ,Biological Transport ,NAD ,Oxidation-Reduction ,Vibrio cholerae ,Enzyme Assays - Abstract
We demonstrate the miniaturization of an enzymatic assay for the determination of NADH oxidation and quinone reduction by the Na
- Published
- 2017
32. How USP18 deals with ISG15-modified proteins: structural basis for the specificity of the protease
- Author
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Anja Basters, Klaus-Peter Knobeloch, and Günter Fritz
- Subjects
0301 basic medicine ,Models, Molecular ,medicine.medical_treatment ,Proteolysis ,Protein domain ,Plasma protein binding ,Receptor, Interferon alpha-beta ,Biology ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Protein Domains ,Interferon ,Endopeptidases ,medicine ,Humans ,Molecular Biology ,Ubiquitins ,Protease ,medicine.diagnostic_test ,Cell Biology ,ISG15 ,Cysteine protease ,030104 developmental biology ,Cytokines ,Signal transduction ,Ubiquitin Thiolesterase ,medicine.drug ,Protein Binding ,Signal Transduction - Abstract
The ubiquitin-specific protease 18 (USP18) has two major functions: (a) it is a highly specific protease that cleaves the ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) from proteins, and (b) independent from its enzymatic activity USP18 interacts with the type I interferon receptor and shuts off downstream signaling. The structures of USP18 and a USP18-ISG15 complex revealed the molecular basis of the unique specificity of the protease and might shed some light into its interaction with the interferon receptor.
- Published
- 2017
33. Structural basis of the specificity of USP18 toward ISG15
- Author
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Annika Röcker, Huib Ovaa, Sandra Hess, Katharina F. Witting, Paul P. Geurink, Anja Basters, Roya Tadayon, Günter Fritz, Paola Storici, Klaus-Peter Knobeloch, and Marta S. Semrau
- Subjects
0301 basic medicine ,Models, Molecular ,Proteases ,Protein Conformation ,medicine.medical_treatment ,Protein domain ,Plasma protein binding ,Bioinformatics ,Crystallography, X-Ray ,Article ,Substrate Specificity ,03 medical and health sciences ,Structure-Activity Relationship ,Protein structure ,Ubiquitin ,Protein Domains ,Structural Biology ,Hydrolase ,Endopeptidases ,medicine ,Animals ,Humans ,Molecular Biology ,Ubiquitins ,Zebrafish ,Protease ,030102 biochemistry & molecular biology ,biology ,Zebrafish Proteins ,ISG15 ,Cell biology ,Kinetics ,030104 developmental biology ,HEK293 Cells ,biology.protein ,Cytokines ,Mutant Proteins ,Crystallization ,Hydrophobic and Hydrophilic Interactions ,Ubiquitin Thiolesterase ,Protein Binding - Abstract
Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin proteases and Ubl proteases, collectively termed DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity toward ubiquitin but specifically deconjugates the interferon-induced Ubl ISG15. To identify the molecular determinants of this specificity, we solved the crystal structures of mouse USP18 alone and in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation, thus revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15, thus providing evidence that USP18's ISG15 specificity is mediated by a small interaction interface. Our results may provide a structural basis for the development of new drugs modulating ISG15 linkage.
- Published
- 2017
34. Crystallization and preliminary analysis of the NqrA and NqrC subunits of the Na+-translocating NADH:ubiquinone oxidoreductase fromVibrio cholerae
- Author
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Günter Fritz, Marco S. Casutt, Georg Vohl, Julia Steuber, Heiko M. Möller, Kay Diederichs, Björn Claussen, Ruslan Nedielkov, and Thomas Vorburger
- Subjects
Flavin Mononucleotide ,Biophysics ,Gene Expression ,Flavin mononucleotide ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Bacterial Proteins ,Structural Biology ,Oxidoreductase ,ddc:570 ,Escherichia coli ,Genetics ,medicine ,Molecular replacement ,Cloning, Molecular ,Quinone Reductases ,Vibrio cholerae ,chemistry.chemical_classification ,Na+-translocating NQR ,covalently bound FMN ,Membrane Proteins ,Condensed Matter Physics ,Recombinant Proteins ,Protein Structure, Tertiary ,Protein Subunits ,Transmembrane domain ,chemistry ,Crystallization Communications ,Structural Homology, Protein ,Membrane protein complex ,Parabacteroides distasonis ,Crystallization - Abstract
The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) fromVibrio choleraeis a membrane protein complex consisting of six different subunits NqrA–NqrF. The major domains of the NqrA and NqrC subunits were heterologously expressed inEscherichia coliand crystallized. The structure of NqrA1–377was solved in space groupsC2221andP21by SAD phasing and molecular replacement at 1.9 and 2.1 Å resolution, respectively. NqrC devoid of the transmembrane helix was co-expressed with ApbE to insert the flavin mononucleotide group covalently attached to Thr225. The structure was determined by molecular replacement using apo-NqrC ofParabacteroides distasonisas search model at 1.8 Å resolution.
- Published
- 2014
35. Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15
- Author
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Anja Basters, Lars Ketscher, Eberhard Krause, Huib Ovaa, Paul P. Geurink, Farid El Oualid, Günter Fritz, Marco S. Casutt, and Klaus-Peter Knobeloch
- Subjects
medicine.medical_treatment ,Spodoptera ,Biochemistry ,Substrate Specificity ,Mice ,Ubiquitin ,Sf9 Cells ,medicine ,Animals ,Enzyme kinetics ,Ubiquitins ,Molecular Biology ,chemistry.chemical_classification ,Isopeptide bond ,Protease ,biology ,Effector ,Microscale thermophoresis ,Cell Biology ,ISG15 ,Ubiquitin ligase ,chemistry ,biology.protein ,Cytokines ,Ubiquitin Thiolesterase ,Protein Binding - Abstract
Protein modification by interferon-stimulated gene 15 (ISG15), an ubiquitin-like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin-specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high-yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3 ± 0.2 μm. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6 ± 0.2 μm and a kcat of 0.23 ± 0.004 s−1, respectively, at pH 7.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross-reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15. Structured digital abstract ●ISG15 and USP18 bind by microscale thermophoresis (View interaction) ●USP18 cleaves ISG15 by enzymatic study (View interaction)
- Published
- 2014
36. Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease
- Author
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Anette Bygum, Regina C. Betz, Maria Wehner, Ana-Maria Oprisoreanu, Janine Altmüller, Sandra Hanneken, Holger Thiele, Laila El Shabrawi-Caelen, F. Buket Basmanav, Leopold Größer, Sabrina Wolf, Christina Fagerberg, Günter Fritz, Arno Rütten, Peter Nürnberg, Laurent Parmentier, Susanne Schoch, Jörg Wenzel, Sandra M. Pasternack, Christian Hafner, and Roland Kruse
- Subjects
Adult ,Keratinocytes ,Male ,Heterozygote ,Adolescent ,Protein Conformation ,Sequence analysis ,Skin Diseases, Papulosquamous ,Biology ,medicine.disease_cause ,Young Adult ,Protein structure ,Hyperpigmentation ,Report ,Genetics ,medicine ,Humans ,Genetics(clinical) ,Exome ,Gene ,Genetics (clinical) ,Exome sequencing ,Skin ,Mutation ,Genodermatosis ,Skin Diseases, Genetic ,Heterozygote advantage ,Sequence Analysis, DNA ,Middle Aged ,medicine.disease ,Molecular biology ,Pedigree ,Glucosyltransferases ,Female ,Genome-Wide Association Study - Abstract
Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4(∗)), c.652C>T (p.Arg218(∗)), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218(∗)) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology.
- Published
- 2014
37. S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation
- Author
-
Günter Fritz, Ludmilla A. Morozova-Roche, Kiran Yanamandra, Sónia S. Leal, Hugo M. Botelho, Cláudio M. Gomes, Isabel Cardoso, and Repositório Científico do Instituto Politécnico do Porto
- Subjects
Amyloid ,Kinetics ,SOD1 ,Biophysics ,chemistry.chemical_element ,Cell Cycle Proteins ,Protein aggregation ,Calcium ,Fibril ,Biochemistry ,Protein Structure, Secondary ,S100 Calcium Binding Protein A6 ,Aggregation ,03 medical and health sciences ,Superoxide Dismutase-1 ,0302 clinical medicine ,Cell Line, Tumor ,Spectroscopy, Fourier Transform Infrared ,Humans ,Benzothiazoles ,SOD ,Protein Structure, Quaternary ,Cytoskeleton ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Superoxide Dismutase ,Amyotrophic Lateral Sclerosis ,S100 Proteins ,Cell Biology ,Protein Aggregation ,Receptor–ligand kinetics ,Thiazoles ,chemistry ,Protein Structure and Folding ,030217 neurology & neurosurgery - Abstract
S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H(I) and H(IV). Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca(2+) exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca(2+) promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca(2+) rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca(2+). Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.
- Published
- 2012
38. Strong pH dependence of coupling efficiency of the Na+ - translocating NADH:quinone oxidoreductase (Na+-NQR) of Vibrio cholerae
- Author
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Julia Steuber, Günter Fritz, Valentin Muras, Björn Claussen, and Charlotte Toulouse
- Subjects
0301 basic medicine ,Models, Molecular ,Protein Conformation ,Sodium ,Clinical Biochemistry ,Respiratory chain ,chemistry.chemical_element ,Lithium ,Photochemistry ,medicine.disease_cause ,Quinone oxidoreductase ,Biochemistry ,Ion ,Membrane Potentials ,Electron Transport ,03 medical and health sciences ,Electron transfer ,medicine ,NADH, NADPH Oxidoreductases ,Molecular Biology ,Vibrio cholerae ,030102 biochemistry & molecular biology ,Chemistry ,Hydrogen-Ion Concentration ,Coupling (electronics) ,030104 developmental biology ,Membrane ,Liposomes - Abstract
The Na+-translocating NADH:quinone oxidoreductase (NQR) is the entry site for electrons into the respiratory chain of Vibrio cholerae, the causative agent of cholera disease. NQR couples the electron transfer from NADH to ubiquinone to the translocation of sodium ions across the membrane. We investigated the pH dependence of electron transfer and generation of a transmembrane voltage (ΔΨ) by NQR reconstituted in liposomes with Na+ or Li+ as coupling cation. ΔΨ formation was followed with the voltage-sensitive dye oxonol. With Na+, ΔΨ was barely influenced by pH (6.5–8.5), while Q reduction activity exhibited a maximum at pH 7.5–8.0. With Li+, ΔΨ was generally lower, and the pH profile of electron transfer activity did not reveal a pronounced maximum. We conclude that the coupling efficiency of NQR is influenced by the nature of the transported cation, and by the concentration of protons. The 3D structure of NQR reveals a transmembrane channel in subunit NqrB. It is proposed that partial uncoupling of the NQR observed with the smaller Li+, or with Na+ at pH 7.5–8.0, is caused by the backflow of the coupling cation through the channel in NqrB.
- Published
- 2016
39. Sodium as Coupling Cation in Respiratory Energy Conversion
- Author
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Günter, Fritz and Julia, Steuber
- Subjects
Electron Transport ,Models, Molecular ,Oxygen Consumption ,Gene Expression Regulation ,Protein Conformation ,Cations ,Sodium ,Animals ,Quinone Reductases ,Sodium-Potassium-Exchanging ATPase ,Energy Metabolism ,Oxidation-Reduction ,Membrane Potentials - Abstract
Among the alkali cations, Na(+) has an extraordinary role in living cells since it is used to charge the battery of life. To this end, sophisticated protein complexes in biological membranes convert chemical energy obtained from oxidation of NADH, or hydrolysis of ATP, into an electrochemical gradient of sodium ions. Cells use this so-called sodium-motive force stored in energy-converting membranes for important processes like uptake of nutrients, motility, or expulsion of toxic compounds. The Na(+) pumps act in concert with other enzymes embedded in the lipid membrane, and together they form the respiratory chain which achieves the oxidation of NADH derived from nutrients under formation of an electrochemical sodium (or proton) gradient. We explain why Na(+) pumps are important model systems for the homologous, proton-translocating complexes, and hope to convince the reader that studying the Na(+)-translocating ATP synthase from the unimpressive bacterium Ilyobacter tartaricus had a big impact on our understanding of energy conversion by human ATP synthase. The Na(+)-translocating systems described here are either driven by the oxidation of NADH, the carrier of redox equivalents of cells, or by the hydrolysis of adenosine 5'-triphosphate, the universal high-energy compound of cells. The electrochemical energy provided by these respiratory Na(+) pumps, the NADH dehydrogenase or the ATPase, drives other Na(+) transport systems like the bacterial flagellum discussed in the last part of this chapter. The flagellar motor does not represent a Na(+) pump, but like ATPase, it operates by a rotational mechanism. By comparing these two Na(+) -translocating, rotary machines, we obtain new insight into the possible mechanisms of Na(+) transport through the stator proteins of the flagellar motor. Na(+) pumps are widespread in pathogenic bacteria where they play an important role in metabolism, making them novel targets for antibiotics.
- Published
- 2016
40. The Mouse-Specific Splice Variant mRAGE_v4 Encodes a Membrane-Bound RAGE That Is Resistant to Shedding and Does Not Contribute to the Production of Soluble RAGE
- Author
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Matteo Bertolotti, Angela Raucci, Günter Fritz, Marco Bianchi, Jaron Liu, Elena Gatti, Stefania Di Maggio, Di Maggio, S, Gatti, E, Liu, J, Bertolotti, M, Fritz, G, Bianchi, MARCO EMILIO, and Raucci, A.
- Subjects
0301 basic medicine ,Gene isoform ,Genetics ,Messenger RNA ,Multidisciplinary ,endocrine system diseases ,ADAM10 ,Alternative splicing ,lcsh:R ,nutritional and metabolic diseases ,lcsh:Medicine ,Biology ,Primary transcript ,Transmembrane protein ,Cell biology ,03 medical and health sciences ,Exon ,030104 developmental biology ,cardiovascular system ,lcsh:Q ,cardiovascular diseases ,Receptor ,lcsh:Science ,human activities - Abstract
The receptor for advanced glycation end-products (RAGE) is involved in the onset and progression of several inflammatory diseases. The RAGE primary transcript undergoes numerous alternative splicing (AS) events, some of which are species-specific. Here, we characterize the mouse-specific mRAGE_v4 splice variant, which is conserved in rodents and absent in primates. mRAGE_v4 derives from exon 9 skipping and encodes a receptor (M-RAGE) that lacks 9 amino acids between the transmembrane and the immunoglobulin (Ig) domains. RNA-Seq data confirm that in mouse lung mRAGE_v4 is the most abundant RAGE mRNA isoform after mRAGE, which codes for full-length RAGE (FL-RAGE), while in heart all RAGE variants are almost undetectable. The proteins M-RAGE and FL-RAGE are roughly equally abundant in mouse lung. Contrary to FL-RAGE, M-RAGE is extremely resistant to shedding because it lacks the peptide motif recognized by both ADAM10 and MMP9, and does not contribute significantly to soluble cRAGE formation. Thus, a cassette exon in RAGE corresponds to a specific function of the RAGE protein-the ability to be shed. Given the differences in RAGE AS variants between rodents and humans, caution is due in the interpretation of results obtained in mouse models of RAGE-dependent human pathologies.
- Published
- 2016
41. Sodium as Coupling Cation in Respiratory Energy Conversion
- Author
-
Günter Fritz and Julia Steuber
- Subjects
0301 basic medicine ,biology ,ATP synthase ,Chemistry ,ATPase ,Sodium-Potassium-Exchanging ATPase ,Inorganic chemistry ,Respiratory chain ,Electron transport chain ,03 medical and health sciences ,030104 developmental biology ,ATP hydrolysis ,biology.protein ,Biophysics ,Lipid bilayer ,Electrochemical gradient - Abstract
Among the alkali cations, Na+ has an extraordinary role in living cells since it is used to charge the battery of life. To this end, sophisticated protein complexes in biological membranes convert chemical energy obtained from oxidation of NADH, or hydrolysis of ATP, into an electrochemical gradient of sodium ions. Cells use this so-called sodium-motive force stored in energy-converting membranes for important processes like uptake of nutrients, motility, or expulsion of toxic compounds. The Na+ pumps act in concert with other enzymes embedded in the lipid membrane, and together they form the respiratory chain which achieves the oxidation of NADH derived from nutrients under formation of an electrochemical sodium (or proton) gradient. We explain why Na+ pumps are important model systems for the homologous, proton-translocating complexes, and hope to convince the reader that studying the Na+-translocating ATP synthase from the unimpressive bacterium Ilyobacter tartaricus had a big impact on our understanding of energy conversion by human ATP synthase. The Na+-translocating systems described here are either driven by the oxidation of NADH, the carrier of redox equivalents of cells, or by the hydrolysis of adenosine 5′-triphosphate, the universal high-energy compound of cells. The electrochemical energy provided by these respiratory Na+ pumps, the NADH dehydrogenase or the ATPase, drives other Na+ transport systems like the bacterial flagellum discussed in the last part of this chapter. The flagellar motor does not represent a Na+ pump, but like ATPase, it operates by a rotational mechanism. By comparing these two Na+ -translocating, rotary machines, we obtain new insight into the possible mechanisms of Na+ transport through the stator proteins of the flagellar motor. Na+ pumps are widespread in pathogenic bacteria where they play an important role in metabolism, making them novel targets for antibiotics.
- Published
- 2016
42. The Catalytic Redox Activity of Prion Protein-CuII is Controlled by Metal Exchange with the ZnII-Thiolate Clusters of Zn7Metallothionein-3
- Author
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Andrea Crameri, Günter Fritz, Milan Vašák, Gabriele Meloni, Peter M. H. Kroneck, David R. Brown, and Paul Davies
- Subjects
Prions ,Stereochemistry ,animal diseases ,Inorganic chemistry ,chemistry.chemical_element ,Nerve Tissue Proteins ,Zinc ,Cleavage (embryo) ,Biochemistry ,Redox ,Catalysis ,Metal ,Humans ,Sulfhydryl Compounds ,Prion protein ,Molecular Biology ,chemistry.chemical_classification ,Reactive oxygen species ,Organic Chemistry ,Copper ,Metallothionein 3 ,nervous system diseases ,chemistry ,visual_art ,Biocatalysis ,visual_art.visual_art_medium ,Molecular Medicine ,Oxidation-Reduction - Abstract
Silencing prion: Copper-catalyzed transformations of prion protein (PrP) lead to the production of reactive oxygen species (ROS), PrP oxidation, and cleavage and aggregation in transmissible spongiphorm encephalopathies. Zn(7) MT-3 efficiently targets Cu(II) bound in different coordination modes to PrP-Cu(II) . By an unusual redox-dependent metal-swap reaction, MT-3 modulates the catalytic redox properties of PrP-Cu(II) .
- Published
- 2012
43. Low-resolution structure determination of Na+-translocating NADH:ubiquinone oxidoreductase fromVibrio choleraebyab initiophasing and electron microscopy
- Author
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Julia Steuber, Christiane Schaffitzel, Vladimir Y. Lunin, N.L. Lunina, Marco S. Casutt, Manfred W. Baumstark, Günter Fritz, and Kèvin Knoops
- Subjects
Models, Molecular ,Ab initio ,Biology ,medicine.disease_cause ,Quinone oxidoreductase ,law.invention ,Protein structure ,Structural Biology ,law ,Oxidoreductase ,medicine ,Vibrio cholerae ,chemistry.chemical_classification ,Electron Transport Complex I ,Resolution (electron density) ,Computational Biology ,General Medicine ,Protein Structure, Tertiary ,Microscopy, Electron ,Crystallography ,chemistry ,Structural Homology, Protein ,Biophysics ,Electron microscope - Abstract
A low-resolution structure of the Na(+)-translocating NADH:ubiquinone oxidoreductase from the human pathogen Vibrio cholerae was determined by ab initio phasing and independently confirmed by electron microscopy. This multi-subunit membrane-protein complex (molecular weight 210 kDa) generates an Na(+) gradient that is essential for substrate uptake, motility, pathogenicity and efflux of antibiotics. The obtained 16 Å resolution electron density-map revealed an asymmetric particle with a central region of low electron density and a putative detergent region, and allowed the identification of the transmembrane regions of the complex.
- Published
- 2012
44. HMGB1 conveys immunosuppressive characteristics on regulatory and conventional T cells
- Author
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Astrid M. Westendorf, Sven Brandau, Stephan Lang, Thomas K. Hoffmann, Christoph Bergmann, Patrick J. Schuler, Clarissa A. Wild, Günter Fritz, and Ramin Lotfi
- Subjects
Cell Survival ,T-Lymphocytes ,Receptor for Advanced Glycation End Products ,Immunology ,Medizin ,Fluorescent Antibody Technique ,Apoptosis ,Enzyme-Linked Immunosorbent Assay ,chemical and pharmacologic phenomena ,Cell Separation ,HMGB1 ,T-Lymphocytes, Regulatory ,RAGE (receptor) ,Immune system ,Immune Tolerance ,Humans ,Immunology and Allergy ,IL-2 receptor ,HMGB1 Protein ,Receptors, Immunologic ,Interleukin-7 receptor ,Receptor ,biology ,Pattern recognition receptor ,hemic and immune systems ,General Medicine ,Flow Cytometry ,Cell biology ,Chemotaxis, Leukocyte ,biology.protein ,TLR4 ,Tumor Escape - Abstract
Objective: The high-mobility group box-1 protein (HMGB1) serves as the prototypic damage-associated molecular pattern molecule, interacting with a variety of defined pattern recognition receptors in the microenvironment of damaged or necrotic tissue. As regulatory T cells (Treg) play a crucial role in autoimmune diseases and tumor immune escape, the previously unexamined role of HMGB1 on the function of Treg is of great interest. Methods: Human CD4 1 CD25 1 CD127 2 Treg and CD4 1 CD25 2 CD127 1 conventional T cells (Tcon) were phenotypically analyzed for their constitutive as well as HMGB1-modulated expression of Toll-like receptors (TLR) and the receptor for advanced glycation end products (RAGE). Furthermore, the influence of recombinant and complexed HMGB1 from necrotic cell supernatant on the function of Treg and Tcon was investigated. Results: Treg express significantly higher levels of RAGE on the cell surface than Tcon, while levels of TLR4 are similar. HMGB1 modulates Treg biology by inducing migration and prolonging survival. Furthermore, HMGB1 enhances IL-10 release and Treg suppressive capacity in a RAGE-dependent manner. In addition, HMGB1 directly suppresses IFNg release of Tcon and inhibits their proliferation via TLR4. Conclusion: HMGB1 directly enhances immune inhibitory functions of Treg via RAGE-mediated mechanisms and limits the number and activity of Tcon. HMGB1 effects on Treg may alter immune reactivity in the setting of chronic inflammatory states such as cancer.
- Published
- 2012
45. The structure of Ca2+-loaded S100A2 at 1.3-Å resolution
- Author
-
Michael Koch and Günter Fritz
- Subjects
Conformational change ,EF hand ,Chemistry ,Cell Biology ,computer.file_format ,Protein Data Bank ,Biochemistry ,S100 protein ,Crystallography ,Protein structure ,Intramolecular force ,Lattice protein ,Target protein ,Molecular Biology ,computer - Abstract
S100A2 is an EF-hand calcium ion (Ca2+)-binding protein that activates the tumour suppressor p53. In order to understand the molecular mechanisms underlying the Ca2+-induced activation of S100A2, the structure of Ca2+-bound S100A2 was determined at 1.3 A resolution by X-ray crystallography. The structure was compared with Ca2+-free S100A2 and with other S100 proteins. Binding of Ca2+ to S100A2 induces small structural changes in the N-terminal EF-hand, but a large conformational change in the C-terminal EF-hand, reorienting helix III by approximately 90°. This movement is accompanied by the exposure of a hydrophobic cavity between helix III and helix IV that represents the target protein interaction site. This molecular reorganization is associated with the breaking and new formation of intramolecular hydrophobic contacts. The target binding site exhibits unique features; in particular, the hydrophobic cavity is larger than in other Ca2+-loaded S100 proteins. The structural data underline that the shape and size of the hydrophobic cavity are major determinants for target specificity of S100 proteins and suggest that the binding mode for S100A2 is different from that of other p53-interacting S100 proteins. Database Structural data are available in the Protein Data Bank database under the accession number 4DUQ Structured digital abstract • S100A2 and S100A2 bind by x-ray crystallography (View interaction)
- Published
- 2012
46. MICROBIOLOGY. Sulfate to go
- Author
-
Günter, Fritz and Peter M H, Kroneck
- Subjects
Archaeal Proteins ,Archaeoglobus fulgidus ,Sulfides ,Energy Metabolism ,Sulfur - Published
- 2015
47. Pattern Recognition with a Fibril-Specific Antibody Fragment Reveals the Surface Variability of Natural Amyloid Fibrils
- Author
-
Senthil Kumar, Isabel Morgado, Marcus Fändrich, Barbara Kieninger, Günter Fritz, Peter Hortschansky, Uwe Horn, Christian Haupt, Magdalena Bereza, and Christoph Röcken
- Subjects
Models, Molecular ,Amyloid ,Peptide ,Biology ,Fibril ,Microscopy, Electron, Transmission ,Structural Biology ,mental disorders ,medicine ,Humans ,Immunoglobulin Fragments ,Molecular Biology ,chemistry.chemical_classification ,Histocytochemistry ,Amyloidosis ,Pattern recognition receptor ,P3 peptide ,Brain ,medicine.disease ,Biochemistry of Alzheimer's disease ,chemistry ,Biochemistry ,Biophysics ,Protein folding ,Protein Binding - Abstract
Amyloid immunotherapy has led to the rise of antibodies, which target amyloid fibrils or structural precursors of fibrils, based on their specific conformational properties. Recently, we reported the biotechnological generation of the B10 antibody fragment, which provides conformation-specific binding to amyloid fibrils. B10 strongly interacts with fibrils from Alzheimer's β amyloid (Aβ) peptide, while disaggregated Aβ peptide or Aβ oligomers are not explicitly recognized. B10 also enables poly-amyloid-specific binding and recognizes amyloid fibrils derived from different types of amyloidosis or different polypeptide chains. Based on our current data, however, we find that B10 does not recognize all tested amyloid fibrils and amyloid tissue deposits. It also does not specifically interact with intrinsically unfolded polypeptide chains or globular proteins even if the latter encompass high β-sheet content or β-solenoid domains. By contrast, B10 binds amyloid fibrils from d-amino acid or l-amino acid peptides and non-proteinaceous biopolymers with highly regular and anionic surface properties, such as heparin and DNA. These data establish that B10 binding does not depend on an amyloid-specific or protein-specific backbone structure. Instead, it involves the recognition of a highly regular and anionic surface pattern. This specificity mechanism is conserved in nature and occurs also within a group of natural amyloid receptors from the innate immune system, the pattern recognition receptors. Our data illuminate the structural diversity of naturally occurring amyloid scaffolds and enable the discrimination of distinct fibril populations in vitro and within diseased tissues.
- Published
- 2011
48. Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity
- Author
-
Ludmilla A. Morozova-Roche, Günter Fritz, Hugo M. Botelho, and Cláudio M. Gomes
- Subjects
Programmed cell death ,Protein structure ,Amyloid ,Chemistry ,Calcium-binding protein ,Cell Biology ,Receptor ,Corpora amylacea ,Molecular Biology ,Biochemistry ,Function (biology) ,RAGE (receptor) ,Cell biology - Abstract
The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca(2+) binding through EF-hand motifs and binding of Zn(2+) and Cu(2+) at additional sites, usually at the homodimer interfaces. Ca(2+) binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.
- Published
- 2010
49. Structural Basis for Ligand Recognition and Activation of RAGE
- Author
-
Brian M. Dattilo, Michael Koch, Joachim Diez, Walter J. Chazin, Günter Fritz, André Schiefner, and Seth Chitayat
- Subjects
Models, Molecular ,Protein Folding ,Magnetic Resonance Spectroscopy ,Molecular Sequence Data ,Receptor for Advanced Glycation End Products ,S100 Calcium Binding Protein beta Subunit ,Plasma protein binding ,Crystallography, X-Ray ,Ligands ,HMGB1 ,Article ,Protein Structure, Secondary ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Animals ,Humans ,Amino Acid Sequence ,Nerve Growth Factors ,Receptors, Immunologic ,Binding site ,Receptor ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Sequence Homology, Amino Acid ,biology ,Chemistry ,S100 Proteins ,Hydrogen-Ion Concentration ,S100 protein binding ,Protein Structure, Tertiary ,Kinetics ,Biochemistry ,Ectodomain ,Docking (molecular) ,biology.protein ,Biophysics ,Protein folding ,030217 neurology & neurosurgery ,Protein Binding - Abstract
SummaryThe receptor for advanced glycation end products (RAGE) is a pattern recognition receptor involved in inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling events upon binding of a variety of ligands, such as glycated proteins, amyloid-β, HMGB1, and S100 proteins. The X-ray crystal structure of the VC1 ligand-binding region of the human RAGE ectodomain was determined at 1.85 Å resolution. The VC1 ligand-binding surface was mapped onto the structure from titrations with S100B monitored by heteronuclear NMR spectroscopy. These NMR chemical shift perturbations were used as input for restrained docking calculations to generate a model for the VC1-S100B complex. Together, the arrangement of VC1 molecules in the crystal and complementary biochemical studies suggest a role for self-association in RAGE function. Our results enhance understanding of the functional outcomes of S100 protein binding to RAGE and provide insight into mechanistic models for how the receptor is activated.
- Published
- 2010
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50. Crystallization and calcium/sulfur SAD phasing of the human EF-hand protein S100A2
- Author
-
Joachim Diez, Michael Koch, Günter Fritz, and Armin Wagner
- Subjects
Models, Molecular ,genetic structures ,Biophysics ,chemistry.chemical_element ,Calcium ,Crystallography, X-Ray ,behavioral disciplines and activities ,Biochemistry ,law.invention ,Structural Biology ,law ,Phase (matter) ,mental disorders ,Genetics ,Structural Communications ,Humans ,EF Hand Motifs ,Crystallization ,Chemotactic Factors ,Anomalous scattering ,EF hand ,Chemistry ,S100 Proteins ,Resolution (electron density) ,Condensed Matter Physics ,Sulfur ,Protein Structure, Tertiary ,Crystallography ,Substructure - Abstract
Human S100A2 is an EF-hand protein and acts as a major tumour suppressor, binding and activating p53 in a Ca2+-dependent manner. Ca2+-bound S100A2 was crystallized and its structure was determined based on the anomalous scattering provided by six S atoms from methionine residues and four calcium ions present in the asymmetric unit. Although the diffraction data were recorded at a wavelength of 0.90 Å, which is usually not assumed to be suitable for calcium/sulfur SAD, the anomalous signal was satisfactory. A nine-atom substructure was determined at 1.8 Å resolution usingSHELXD, andSHELXEwas used for density modification and phase extension to 1.3 Å resolution. The electron-density map obtained was well interpretable and could be used for automated model building byARP/wARP.
- Published
- 2010
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