48 results on '"Weiwad M"'
Search Results
2. Rat PP5 co-crystallized with P5SA-2
- Author
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Haslbeck, V., primary, Helmuth, M., additional, Alte, F., additional, Popowicz, G., additional, Schmidt, W., additional, Weiwad, M., additional, Fischer, G., additional, Gemmecker, G., additional, Sattler, M., additional, Striggow, F., additional, Groll, M., additional, and Richter, K., additional
- Published
- 2014
- Full Text
- View/download PDF
3. Solution structure of the PPIase domain of human aryl-hydrocarbon receptor-interacting protein (AIP)
- Author
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Linnert, M., primary, Lin, Y., additional, Manns, A., additional, Haupt, K., additional, Paschke, A., additional, Fischer, G., additional, Weiwad, M., additional, and Luecke, C., additional
- Published
- 2012
- Full Text
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4. Insights into Immunophilin Structure and Function
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Lucke, C., primary and Weiwad, M., additional
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- 2011
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5. Solution structure of the FK506-binding domain of human FKBP38
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Maestre-Martinez, M., primary, Edlich, F., additional, Jarczowski, F., additional, Weiwad, M., additional, Fischer, G., additional, and Luecke, C., additional
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- 2006
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6. Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase
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Weiwad, M., Kullertz, G., Schutkowski, M., and Fischer, G.
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- 2000
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7. Calcineurin inhibitors: status quo and perspectives
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Erdmann Frank and Weiwad Matthias
- Subjects
calcineurin ,drug discovery ,immunosuppression ,inhibitor ,nfat ,Biology (General) ,QH301-705.5 - Abstract
Despite the fact that cyclosporin A (CsA) and tacrolimus (FK506) are very potent drugs in the treatment of serious autoimmune diseases and in the prevention of graft vs. host reactions or tissue rejections after allo- or xenotransplantations, modern transplantation medicine attempts to develop alternative medication regimes without these calcineurin inhibitors. The primary motivation for this endeavor is the high incidence of dramatic side effects upon immunosuppressive therapy. CsA and FK506 target not only the calcineurin/NFAT pathway, but they also bind and inhibit members of distinct peptidyl-prolyl cis/trans isomerase families, which are involved in numerous important signal transduction pathways. Therefore, the development of a potent calcineurin inhibitor that discriminates between calcineurin and other protein phosphatases and peptidyl-prolyl cis/trans isomerases, respectively, should improve the drug safety in clinical use and represent a valuable tool in basic research to investigate calcineurin modulated pathways. This review gives a current overview about novel calcineurin inhibitors, which were identified by screening of compound libraries and in natural materials or were derived from known inhibitors in the past decades. Thereby, we focus on their structure, properties and biological effects.
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- 2011
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8. A chemical compound inhibiting the Aha1-Hsp90 chaperone complex.
- Author
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Stiegler SC, Rübbelke M, Korotkov VS, Weiwad M, John C, Fischer G, Sieber SA, Sattler M, and Buchner J
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- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases metabolism, Chaperonins chemistry, Chaperonins genetics, Chaperonins metabolism, HSP90 Heat-Shock Proteins chemistry, HSP90 Heat-Shock Proteins genetics, HSP90 Heat-Shock Proteins metabolism, Multiprotein Complexes chemistry, Multiprotein Complexes genetics, Multiprotein Complexes metabolism, Nuclear Magnetic Resonance, Biomolecular, Protein Domains, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Adenosine Triphosphatases antagonists & inhibitors, Chaperonins antagonists & inhibitors, Enzyme Inhibitors chemistry, HSP90 Heat-Shock Proteins antagonists & inhibitors, Multiprotein Complexes antagonists & inhibitors, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae Proteins antagonists & inhibitors
- Abstract
The eukaryotic Hsp90 chaperone machinery comprises many co-chaperones and regulates the conformation of hundreds of cytosolic client proteins. Therefore, it is not surprising that the Hsp90 machinery has become an attractive therapeutic target for diseases such as cancer. The compounds used so far to target this machinery affect the entire Hsp90 system. However, it would be desirable to achieve a more selective targeting of Hsp90-co-chaperone complexes. To test this concept, in this-proof-of-principle study, we screened for modulators of the interaction between Hsp90 and its co-chaperone Aha1, which accelerates the ATPase activity of Hsp90. A FRET-based assay that monitored Aha1 binding to Hsp90 enabled identification of several chemical compounds modulating the effect of Aha1 on Hsp90 activity. We found that one of these inhibitors can abrogate the Aha1-induced ATPase stimulation of Hsp90 without significantly affecting Hsp90 ATPase activity in the absence of Aha1. NMR spectroscopy revealed that this inhibitory compound binds the N-terminal domain of Hsp90 close to its ATP-binding site and overlapping with a transient Aha1-interaction site. We also noted that this inhibitor does not dissociate the Aha1-Hsp90 complex but prevents the specific interaction with the N-terminal domain of Hsp90 required for catalysis. In consequence, the inhibitor affected the activation and processing of Hsp90-Aha1-dependent client proteins in vivo We conclude that it is possible to abrogate a specific co-chaperone function of Hsp90 without inhibiting the entire Hsp90 machinery. This concept may also hold true for other co-chaperones of Hsp90., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2017
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9. Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.
- Author
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Seufert F, Kuhn M, Hein M, Weiwad M, Vivoli M, Norville IH, Sarkar-Tyson M, Marshall LE, Schweimer K, Bruhn H, Rösch P, Harmer NJ, Sotriffer CA, and Holzgrabe U
- Subjects
- Dose-Response Relationship, Drug, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors chemistry, Molecular Docking Simulation, Molecular Structure, Peptidylprolyl Isomerase metabolism, Structure-Activity Relationship, Burkholderia pseudomallei enzymology, Drug Design, Enzyme Inhibitors pharmacology, Legionella pneumophila enzymology, Peptidylprolyl Isomerase antagonists & inhibitors
- Abstract
The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
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- 2016
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10. Inhibitors of macrophage infectivity potentiator-like PPIases affect neisserial and chlamydial pathogenicity.
- Author
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Reimer A, Seufert F, Weiwad M, Ebert J, Bzdyl NM, Kahler CM, Sarkar-Tyson M, Holzgrabe U, Rudel T, and Kozjak-Pavlovic V
- Subjects
- Bacterial Adhesion drug effects, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cells, Cultured, Chlamydia trachomatis immunology, Chlamydia trachomatis metabolism, Gene Knockout Techniques, Humans, Microbial Viability drug effects, Neisseria gonorrhoeae immunology, Neisseria gonorrhoeae metabolism, Neisseria gonorrhoeae physiology, Neisseria meningitidis immunology, Neisseria meningitidis metabolism, Neisseria meningitidis physiology, Virulence drug effects, Virulence Factors genetics, Virulence Factors metabolism, Bacterial Proteins antagonists & inhibitors, Chlamydia trachomatis pathogenicity, Neisseria gonorrhoeae pathogenicity, Neisseria meningitidis pathogenicity, Virulence Factors antagonists & inhibitors
- Abstract
The pathogenic bacteria Chlamydia trachomatis, Neisseria gonorrhoeae and Neisseria meningitidis express the surface-exposed macrophage infectivity potentiator (MIP)-like protein, which plays a role in their pathogenicity. MIP exhibits a peptidyl-prolyl isomerase (PPIase) activity that is inhibited by rapamycin and FK506. In this study, pipecolic acid derivatives were tested for their activity against the chlamydial and neisserial MIP. Two MIP inhibitors were identified, PipN3 and PipN4, that affected the developmental cycle of C. trachomatis in HeLa cells. Furthermore, we could show that deletion of neisserial MIP or addition of the two MIP inhibitors affected the survival of N. gonorrhoeae in the presence of neutrophils. Furthermore, both compounds inhibited the adherence, invasion and/or survival of N. meningitidis in epithelial cells. These results confirm the importance of MIP-like proteins in infection and indicate the relevance of pipecolic acid derivatives as antimicrobials against C. trachomatis, N. gonorrhoeae and N. meningitidis., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)
- Published
- 2016
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11. TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics.
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Zhu J, Bailly A, Zwiewka M, Sovero V, Di Donato M, Ge P, Oehri J, Aryal B, Hao P, Linnert M, Burgardt NI, Lücke C, Weiwad M, Michel M, Weiergräber OH, Pollmann S, Azzarello E, Mancuso S, Ferro N, Fukao Y, Hoffmann C, Wedlich-Söldner R, Friml J, Thomas C, and Geisler M
- Subjects
- Actin Cytoskeleton metabolism, Arabidopsis genetics, Arabidopsis Proteins genetics, Biological Transport genetics, Biological Transport physiology, Gene Expression Regulation, Plant genetics, Gene Expression Regulation, Plant physiology, Tacrolimus Binding Proteins genetics, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Indoleacetic Acids metabolism, Tacrolimus Binding Proteins metabolism
- Abstract
Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity., (© 2016 American Society of Plant Biologists. All rights reserved.)
- Published
- 2016
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12. The ɛ-Amino Group of Protein Lysine Residues Is Highly Susceptible to Nonenzymatic Acylation by Several Physiological Acyl-CoA Thioesters.
- Author
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Simic Z, Weiwad M, Schierhorn A, Steegborn C, and Schutkowski M
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- Acylation, Amines chemistry, Amines metabolism, Crystallography, X-Ray, Cyclophilin A chemistry, Cyclophilin A metabolism, Humans, Kinetics, Lysine chemistry, Mitochondria metabolism, Molecular Conformation, Peptides chemistry, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Sirtuin 3 chemistry, Sirtuin 3 genetics, Sirtuins chemistry, Sirtuins genetics, Sirtuins metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Thermodynamics, Acyl Coenzyme A metabolism, Lysine metabolism, Peptides metabolism, Sirtuin 3 metabolism
- Abstract
Mitochondrial enzymes implicated in the pathophysiology of diabetes, cancer, and metabolic syndrome are highly regulated by acetylation. However, mitochondrial acetyltransferases have not been identified. Here, we show that acetylation and also other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue. In the case of a peptide derived from carbamoyl phosphate synthetase 1, the rates of succinylation and glutarylation were up to 150 times than for acetylation. These results were confirmed by using the protein substrate cyclophilin A (CypA). Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl groups from CypA, whereas SIRT5 is an effective protein desuccinylase. Thus, the acylation landscape on lysine residues might largely depend on the enzymatic activity of specific sirtuins, and the availability and reactivity of acyl-CoA compounds., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2015
- Full Text
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13. Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner.
- Author
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Burgardt NI, Schmidt A, Manns A, Schutkowski A, Jahreis G, Lin YJ, Schulze B, Masch A, Lücke C, and Weiwad M
- Subjects
- Amino Acid Motifs, Calmodulin genetics, Catalytic Domain, Humans, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, NIMA-Interacting Peptidylprolyl Isomerase, Peptidylprolyl Isomerase chemistry, Peptidylprolyl Isomerase genetics, Polymerization, Protein Binding, Calcium metabolism, Calmodulin metabolism, Peptidylprolyl Isomerase metabolism, Tubulin chemistry, Tubulin metabolism
- Abstract
Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca(2+)/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with (15)N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca(2+)-dependent manner with the Par17 N terminus. The reverse experiment with (15)N-labeled Ca(2+)/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca(2+)/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK(796-815) complex. In vitro tubulin polymerization assays furthermore showed that Ca(2+)/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca(2+)/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca(2+)/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca(2+) signaling with microtubule function., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2015
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14. Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.
- Author
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Haslbeck V, Drazic A, Eckl JM, Alte F, Helmuth M, Popowicz G, Schmidt W, Braun F, Weiwad M, Fischer G, Gemmecker G, Sattler M, Striggow F, Groll M, and Richter K
- Subjects
- Animals, Caenorhabditis elegans Proteins metabolism, Crystallography, X-Ray, Drug Evaluation, Preclinical methods, Enzyme Activation drug effects, HSC70 Heat-Shock Proteins genetics, HSC70 Heat-Shock Proteins metabolism, Mutation, Nuclear Magnetic Resonance, Biomolecular, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins chemistry, Phosphoprotein Phosphatases antagonists & inhibitors, Phosphoprotein Phosphatases chemistry, Protein Domains, Rats, Nuclear Proteins metabolism, Phosphoprotein Phosphatases metabolism, Small Molecule Libraries pharmacology
- Abstract
Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds., (© 2015 Authors.)
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- 2015
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15. Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila.
- Author
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Rasch J, Theuerkorn M, Ünal C, Heinsohn N, Tran S, Fischer G, Weiwad M, and Steinert M
- Abstract
Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.
- Published
- 2015
- Full Text
- View/download PDF
16. Artificial accelerators of the molecular chaperone Hsp90 facilitate rate-limiting conformational transitions.
- Author
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Zierer BK, Weiwad M, Rübbelke M, Freiburger L, Fischer G, Lorenz OR, Sattler M, Richter K, and Buchner J
- Subjects
- Fluorescence Resonance Energy Transfer, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, HSP90 Heat-Shock Proteins chemistry
- Abstract
The molecular chaperone Hsp90 undergoes an ATP-driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well-established small-molecule inhibitors of Hsp90 compete with ATP-binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET-based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co-chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate-limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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17. Assessment of cell death studies by monitoring hydrogen peroxide in cell culture.
- Author
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Hirsch I, Prell E, and Weiwad M
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- 3T3 Cells, Animals, Cell Death drug effects, Cell Survival drug effects, Culture Media chemistry, Mice, Time Factors, Cell Culture Techniques methods, Hydrogen Peroxide analysis, Hydrogen Peroxide pharmacology
- Abstract
Hydrogen peroxide (H2O2) has been widely used to study the oxidative stress response. However, H2O2 is unstable and easily decomposes into H2O and O2. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H2O2 concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H2O2, therefore acting as a protection against cell death by accidental necrosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
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18. ERp29 deficiency affects sensitivity to apoptosis via impairment of the ATF6-CHOP pathway of stress response.
- Author
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Hirsch I, Weiwad M, Prell E, and Ferrari DM
- Subjects
- Animals, Apoptosis drug effects, Endoplasmic Reticulum Stress drug effects, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Hydrogen Peroxide pharmacology, Mice, Mice, Inbred C57BL, Mice, Knockout, Oxidants pharmacology, Protein Synthesis Inhibitors pharmacology, Signal Transduction, Skin cytology, Thyroid Gland cytology, Tunicamycin pharmacology, Unfolded Protein Response drug effects, Unfolded Protein Response physiology, Activating Transcription Factor 6 metabolism, Apoptosis genetics, Endoplasmic Reticulum Stress physiology, Heat-Shock Proteins genetics, Transcription Factor CHOP metabolism
- Abstract
Endoplasmic reticulum protein 29 (ERp29) belongs to the redox-inactive PDI-Dβ-subfamily of PDI-proteins. ERp29 is expressed in all mammalian tissues examined. Especially high levels of expression were observed in secretory tissues and in some tumors. However, the biological role of ERp29 remains unclear. In the present study we show, by using thyrocytes and primary dermal fibroblasts from adult ERp29(-/-) mice, that ERp29 deficiency affects the activation of the ATF6-CHOP-branch of unfolded protein response (UPR) without influencing the function of other UPR branches, like the ATF4-eIF2α-XBP1 signaling pathway. As a result of impaired ATF6 activation, dermal fibroblasts and adult thyrocytes from ERp29(-/-) mice display significantly lower apoptosis sensitivities when treated with tunicamycin and hydrogen peroxide. However, in contrast to previous reports, we could demonstrate that ERp29 deficiency does not alter thyroglobulin expression levels. Therefore, our study suggests that ERp29 acts as an escort factor for ATF6 and promotes its transport from ER to Golgi apparatus under ER stress conditions.
- Published
- 2014
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19. ¹H, ¹³C and ¹⁵N resonance assignments of human parvulin 17.
- Author
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Lin YJ, Schmidt A, Burgardt NI, Thiele A, Weiwad M, and Lücke C
- Subjects
- Amino Acid Sequence, Carbon Isotopes, Humans, NIMA-Interacting Peptidylprolyl Isomerase, Nitrogen Isotopes, Nuclear Magnetic Resonance, Biomolecular, Peptidylprolyl Isomerase chemistry, Protons
- Abstract
A 25-residue elongation at the N-terminus endows parvulin 17 (Par17) with altered functional properties compared to parvulin 14 (Par14), such as an enhanced influence on microtubule assembly. Therefore the three-dimensional structure of this N-terminal elongation is of particular interest. Here, we report the nearly complete (1)H, (13)C and (15)N chemical shift assignments of Par17. Subsequent chemical shift index analysis indicated that Par17 features a parvulin-type PPIase domain at the C-terminus, analogous to Par14, and an unstructured N-terminus encompassing the first 60 residues. Hence the N-terminus of Par17 apparently adopts a functionally-relevant structure only in presence of the respective interaction partner(s).
- Published
- 2013
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20. The FKBP-type domain of the human aryl hydrocarbon receptor-interacting protein reveals an unusual Hsp90 interaction.
- Author
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Linnert M, Lin YJ, Manns A, Haupt K, Paschke AK, Fischer G, Weiwad M, and Lücke C
- Subjects
- Amino Acid Sequence, Humans, Intracellular Signaling Peptides and Proteins genetics, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Peptidylprolyl Isomerase chemistry, Peptidylprolyl Isomerase genetics, Peptidylprolyl Isomerase metabolism, Protein Conformation, Protein Interaction Domains and Motifs, Sequence Homology, Amino Acid, Structural Homology, Protein, Tacrolimus metabolism, Tacrolimus Binding Protein 1A chemistry, Tacrolimus Binding Protein 1A genetics, Tacrolimus Binding Protein 1A metabolism, Tacrolimus Binding Proteins chemistry, Tacrolimus Binding Proteins genetics, Tacrolimus Binding Proteins metabolism, HSP90 Heat-Shock Proteins chemistry, HSP90 Heat-Shock Proteins metabolism, Intracellular Signaling Peptides and Proteins chemistry, Intracellular Signaling Peptides and Proteins metabolism
- Abstract
The aryl hydrocarbon receptor-interacting protein (AIP) has been predicted to consist of an N-terminal FKBP-type peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal tetratricopeptide repeat (TPR) domain, as typically found in FK506-binding immunophilins. AIP, however, exhibited no inherent FK506 binding or PPIase activity. Alignment with the prototypic FKBP12 showed a high sequence homology but indicated inconsistencies with regard to the secondary structure prediction derived from chemical shift analysis of AIP(2-166). NMR-based structure determination of AIP(2-166) now revealed a typical FKBP fold with five antiparallel β-strands forming a half β-barrel wrapped around a central α-helix, thus permitting AIP to be also named FKBP37.7 according to FKBP nomenclature. This PPIase domain, however, features two structure elements that are unusual for FKBPs: (i) an N-terminal α-helix, which additionally stabilizes the domain, and (ii) a rather long insert, which connects the last two β-strands and covers the putative active site. Diminution of the latter insert did not generate PPIase activity or FK506 binding capability, indicating that the lack of catalytic activity in AIP is the result of structural differences within the PPIase domain. Compared to active FKBPs, a diverging conformation of the loop connecting β-strand C' and the central α-helix apparently is responsible for this inherent lack of catalytic activity in AIP. Moreover, Hsp90 was identified as potential physiological interaction partner of AIP, which revealed binding contacts not only at the TPR domain but uncommonly also at the PPIase domain.
- Published
- 2013
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21. PPARα modulates the TSH β-subunit mRNA expression in thyrotrope TαT1 cells and in a mouse model.
- Author
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Wege N, Schutkowski A, König B, Brandsch C, Weiwad M, and Stangl GI
- Subjects
- Animals, Cell Line, Fatty Acids, Nonesterified genetics, Fatty Acids, Nonesterified metabolism, Fibric Acids pharmacology, Gene Expression Regulation drug effects, Humans, Male, Mice, Mice, Knockout, Mice, Transgenic, Neurokinin B analogs & derivatives, Neurokinin B genetics, PPAR alpha agonists, PPAR alpha genetics, Pituitary Gland drug effects, Pituitary Gland metabolism, Pyrimidines pharmacology, RNA, Messenger metabolism, Retinoid X Receptor alpha genetics, Thyroid Hormone Receptors beta genetics, Thyrotrophs cytology, Thyrotrophs drug effects, Thyroxine blood, Thyroxine genetics, Triiodothyronine blood, Triiodothyronine genetics, Fasting physiology, PPAR alpha metabolism, Thyrotrophs physiology, Thyrotropin, beta Subunit genetics
- Abstract
Scope: Fasting leads to a significant downregulation of the hypothalamus-pituitary-thyroid axis, and peroxisome proliferator-activated receptor (PPAR) α is a key transcription factor in mediating a magnitude of adaptive responses to fasting. In this study, we examined the role of PPARα in regulation of the hypothalamus-pituitary-thyroid axis., Methods and Results: Thyroid-stimulating hormone β-subunit (TSHβ) mRNA abundance was being reduced in response to treatment of TαT1 cells with PPARα agonists (p < 0.05), indicating an inhibitory transcriptional regulation of TSHβ by PPARα. As expected, fasting significantly downregulated TSHβ mRNA expression in a two-factorial study with fed or fasted wild-type (WT) and PPARα knockout mice (p < 0.05). In contrast to the in vitro data, fasted PPARα knockout mice revealed lower mRNA concentrations of pituitary TSHβ (-64%) and TSH-regulated thyroid genes, and lower plasma concentrations of thyroxine (T4, -25%), triiodothyronine (T3, -25%), free T4 (-60%), and free T3 (-35%) than fasted WT mice (p < 0.05). Those differences were not observed in fed mice., Conclusions: Data from thyrotrope cells revealed that PPARα could contribute to the fasting-associated downregulation of the TSHβ mRNA expression. In a mouse model, fasting led to a significant reduction in TSHβ mRNA level, but unexpectedly this effect was stronger in mice lacking PPARα than in WT mice., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2013
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22. NMR assignments of the FKBP-type PPIase domain of the human aryl-hydrocarbon receptor-interacting protein (AIP).
- Author
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Linnert M, Haupt K, Lin YJ, Kissing S, Paschke AK, Fischer G, Weiwad M, and Lücke C
- Subjects
- Humans, Protein Structure, Secondary, Protein Structure, Tertiary, Intracellular Signaling Peptides and Proteins chemistry, Nuclear Magnetic Resonance, Biomolecular, Peptidylprolyl Isomerase chemistry, Tacrolimus Binding Proteins chemistry
- Abstract
The aryl-hydrocarbon receptor-interacting protein (AIP) interacts with several protein binding partners and has been associated with pituitary tumor development. Here, we report nearly complete (1)H, (13)C and (15)N chemical shift assignments for the N-terminal AIP(2-166) segment, which has been predicted to represent a FKBP-type PPIase domain. Sequence alignment with the prototypic FKBP12, however, reveals disagreements between the AIP chemical shift index consensus and the corresponding FKBP12 secondary structure elements.
- Published
- 2012
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- View/download PDF
23. NMR assignments of the FKBP-type PPIase domain of FKBP42 from Arabidopsis thaliana.
- Author
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Burgardt NI, Linnert M, Weiwad M, Geisler M, and Lücke C
- Subjects
- Protein Structure, Secondary, Protein Structure, Tertiary, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Nuclear Magnetic Resonance, Biomolecular, Peptidylprolyl Isomerase chemistry, Tacrolimus Binding Proteins chemistry
- Abstract
The Atfkbp42 gene is associated with reduced and disoriented growth of Arabidopsis thaliana. Resonance assignments are reported for the FKBP-type PPIase domain of AtFKBP42. Signal intensities reveal an additional structure element that is atypical for such FKBP domains.
- Published
- 2012
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- View/download PDF
24. Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation.
- Author
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Henrichs S, Wang B, Fukao Y, Zhu J, Charrier L, Bailly A, Oehring SC, Linnert M, Weiwad M, Endler A, Nanni P, Pollmann S, Mancuso S, Schulz A, and Geisler M
- Subjects
- Arabidopsis metabolism, Biological Transport, Gene Expression Regulation, Plant physiology, Phosphoproteins metabolism, Phosphorylation, Plants, Genetically Modified, Protein Serine-Threonine Kinases metabolism, Proteomics, Quercetin metabolism, Tacrolimus Binding Proteins metabolism, Nicotiana metabolism, ATP-Binding Cassette Transporters metabolism, Arabidopsis Proteins metabolism, Indoleacetic Acids metabolism
- Abstract
Polar transport of the plant hormone auxin is controlled by PIN- and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-IP) and shotgun LC-MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. In-vitro and yeast expression analyses indicated that PID specifically modulates ABCB1-mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co-transfection in tobacco revealed that PID enhances ABCB1-mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho-proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter-active impact on ABCB1 activity that is coordinated by TWD1-PID interaction.
- Published
- 2012
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- View/download PDF
25. Collagen IV-derived peptide binds hydrophobic cavity of Legionella pneumophila Mip and interferes with bacterial epithelial transmigration.
- Author
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Ünal C, Schwedhelm KF, Thiele A, Weiwad M, Schweimer K, Frese F, Fischer G, Hacker J, Faber C, and Steinert M
- Subjects
- Cell Line, Epithelial Cells microbiology, Humans, Immunoprecipitation, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Dynamics Simulation, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Interaction Mapping, Bacterial Proteins metabolism, Collagen Type IV metabolism, Host-Pathogen Interactions, Legionella pneumophila pathogenicity, Peptidylprolyl Isomerase metabolism, Transendothelial and Transepithelial Migration
- Abstract
The Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to bacterial dissemination within infected lung tissue. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), binds specifically to collagen IV. We identified a surface-exposed Mip-binding sequence in the NC1 domain of human collagen IV α1. The corresponding collagen IV-derived peptide (P290) co-precipitated with Mip and competitively inhibited the Mip-collagen IV binding. Transmigration of Legionella pneumophila across a barrier of NCI-H292 lung epithelial cells and extracellular matrix was efficiently inhibited by P290. This significantly reduced transmigration was comparable to the inefficient transmigration of PPIase-negative Mip mutant or rapamycin-treated L. pneumophila. Based on NMR data and docking studies a model for the mode of interaction of P290 and Mip was developed. The amino acids of the hydrophobic cavity of Mip, D142 and to a lesser extent Y185 were identified to be part of the interaction surface. In the complex structure of Mip(77-213) and P290, both amino acid residues form hydrogen bonds to P290. Utilizing modelling, molecular dynamics (MD) simulations and structural data of human PPIase FKBP12, the most related human orthologue of Mip, we were able to propose optimized P290 variants with increased binding specificity and selectivity for the putative bacterial drug target Mip., (© 2011 Blackwell Publishing Ltd.)
- Published
- 2011
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26. Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity.
- Author
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Thiele A, Krentzlin K, Erdmann F, Rauh D, Hause G, Zerweck J, Kilka S, Pösel S, Fischer G, Schutkowski M, and Weiwad M
- Subjects
- Animals, Brain metabolism, Cattle, Colchicine pharmacology, Humans, NIMA-Interacting Peptidylprolyl Isomerase, Peptidylprolyl Isomerase genetics, Polymerization, Protein Binding, Tubulin Modulators pharmacology, Microtubules metabolism, Peptide Fragments metabolism, Peptidylprolyl Isomerase chemistry, Peptidylprolyl Isomerase metabolism, Tubulin metabolism
- Abstract
The parvulin-type peptidyl-prolyl cis/trans isomerases (PPIases) have been shown to be involved in tumor progression and the pathogenesis of Alzheimer's disease and were therefore a subject of intense research. Here, we describe a role for parvulin 17 in microtubule assembly. Co-precipitation experiments and sedimentation assays demonstrated that parvulin 17 interacts with tubulin in a GTP-dependent manner and thereby promotes the formation of microtubules, as shown by transmission electron microscopy and a microtubule polymerization assay. The microtubule-assembly-promoting properties of parvulin 17 seem to depend on its PPIase activity. Thus, catalytic deficient variants of parvulin 17 were not able to promote microtubule formation. Accordingly, inhibitors of parvulin 17 activity also prevent parvulin-catalyzed tubulin polymerization. The analysis of tubulin interaction sites on parvulin using peptide microarrays revealed that tubulin interacts with the substrate binding pocket of parvulin. Additionally, β-tubulin peptide scan on microarrays demonstrates interaction of parvulin 17 with an Arg-Pro-Asp motif corresponding to proline residue 87 of β-tubulin. Confocal laser scanning microscopy points to a function of parvulin 17 in microtubule dynamics as well. Parvulin 17 is predominantly found in the cytosol and colocalizes with microtubules., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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27. Pipecolic acid derivatives as small-molecule inhibitors of the Legionella MIP protein.
- Author
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Juli C, Sippel M, Jäger J, Thiele A, Weiwad M, Schweimer K, Rösch P, Steinert M, Sotriffer CA, and Holzgrabe U
- Subjects
- Animals, Bacterial Proteins chemistry, Binding Sites, Cell Line, Colony Count, Microbial, Guinea Pigs, Legionella pneumophila enzymology, Legionnaires' Disease drug therapy, Magnetic Resonance Spectroscopy, Models, Molecular, Peptidylprolyl Isomerase chemistry, Pipecolic Acids chemistry, Pipecolic Acids pharmacology, Stereoisomerism, Structure-Activity Relationship, Tacrolimus Binding Protein 1A chemistry, Bacterial Proteins antagonists & inhibitors, Legionella pneumophila drug effects, Peptidylprolyl Isomerase antagonists & inhibitors, Pipecolic Acids chemical synthesis
- Abstract
The macrophage infectivity potentiator (MIP) protein is a major virulence factor of Legionella pneumophila, the causative agent of Legionnaires' disease. MIP belongs to the FK506-binding proteins (FKBP) and is necessary for optimal intracellular survival and lung tissue dissemination of L. pneumophila. We aimed to identify new small-molecule inhibitors of MIP by starting from known FKBP12 ligands. Computational analysis, synthesis, and biological testing of pipecolic acid derivatives revealed a promising scaffold for new MIP inhibitors.
- Published
- 2011
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28. The novel calcineurin inhibitor CN585 has potent immunosuppressive properties in stimulated human T cells.
- Author
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Erdmann F, Weiwad M, Kilka S, Karanik M, Pätzel M, Baumgrass R, Liebscher J, and Fischer G
- Subjects
- Calcineurin metabolism, Cell Proliferation drug effects, Cytokines biosynthesis, Drug Evaluation, Preclinical, Enzyme Inhibitors chemistry, Enzyme Inhibitors immunology, Enzyme Inhibitors metabolism, Humans, Immunization, Immunosuppressive Agents chemistry, Immunosuppressive Agents immunology, Immunosuppressive Agents metabolism, Intracellular Space drug effects, Intracellular Space metabolism, Jurkat Cells, Leukocytes, Mononuclear immunology, NFATC Transcription Factors metabolism, Phosphorylation drug effects, Pyrimidines chemistry, Pyrimidines immunology, Pyrimidines metabolism, Substrate Specificity, T-Lymphocytes cytology, Calcineurin Inhibitors, Enzyme Inhibitors pharmacology, Immunosuppressive Agents pharmacology, Pyrimidines pharmacology, T-Lymphocytes drug effects, T-Lymphocytes immunology
- Abstract
The Ca2+/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K(i) value of 3.8 mum. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.
- Published
- 2010
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29. High density peptide microarrays for proteome-wide fingerprinting of kinase activities in cell lysates.
- Author
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Thiele A, Weiwad M, Zerweck J, Fischer G, and Schutkowski M
- Subjects
- Amino Acid Sequence, Binding Sites, Cell Line, Enzymes, Immobilized chemistry, Enzymes, Immobilized metabolism, Humans, Molecular Sequence Data, Peptide Fragments chemistry, Peptidylprolyl Isomerase chemistry, Peptidylprolyl Isomerase metabolism, Phosphorylation, Phosphotransferases chemistry, Proteome chemistry, Cell Extracts, Peptide Fragments metabolism, Peptide Mapping methods, Phosphotransferases metabolism, Protein Array Analysis methods, Proteome metabolism
- Abstract
Protein function is highly regulated in pathways that are responsible for complex biochemical mechanisms such as growth, metabolism, and signal transduction. One of the most important mechanisms is posttranslational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation, glycosylation, and sumoylation resulting in a more than 100-fold higher complexity (Geiss-Friedlander and Melchior, Nat Rev Mol Cell Biol 8, 947-956, 2007; Hunter, Mol Cell 28, 730-738, 2007). This chapter presents a very efficient way to detect potential phosphorylation sites in protein families using overlapping peptides covering the complete primary structures (peptide scans) immobilized on glass slides. Results of kinase activity fingerprinting of cell lysates using peptide microarrays displaying peptide scans through all human peptidyl-prolyl-cis/trans-isomerases are shown.
- Published
- 2010
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30. Identification of a disulfide bridge essential for transport function of the human proton-coupled amino acid transporter hPAT1.
- Author
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Dorn M, Weiwad M, Markwardt F, Laug L, Rudolph R, Brandsch M, and Bosse-Doenecke E
- Subjects
- Amino Acid Sequence, Amino Acid Transport Systems physiology, Animals, Binding Sites, Disulfides, Dithiothreitol pharmacology, Humans, Microscopy, Fluorescence methods, Models, Biological, Molecular Sequence Data, Mutagenesis, Site-Directed, Oocytes metabolism, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Symporters physiology, Xenopus laevis, Amino Acid Transport Systems chemistry, Symporters chemistry
- Abstract
The proton-coupled amino acid transporter 1 (PAT1, SLC36A1) mediates the uptake of small neutral amino acids at the apical membrane of intestinal epithelial cells after protein digestion. The transporter is currently under intense investigation, because it is a possible vehicle for oral drug delivery. Structural features of the protein such as the number of transmembrane domains, the substrate binding site, or essential amino acids are still unknown. In the present study we use mutagenesis experiments and biochemical approaches to determine the role of the three putative extracellular cysteine residues on transport function and their possible involvement in the formation of a disulfide bridge. As treatment with the reducing reagent dithiothreitol impaired transport function of hPAT1 wild type protein, substitution of putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 by alanine or serine was performed. Replacement of the two highly conserved cysteine residues Cys-180 and Cys-329 abolished the transport function of hPAT1 in Xenopus laevis oocytes. Studies of wild type and mutant transporters expressed in human retinal pigment epithelial (HRPE) cells suggested that the binding of the substrate was inhibited in these mutants. Substitution of the third putative extracellular nonconserved cysteine residue Cys-473 did not affect transport function. All mutants were expressed at the plasma membrane. Biotinylation of free sulfhydryl groups using maleimide-PEG(11)-biotin and SDS-PAGE analysis under reducing and nonreducing conditions provided direct evidence for the existence of an essential disulfide bond between Cys-180 and Cys-329. This disulfide bridge is very likely involved in forming or stabilizing the substrate binding site.
- Published
- 2009
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31. The role of N-glycosylation in transport function and surface targeting of the human solute carrier PAT1.
- Author
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Dorn M, Jaehme M, Weiwad M, Markwardt F, Rudolph R, Brandsch M, and Bosse-Doenecke E
- Subjects
- Animals, Fluorescent Antibody Technique, Glycosylation, Humans, Models, Biological, Mutagenesis, Site-Directed, Oocytes metabolism, Patch-Clamp Techniques, Protein Transport physiology, Amino Acid Transport Systems metabolism, Symporters metabolism
- Abstract
In the present study we show in the Xenopus laevis expression system that the proton-coupled amino acid transporter 1 (PAT1, SLC36A1) is glycosylated at asparagine residues N174, N183 and N470. To determine the functional role of N-glycosylation, glycosylation-deficient mutants were analyzed by two-electrode voltage-clamp measurements after expression in X. laevis oocytes. Single replacements of asparagine residues had no effect on transport activity. However, multiple substitutions resulted in a decreased transport rate, leaving K(t) unchanged. Immunofluorescence localisation revealed a diminished plasma membrane expression of glycosylation-defective mutants. This indicates that N-glycans are not required for transport function, but are important for membrane targeting.
- Published
- 2009
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- View/download PDF
32. Reassessment of the role of FKBP38 in the Rheb/mTORC1 pathway.
- Author
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Uhlenbrock K, Weiwad M, Wetzker R, Fischer G, Wittinghofer A, and Rubio I
- Subjects
- Cells, Cultured, Humans, Insulin pharmacology, Mechanistic Target of Rapamycin Complex 1, Monomeric GTP-Binding Proteins genetics, Monomeric GTP-Binding Proteins metabolism, Multiprotein Complexes, Neuropeptides genetics, Neuropeptides metabolism, Phosphorylation drug effects, Protein Binding, Proteins, RNA, Small Interfering pharmacology, Ras Homolog Enriched in Brain Protein, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Recombinant Fusion Proteins physiology, Ribosomal Protein S6 Kinases metabolism, Signal Transduction drug effects, Signal Transduction genetics, Signal Transduction physiology, TOR Serine-Threonine Kinases, Tacrolimus Binding Proteins antagonists & inhibitors, Tacrolimus Binding Proteins genetics, Transcription Factors metabolism, Transcriptional Activation drug effects, Transfection, Monomeric GTP-Binding Proteins physiology, Neuropeptides physiology, Tacrolimus Binding Proteins physiology, Transcription Factors physiology
- Abstract
The small G-protein Rheb regulates cell growth via the mTORC1 complex by incompletely understood mechanisms. Recent studies document that Rheb activates mTORC1 via direct, GTP-dependent interaction with the peptidyl-prolyl-cis/trans-isomerase FKBP38, which is proposed to act as an inhibitor of mTORC1. We have conducted a comprehensive biochemical characterization of the Rheb/FKBP38 interaction. Using three different in vitro assays we did not detect an interaction between Rheb and FKBP38. Cell biological experiments illustrate that FKBP38 plays only a very minor, if any, role in mTORC1 activation. Our data document that FKBP38 is not the long-sought Rheb effector linking Rheb to mTORC1 activation.
- Published
- 2009
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- View/download PDF
33. The proline-rich N-terminal sequence of calcineurin Abeta determines substrate binding.
- Author
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Kilka S, Erdmann F, Migdoll A, Fischer G, and Weiwad M
- Subjects
- Amino Acid Sequence, Binding Sites genetics, Blotting, Western, Calcineurin genetics, Catalysis, Catalytic Domain genetics, Cell Line, Cell Line, Tumor, HeLa Cells, Humans, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Jurkat Cells, Kinetics, Luciferases genetics, Luciferases metabolism, Microscopy, Confocal, Models, Molecular, Molecular Sequence Data, NFATC Transcription Factors genetics, NFATC Transcription Factors metabolism, Phosphorylation, Proline genetics, Protein Multimerization, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Substrate Specificity, Transfection, ets-Domain Protein Elk-1 genetics, ets-Domain Protein Elk-1 metabolism, Calcineurin chemistry, Calcineurin metabolism
- Abstract
Three different genes of catalytic subunit A of the Ca(2+)-dependent serine/threonine protein phosphatase calcineurin (CaN) are encoded in the human genome forming heterodimers with regulatory subunit B. Even though physiological roles of CaN have been investigated extensively, less is known about the specific functions of the different catalytic isoforms. In this study, all human CaN holoenzymes containing either the alpha, beta, or gamma isoform of the catalytic subunit (CaN alpha, beta, or gamma, respectively) were expressed for the first time. Comparative kinetic analysis of the dephosphorylation of five specific CaN substrates provided evidence that the distinct isoforms of the catalytic subunit confer substrate specificities to the holoenzymes. CaN alpha dephosphorylates the transcription factor Elk-1 with 7- and 2-fold higher catalytic efficiencies than the beta and gamma isoforms, respectively. CaN gamma exhibits the highest k(cat)/K(m) value for DARPP-32, whereas the catalytic efficiencies for the dephosphorylation of NFAT and RII peptide were 3- and 5-fold lower, respectively, when compared with the other isoforms. Elk-1 and NFAT reporter gene activity measurements revealed even more pronounced substrate preferences of CaNA isoforms. Moreover, kinetic analysis demonstrated that CaN beta exhibits for all tested protein substrates the lowest K(m) values. Enzymatic characterization of the CaN beta(P14G/P18G) variant as well as the N-terminal truncated form CaN beta(22-524) revealed that the proline-rich sequence of CaN beta is involved in substrate recognition. CaN beta(22-524) exhibits an at least 4-fold decreased substrate affinity and a 5-fold increased turnover number. Since this study demonstrates that all CaN isoforms display the same cytoplasmic subcellular distribution and are expressed in each tested cell line, differences in substrate specificities may determine specific physiological functions of the distinct isoforms.
- Published
- 2009
- Full Text
- View/download PDF
34. High-density peptide microarrays for reliable identification of phosphorylation sites and upstream kinases.
- Author
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Thiele A, Zerweck J, Weiwad M, Fischer G, and Schutkowski M
- Subjects
- Amino Acid Motifs, Animals, Binding Sites, Fluorescence, High-Throughput Screening Assays instrumentation, Humans, Models, Biological, Peptides analysis, Peptides metabolism, Phosphorylation, Protein Array Analysis instrumentation, Radioisotopes metabolism, High-Throughput Screening Assays methods, Protein Array Analysis methods, Protein Kinases metabolism
- Abstract
The human genome encodes about 25,000 genes. This number seems to be very small compared to the multitude of different protein functions in highly regulated pathways that are responsible for complex biochemical mechanisms like growth, metabolism, signal transduction and reproduction. Obviously, there are mechanisms creating additional protein diversity. The most important mechanism is post-translational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation and sumoylation resulting in an about 100-fold higher complexity (1, 2). This chapter presents a very efficient way to detect potential phosphorylation sites in proteins using overlapping peptide scans immobilized on glass slides. Results from 35 different human kinases using peptide microarrays displaying overlapping peptide scans through either all human cyclophilins or all human FK506-binding proteins are shown. Additionally, detection of phosphorylation sites in a proteome-wide manner is demonstrated using peptide microarrays displaying cytomegalovirus proteome in the form of more than 17,000 overlapping peptides.
- Published
- 2009
- Full Text
- View/download PDF
35. A novel calmodulin-Ca2+ target recognition activates the Bcl-2 regulator FKBP38.
- Author
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Edlich F, Maestre-Martínez M, Jarczowski F, Weiwad M, Moutty MC, Malesević M, Jahreis G, Fischer G, and Lücke C
- Subjects
- Apoptosis physiology, Calcium chemistry, Calmodulin chemistry, Calmodulin genetics, Cell-Free System chemistry, Cell-Free System metabolism, Humans, Multiprotein Complexes chemistry, Multiprotein Complexes genetics, Neurons chemistry, Neurons metabolism, Protein Binding physiology, Protein Structure, Tertiary physiology, Proto-Oncogene Proteins c-bcl-2 chemistry, Proto-Oncogene Proteins c-bcl-2 genetics, Tacrolimus Binding Proteins chemistry, Tacrolimus Binding Proteins genetics, Calcium metabolism, Calmodulin metabolism, Multiprotein Complexes metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism, Tacrolimus Binding Proteins metabolism
- Abstract
The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290-Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.
- Published
- 2007
- Full Text
- View/download PDF
36. Hsp90-mediated inhibition of FKBP38 regulates apoptosis in neuroblastoma cells.
- Author
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Erdmann F, Jarczowski F, Weiwad M, Fischer G, and Edlich F
- Subjects
- Binding Sites, Calcium metabolism, Calmodulin metabolism, Cell Death, Cell Line, Tumor, Humans, Iodoacetic Acid pharmacology, Tacrolimus Binding Proteins metabolism, Apoptosis, HSP90 Heat-Shock Proteins metabolism, Neuroblastoma metabolism, Tacrolimus Binding Proteins antagonists & inhibitors
- Abstract
The FK506-binding protein 38 (FKBP38) is a pro-apoptotic regulator of Bcl-2 in neuroblastoma cells. Hsp90 inhibits the pro-apoptotic FKBP38/CaM/Ca(2+) complex and thus prevents interactions between FKBP38 and Bcl-2. Here we show that Hsp90 increases cell survival rates of neuroblastoma cells after apoptosis induction. Depletion of FKBP38 by short interference RNA significantly decreased the anti-apoptotic effect of Hsp90 expression. In addition, the influence of high cellular Hsp90 levels was only observed in post-stimulation apoptosis that is sensitive to selective FKBP38 active site inhibition. Similar anti-apoptotic effects in neuroblastoma cells were observed after stimulation of endogenous Hsp90 expression. Hence, the inhibition of FKBP38 by Hsp90 participates in programmed cell death control of neuroblastoma cells.
- Published
- 2007
- Full Text
- View/download PDF
37. The Bcl-2 regulator FKBP38-calmodulin-Ca2+ is inhibited by Hsp90.
- Author
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Edlich F, Erdmann F, Jarczowski F, Moutty MC, Weiwad M, and Fischer G
- Subjects
- Amino Acid Motifs, Binding Sites, Calcium metabolism, Calmodulin metabolism, Cell-Free System chemistry, Cell-Free System metabolism, Enzyme Activation physiology, HSP90 Heat-Shock Proteins metabolism, Humans, Multiprotein Complexes chemistry, Multiprotein Complexes metabolism, Protein Binding physiology, Protein Structure, Quaternary, Proto-Oncogene Proteins c-bcl-2 metabolism, Tacrolimus Binding Proteins metabolism, Apoptosis physiology, Calcium chemistry, Calmodulin chemistry, HSP90 Heat-Shock Proteins chemistry, Proto-Oncogene Proteins c-bcl-2 chemistry, Tacrolimus Binding Proteins chemistry
- Abstract
FKBP38 is a negative effector of the anti-apoptotic Bcl-2 protein in neuroblastoma cells. The interaction with Bcl-2 and the enzyme activity of FKBP38 depend on prior binding of calmodulin-Ca(2+) (CaM-Ca(2+)) at high Ca(2+) concentrations. The FKBP38 protein structure contains three tetratricopeptide repeat (TPR) motifs corresponding to the Hsp90 interaction sites of other immunophilins. In this study we show that the TPR domain of FKBP38 interacts with the C-terminal domain of Hsp90, but only if the FKBP38-CaM-Ca(2+) complex is preformed. Hence, FKBP38 is the first example of a TPR-containing immunophilin that interacts cofactor-dependently with Hsp90. In the ternary Hsp90-FKBP38-CaM-Ca(2+) complex the active site of FKBP38 is blocked, thus preventing interactions with Bcl-2. The dual control of the active site cleft of FKBP38 by CaM-Ca(2+) and Hsp90 highlights the importance of the enzyme activity of the FKBP38-CaM-Ca(2+) complex in the regulation of programmed cell death.
- Published
- 2007
- Full Text
- View/download PDF
38. Comparative analysis of calcineurin inhibition by complexes of immunosuppressive drugs with human FK506 binding proteins.
- Author
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Weiwad M, Edlich F, Kilka S, Erdmann F, Jarczowski F, Dorn M, Moutty MC, and Fischer G
- Subjects
- Amino Acid Sequence, Animals, Calcineurin metabolism, Calmodulin physiology, Cattle, Enzyme Inhibitors pharmacology, Humans, Immunosuppressive Agents metabolism, Jurkat Cells, Molecular Sequence Data, Multienzyme Complexes physiology, Protein Binding physiology, Protein Structure, Tertiary physiology, Sirolimus metabolism, Sirolimus pharmacology, Substrate Specificity, Tacrolimus metabolism, Tacrolimus pharmacology, Tacrolimus Binding Protein 1A physiology, Tacrolimus Binding Proteins metabolism, Calcineurin Inhibitors, Enzyme Inhibitors metabolism, Immunosuppressive Agents pharmacology, Multienzyme Complexes metabolism, Tacrolimus Binding Proteins physiology
- Abstract
Multiple intracellular receptors of the FK506 binding protein (FKBP) family of peptidylprolyl cis/trans-isomerases are potential targets for the immunosuppressive drug FK506. Inhibition of the protein phosphatase calcineurin (CaN), which has been implicated in the FK506-mediated blockade of T cell proliferation, was shown to involve a gain of function in the FKBP12/FK506 complex. We studied the potential of six human FKBPs to contribute to CaN inhibition by comparative examination of inhibition constants of the respective FK506/FKBP complexes. Interestingly, these FKBPs form tight complexes with FK506, exhibiting comparable dissociation constants, but the resulting FK506/FKBP complexes differ greatly in their affinity for CaN, with IC50 values in the range of 0.047-17 microM. The different capacities of FK506/FKBP complexes to affect CaN activity are partially caused by substitutions corresponding to the amino acid side chains K34 and I90 of FKBP12. Only the FK506 complexes of FKBP12, FKBP12.6, and FKBP51 showed high affinity to CaN; small interfering RNA against these FKBP allowed defining the contribution of individual FKBP in an NFAT reporter gene assay. Our results allow quantitative correlation between FK506-mediated CaN effects and the abundance of the different FKBPs in the cell.
- Published
- 2006
- Full Text
- View/download PDF
39. The specific FKBP38 inhibitor N-(N',N'-dimethylcarboxamidomethyl)cycloheximide has potent neuroprotective and neurotrophic properties in brain ischemia.
- Author
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Edlich F, Weiwad M, Wildemann D, Jarczowski F, Kilka S, Moutty MC, Jahreis G, Lücke C, Schmidt W, Striggow F, and Fischer G
- Subjects
- Calcium metabolism, Cell Line, Tumor, Cycloheximide chemistry, Humans, Kinetics, Ligands, Models, Chemical, Neurodegenerative Diseases, Neurons metabolism, Brain pathology, Brain Ischemia pathology, Cycloheximide analogs & derivatives, Cycloheximide pharmacology, Nerve Growth Factors metabolism, Neuroprotective Agents pharmacology, Tacrolimus Binding Proteins antagonists & inhibitors
- Abstract
FK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerases (PPIase) display potent neuroprotective and neuroregenerative properties in various neurodegeneration models, showing the importance of neuroimmunophilins as targets for the treatment of acute and chronic neurodegenerative diseases. However, the PPIase activity targeted by active site-directed ligands remains unknown so far. Here we show that neurotrophic FKBP ligands, such as GPI1046 and N-[methyl(ethoxycarbonyl)]cycloheximide, inhibit the calmodulin/Ca(2+) (CaM/Ca(2+))-regulated FKBP38 with up to 80-fold higher affinity than FKBP12. In contrast, the non-neurotrophic rapamycin inhibits FKBP38.CaM/Ca(2+) 500-fold less affine than other neuroimmunophillins. In the context of the high expression of FKBP38 in neuroblastoma cells, these data suggest that FKBP38.CaM/Ca(2+) inhibition can mediate neurotrophic properties of FKBP ligands. The FKBP38-specific cycloheximide derivative, N-(N',N'-dimethylcarboxamidomethyl)cycloheximide (DM-CHX) was synthesized and used in a rat model of transient focal cerebral ischemia. Accordingly, DM-CHX caused neuronal protection as well as neural stem cell proliferation and neuronal differentiation at a dosage of 27.2 mug/kg. These effects were still dominant, if DM-CHX was applied 2-6 h post-insult. In parallel, sustained motor behavior deficits of diseased animals were improved by drug administration, revealing a potential therapeutic relevance. Thus, our results demonstrate that FKBP38 inhibition by DM-CHX regulates neuronal cell death and proliferation, providing a promising strategy for the treatment of acute and/or chronic neurodegenerative diseases.
- Published
- 2006
- Full Text
- View/download PDF
40. Solution structure of the FK506-binding domain of human FKBP38.
- Author
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Maestre-Martínez M, Edlich F, Jarczowski F, Weiwad M, Fischer G, and Lücke C
- Subjects
- Binding Sites, Humans, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Structure, Tertiary, Solutions, Tacrolimus Binding Proteins genetics, Tacrolimus Binding Proteins metabolism, Tacrolimus Binding Proteins chemistry
- Published
- 2006
- Full Text
- View/download PDF
41. Bcl-2 regulator FKBP38 is activated by Ca2+/calmodulin.
- Author
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Edlich F, Weiwad M, Erdmann F, Fanghänel J, Jarczowski F, Rahfeld JU, and Fischer G
- Subjects
- Apoptosis physiology, Binding Sites, Calmodulin chemistry, Cell Line, Tumor, Circular Dichroism, Humans, Peptidylprolyl Isomerase metabolism, Tacrolimus Binding Proteins antagonists & inhibitors, Tacrolimus Binding Proteins chemistry, Tacrolimus Binding Proteins metabolism, Calcium physiology, Calmodulin physiology, Proto-Oncogene Proteins c-bcl-2 metabolism, Tacrolimus Binding Proteins physiology
- Abstract
FKBP-type peptidyl prolyl cis/trans isomerases (PPIases) are folding helper enzymes involved in the control of functional regrowth of damaged sciatic, cortical cholinergic, dopaminergic and 5-HT neurones. Here, we show that the constitutively inactive human FK506-binding protein 38 (FKBP38) is capable of responding directly to intracellular Ca2+ rise through formation of a heterodimeric Ca2+/calmodulin/FKBP38 complex. Only complex formation creates an enzymatically active FKBP, displaying affinity for Bcl-2 mediated through the PPIase site. Association between Bcl-2 and the active site of Ca2+/calmodulin/FKBP38 regulates Bcl-2 function and thereby participates in the promotion of apoptosis in neuronal tissues. FKBP38 proapoptotic function mediated by this interaction is abolished by either potent inhibitors of the PPIase activity of the Ca2+/calmodulin/FKBP38 complex or RNA interference-mediated depletion of FKBP38, promoting neuronal cell survival.
- Published
- 2005
- Full Text
- View/download PDF
42. Human alpha-defensins neutralize anthrax lethal toxin and protect against its fatal consequences.
- Author
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Kim C, Gajendran N, Mittrücker HW, Weiwad M, Song YH, Hurwitz R, Wilmanns M, Fischer G, and Kaufmann SH
- Subjects
- Animals, Antigens, Bacterial, Female, Furin antagonists & inhibitors, Humans, Kinetics, MAP Kinase Kinase 3 metabolism, Macrophages metabolism, Matrix Metalloproteinase Inhibitors, Mice, Mice, Inbred BALB C, Spores, Bacterial drug effects, Survival Analysis, Tetrazolium Salts, Thiazoles, alpha-Defensins metabolism, Bacterial Toxins antagonists & inhibitors, Macrophages drug effects, Signal Transduction drug effects, alpha-Defensins pharmacology
- Abstract
Anthrax caused by Bacillus anthracis represents a major bioterroristic threat. B. anthracis produces lethal toxin (LeTx), a combination of lethal factor (LF) and protective antigen that plays a major role in anthrax pathogenesis. We demonstrate that human neutrophil alpha-defensins are potent inhibitors of LF. The inhibition of LF by human neutrophil protein (HNP-1) was noncompetitive. HNP-1 inhibited cleavage of a mitogen-activated protein kinase kinase and restored impaired mitogen-activated protein kinase signaling in LeTx-treated macrophages. HNP-1 rescued murine macrophages from B. anthracis-induced cytotoxicity, and in vivo treatment with HNP-1-3 protected mice against the fatal consequences of LeTx.
- Published
- 2005
- Full Text
- View/download PDF
43. A reassessment of the inhibitory capacity of human FKBP38 on calcineurin.
- Author
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Weiwad M, Edlich F, Erdmann F, Jarczowski F, Kilka S, Dorn M, Pechstein A, and Fischer G
- Subjects
- Calcineurin analysis, Calcineurin metabolism, Calcium metabolism, Cell Line, Humans, Immunoprecipitation, Proto-Oncogene Proteins c-bcl-2 metabolism, Recombinant Proteins genetics, Recombinant Proteins pharmacology, Signal Transduction, Tacrolimus metabolism, Tacrolimus pharmacology, Tacrolimus Binding Proteins genetics, Tacrolimus Binding Proteins metabolism, Transfection, Calcineurin Inhibitors, Tacrolimus Binding Proteins pharmacology
- Abstract
The microbial peptidomacrolide FK506 affects many eukaryotic developmental and cell signaling programs via calcineurin inhibition. Prior formation of a complex between FK506 and intracellular FK506-binding proteins (FKBPs) is the precondition for the interaction with calcineurin. A puzzling difference has emerged between the mammalian multidomain protein hFKBP38 and other FKBPs. It was shown that hFKBP38 not only binds to calcineurin but also inhibits the protein phosphatase activity of calcineurin on its own [Shirane, M. and Nakayama, K.I. (2003) Nature Cell Biol. 5, 28-37]. Inherent calcineurin inhibition by hFKBP38 would completely eliminate the need for FK506 in controlling many signal transduction pathways. To address this issue, we have characterized the functional and physical interactions between calcineurin and hFKBP38. A recombinant hFKBP38 variant and endogenous hFKBP38 were tested both in vitro and in vivo. The proteins neither directly inhibited calcineurin activity nor affected NFAT reporter gene activity in SH-SY5Y and Jurkat cells. In addition, a direct physical interaction between calcineurin and hFKBP38 was not detected in co-immunoprecipitation experiments. However, hFKBP38 indirectly affected the subcellular distribution of calcineurin by interaction with typical calcineurin ligands, as exemplified by the anti-apoptotic protein Bcl-2. Our data suggest that hFKBP38 cannot substitute for the FKBP/FK506 complex in signaling pathways controlled by the protein phosphatase activity of calcineurin.
- Published
- 2005
- Full Text
- View/download PDF
44. Catalysis of proline-directed protein phosphorylation by peptidyl-prolyl cis/trans isomerases.
- Author
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Weiwad M, Werner A, Rücknagel P, Schierhorn A, Küllertz G, and Fischer G
- Subjects
- Aspergillus oryzae enzymology, Catalysis, Mitogen-Activated Protein Kinase 1 metabolism, Phosphorylation, Peptidylprolyl Isomerase metabolism, Proline metabolism, Ribonuclease T1 metabolism
- Abstract
Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The cis/trans isomer specificity underlying ERK2-catalyzed phosphate transfer leads to a complete discrimination of the cis Ser(Thr)-Pro conformer of oligopeptide substrates. We investigated in vitro the ERK2-catalyzed phosphorylation of Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the cis Ser54-Pro and the trans Ser72-Pro moiety. Despite trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency kcat/KM of 425 M(-1) s(-1) and 1228 M(-1) s(-1), respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl cis/trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl cis/trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.
- Published
- 2004
- Full Text
- View/download PDF
45. The human nuclear SRcyp is a cell cycle-regulated cyclophilin.
- Author
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Dubourg B, Kamphausen T, Weiwad M, Jahreis G, Feunteun J, Fischer G, and Modjtahedi N
- Subjects
- Amino Acid Sequence, Animals, Arginine chemistry, Blotting, Western, Cell Cycle, Cell Line, Tumor, Cyclophilins chemistry, Cytoplasm metabolism, Fluorescent Antibody Technique, Indirect, Gene Expression Regulation, Humans, Mice, Mitosis, Models, Genetic, Molecular Sequence Data, Peptides chemistry, Phosphorylation, Plasmids metabolism, Precipitin Tests, Protein Structure, Tertiary, Protein Tyrosine Phosphatases metabolism, Purines pharmacology, Rats, Receptor-Like Protein Tyrosine Phosphatases, Class 2, Recombinant Proteins chemistry, Roscovitine, Sequence Homology, Amino Acid, Serine chemistry, Transfection, Cell Nucleus metabolism, Cyclophilins genetics, Cyclophilins physiology
- Abstract
Cyclophilins of the Moca family (Cavarec, L., Kamphausen, T., Dubourg, B., Callebaut, I., Lemeunier, F., Metivier, D., Feunteun, J., Fischer, G., and Modjtahedi, N. (2002) J. Biol. Chem. 277, 41171-41182) are found only in organisms of the animal kingdom and share several structural and enzymatic features. The presence of serine/arginine (S/R) dipeptide repeats in their C-terminal tail suggests that these enzymes belong to the SR protein family involved in the regulation of gene expression. The function of this group of cyclophilins is currently unknown. However, their C-terminal tails contain a highly conserved polypeptide signature segment (the moca domain), which may well be involved in the functional regulation of these proteins. We report here the identification of five Cdc2-type phosphorylation sites gathered in and around the moca domain of SRcyp, a human cyclophilin belonging to the Moca family. The segment of SRcyp containing the identified sites is specifically phosphorylated in mitotic cells. This mitosis-specific phosphorylation was inhibited by treatment of the cells with roscovitine, a specific inhibitor of cyclin-dependent kinases, suggesting that the unknown activity of the moca domain of SRcyp requires mitotic regulation by the Cdc2-cyclin B kinase complex. The Cdc2-cyclin B complex was found to phosphorylate four of the five identified phosphorylation sites in vitro, providing further support for this possibility. Like many factors stored in nuclear speckles and involved in the regulation of gene expression, this nuclear cyclophilin displays a predominantly diffuse cytoplasmic distribution at the onset of mitosis. Only in late telophase is SRcyp recruited to the newly formed nuclei. The transit of SRcyp through mitotic interchromatin granule clusters, before re-entering the nucleus, suggests that the timing of the appearance of this cyclophilin in the telophasic nuclei is tightly coordinated with post-mitotic events. Human SRcyp is the first cell cycle-regulated cyclophilin to be described.
- Published
- 2004
- Full Text
- View/download PDF
46. Substitution in position 3 of cyclosporin A abolishes the cyclophilin-mediated gain-of-function mechanism but not immunosuppression.
- Author
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Baumgrass R, Zhang Y, Erdmann F, Thiel A, Weiwad M, Radbruch A, and Fischer G
- Subjects
- Amino Acid Substitution, Antifungal Agents chemistry, Calcineurin genetics, Cyclophilins genetics, Cyclophilins metabolism, Cyclosporine chemistry, Gene Deletion, Humans, Immunosuppression Therapy, Immunosuppressive Agents chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Tacrolimus Binding Proteins genetics, Tacrolimus Binding Proteins metabolism, Antifungal Agents pharmacology, Calcineurin Inhibitors, Cyclosporine pharmacology, Immunosuppressive Agents pharmacology, Saccharomyces cerevisiae drug effects
- Abstract
Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine(3)]CsA was shown to inhibit calcineurin, either in its free form with an IC(50) value of 10 microm, or in its complex form with cyclophilin 18 with an IC(50) of 500 nm. [Dimethylaminoethylthiosarcosine(3)]CsA ([Dat-Sar(3)]CsA) was found to inhibit calcineurin on its own, with an IC(50) value of 1.0 microm, but was not able to inhibit calcineurin after forming the [Dat-Sar(3)]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar(3)]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar(3)]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Delta12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar(3)]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Delta12 strain.
- Published
- 2004
- Full Text
- View/download PDF
47. Phosphorylation of the N-terminal domain regulates subcellular localization and DNA binding properties of the peptidyl-prolyl cis/trans isomerase hPar14.
- Author
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Reimer T, Weiwad M, Schierhorn A, Ruecknagel PK, Rahfeld JU, Bayer P, and Fischer G
- Subjects
- Blotting, Western, Cell Cycle, Cell Nucleus metabolism, Chromatin metabolism, DNA chemistry, Dichlororibofuranosylbenzimidazole pharmacology, Dose-Response Relationship, Drug, Glutamic Acid chemistry, HeLa Cells, Heparin chemistry, Humans, Kinetics, Mass Spectrometry, Microscopy, Fluorescence, Mutation, NIMA-Interacting Peptidylprolyl Isomerase, Nucleic Acid Synthesis Inhibitors pharmacology, Phosphorylation, Plasmids metabolism, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Serine chemistry, Sodium Chloride pharmacology, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Subcellular Fractions, Time Factors, Transfection, DNA metabolism, Peptidylprolyl Isomerase chemistry, Peptidylprolyl Isomerase metabolism
- Abstract
Human parvulin 14 (hPar14) is a folding helper enzyme belonging to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases). This enzyme is thought to play a role in cell-cycle and chromatin remodeling. Although hPar14 was nuclearly localized and bound to double-stranded DNA, the molecular basis of the subcellular localization and the functional regulation remained unknown. Here we show that subcellular localization and DNA-binding ability of hPar14 is regulated by posttranslational modification of its N-terminal domain. As proved by MALDI-TOF mass spectrometry and MS/MS fragmentation, hPar14 is phosphorylated at Ser19 in vitro and in vivo. In human HeLa cells the protein is most likely modified by casein kinase 2 (CK2). Phosphorylation of hPar14 is inhibited both in vitro and in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific inhibitor of CK2 activity. Mutation of Ser19 to Ala abolishes phosphorylation and alters the subcellular localization of hPar14 from predominantly nuclear to significantly cytoplasmic. Immunostaining shows that a Glu19 mutant of hPar14, which mimics the phosphorylated state of Ser19, is localized around the nuclear envelope, but does not penetrate into the nucleoplasm. In contrast to wild-type hPar14, the in vitro DNA-binding affinity of the Glu19 mutant is strongly reduced, suggesting that only the dephosphorylated protein is the active DNA-binding form of hPar14 in the nucleus.
- Published
- 2003
- Full Text
- View/download PDF
48. Reversible inhibition of calcineurin by the polyphenolic aldehyde gossypol.
- Author
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Baumgrass R, Weiwad M, Erdmann F, Liu JO, Wunderlich D, Grabley S, and Fischer G
- Subjects
- Amino Acid Sequence, Animals, CD4-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes metabolism, Cattle, Cells, Cultured, Cyclic AMP-Dependent Protein Kinases chemistry, DNA-Binding Proteins metabolism, Genes, Reporter, Humans, Luciferases genetics, Lymphocyte Activation, Molecular Sequence Data, NFATC Transcription Factors, Phosphorylation, Protein Transport, Substrate Specificity, Transcription Factors metabolism, Calcineurin Inhibitors, Enzyme Inhibitors pharmacology, Gossypol pharmacology, Nuclear Proteins
- Abstract
The reversible inhibition of calcineurin (CaN), which is the only Ca(2+)/calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of (33)P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC(50) value of 15 microm. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC(50) of 9 and 6 microm, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18.CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.
- Published
- 2001
- Full Text
- View/download PDF
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