9 results on '"Cyril Barinka"'
Search Results
2. Characterization of the class IIa histone deacetylases substrate specificity
- Author
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Zsofia Kutil, Marat Meleshin, Petra Baranova, Barbora Havlinova, Mike Schutkowski, and Cyril Barinka
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Histone Deacetylase Inhibitors ,Genetics ,Amino Acid Sequence ,Peptides ,Molecular Biology ,Biochemistry ,Histone Deacetylases ,Substrate Specificity ,Biotechnology - Abstract
Class IIa histone deacetylases (HDACs) play critical roles in vertebrate development and physiology, yet direct evidence of their intrinsic deacetylase activity and on substrate specificity regarding the peptide sequence is still missing. In this study, we designed and synthesized a combinatorial peptide library allowing us to profile class IIa HDACs sequence specificity at positions +3 through -3 from the central lysine modified by the well-accepted trifluoroacetyl function. Our data revealed a strong preference for bulky aromatic acids directly flanking the central trifluoroacetyllysine, while all class IIa HDACs disfavor positively charged residues and proline at the +1/-1 positions. The chemical nature of amino acid residues N-terminally to the central trifluoroacetyllysine has a more profound effect on substrate recognition as compared to residues located C-terminally. These findings were validated by designing selected favored and disfavored peptide sequences, with the favored ones are accepted with catalytic efficacy of 75 000 and 525 000 M
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- 2022
3. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries
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Ivan Snajdr, Pavel Majer, Zsofia Kutil, Barbora Havlinova, Cyril Barinka, Zora Novakova, Martin Hadzima, David Rauh, Lubica Skultetyova, Marat Meleshin, Mike Schutkowski, Jana Mikešová, Jiri Pavlicek, and Petra Baranova
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0301 basic medicine ,Static Electricity ,Peptide ,macromolecular substances ,Histone Deacetylase 6 ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,0302 clinical medicine ,Catalytic Domain ,Hydrolase ,Genetics ,Humans ,Molecular Biology ,chemistry.chemical_classification ,Lysine ,HDAC6 ,Peptide Fragments ,Cytosol ,HEK293 Cells ,030104 developmental biology ,chemistry ,Substrate specificity ,DNA microarray ,030217 neurology & neurosurgery ,Protein Binding ,Biotechnology - Abstract
Histone deacetylase 6 (HDAC6) is a multidomain cytosolic hydrolase acting mostly on nonhistone protein substrates. Investigations of the substrate specificity of HDAC6 are confounded by the presence of 2 catalytically active deacetylase domains (DD1 and DD2). In this study, acetylome peptide microarrays and peptide libraries were used to map the substrate specificity of DD1 and DD2 of human HDAC6. The results show that DD1 is solely responsible for the deacetylation of substrates harboring the acetyllysine at their C terminus, whereas DD2 exclusively deacetylates peptides with an internal acetyllysine residue. Also, statistical analysis of the deacetylation data revealed amino acid preferences at individual positions flanking the acetyllysine, where glycine and arginine residues are favored at positions N-terminal to the central acetyllysine; negatively charged glutamate is strongly disfavored throughout the sequence. Finally, the deacylation activity of HDAC6 was profiled by using a panel of acyl derivatives of the optimized peptide substrate and showed that HDAC6 acts as a proficient deformylase. Our data thus offer a detailed insight into the substrate preferences of the individual HDAC6 domains at the peptide level, and these findings can in turn help in elucidating the biologic roles of the enzyme and facilitate the development of new domain-specific inhibitors as research tools or therapeutic agents.-Kutil, Z., Skultetyova, L., Rauh, D., Meleshin, M., Snajdr, I., Novakova, Z., Mikesova, J., Pavlicek, J., Hadzima, M., Baranova, P., Havlinova, B., Majer, P., Schutkowski, M., Barinka, C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.
- Published
- 2018
4. The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity
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Jan Konvalinka, Jakub Ptacek, Jana Nedvedova, Barbora Havlinova, Cyril Barinka, and Michal Navrátil
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0301 basic medicine ,chemistry.chemical_classification ,Circular dichroism ,030102 biochemistry & molecular biology ,Metallopeptidase ,Chemistry ,Mutant ,chemistry.chemical_element ,Calcium ,Biochemistry ,03 medical and health sciences ,030104 developmental biology ,Enzyme ,Glutamate carboxypeptidase II ,Biophysics ,Secretion ,Binding site ,Molecular Biology - Abstract
Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca2+ -binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca2+ coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches. Our data unequivocally show that even a slight disruption of the Ca2+ -binding site destabilizes the three-dimensional fold of GCPII and is associated with impaired secretion, a high propensity to form nonphysiological oligomers, and an inability to bind active site-targeted ligands. Additionally, the Ca2+ -binding site is critical for maintenance of the native homodimeric quaternary arrangement of GCPII, which is indispensable for its enzymatic activity. Overall, our results offer a clear picture of the importance of Ca2+ for the structural integrity and hydrolytic activity of human GCPII and by extension homologous members of the M28 zinc-dependent metallopeptidase family.
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- 2018
5. Design of composite inhibitors targeting glutamate carboxypeptidase II: the importance of effector functionalities
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Cyril Barinka, Zora Novakova, Jacek Lubkowski, Clifford E. Berkman, Jessie R. Nedrow, Joeseph K. Choi, Cindy J. Choy, and Jiri Cerny
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Glutamate Carboxypeptidase II ,Models, Molecular ,0301 basic medicine ,Molecular model ,Stereochemistry ,Protein Data Bank (RCSB PDB) ,Biology ,Crystallography, X-Ray ,Biochemistry ,Article ,Structure-Activity Relationship ,03 medical and health sciences ,Hydrolase ,Glutamate carboxypeptidase II ,Humans ,Structure–activity relationship ,Phosphoric Acids ,Enzyme Inhibitors ,Molecular Biology ,Dose-Response Relationship, Drug ,Molecular Structure ,Effector ,Hydrogen Bonding ,Phosphoramidate ,Cell Biology ,computer.file_format ,Protein Data Bank ,Amides ,030104 developmental biology ,Drug Design ,Antigens, Surface ,computer - Abstract
UNLABELLED Inhibitors targeting human glutamate carboxypeptidase II (GCPII) typically consist of a P1' glutamate-derived binding module, which warrants the high affinity and specificity, linked to an effector function that is positioned within the entrance funnel of the enzyme. Here we present a comprehensive structural and computational study aimed at dissecting the importance of the effector function for GCPII binding and affinity. To this end we determined crystal structures of human GCPII in complex with a series of phosphoramidate-based inhibitors harboring effector functions of diverse physicochemical characteristics. Our data show that higher binding affinities of phosphoramidates, compared to matching phosphonates, are linked to the presence of additional hydrogen bonds between Glu424 and Gly518 of the enzyme and the amide group of the phosphoramidate. While the positioning of the P1' glutamate-derived module within the S1' pocket of GCPII is invariant, interaction interfaces between effector functions and residues lining the entrance funnel are highly varied, with the positively charged arginine patch defined by Arg463, Arg534 and Arg536 being the only 'hot-spot' common to several studied complexes. This variability stems in part from the fact that the effector/GCPII interfaces generally encompass isolated areas of nonpolar residues within the entrance funnel and resulting van der Waals contacts lack the directionality typical for hydrogen bonding interactions. The presented data unravel a complexity of binding modes of inhibitors within non-prime site(s) of GCPII and can be exploited for the design of novel GCPII-specific compounds. PDB ID CODES Atomic coordinates of the present structures together with the experimental structure factor amplitudes were deposited at the RCSB Protein Data Bank under accession codes 4P44 (complex with JRB-4-81), 4P45 (complex with JRB-4-73), 4P4B (complex with CTT54), 4P4D (complex with MP1C), 4P4E (complex with MP1D), 4P4F (complex with NC-2-40), 4P4I (complex with T33) and 4P4J (complex with T33D).
- Published
- 2015
6. Tissue expression and enzymologic characterization of human prostate specific membrane antigen and its rat and pig orthologs
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Josef Zamecnik, Jan Konvalinka, Pavel Šácha, Klára Hlouchová, Petra Mlčochová, Vratislav Horak, Cyril Barinka, and Miroslava Rovenska
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Male ,Pathology ,medicine.medical_specialty ,Swine ,medicine.drug_class ,Urology ,Molecular Sequence Data ,Cell ,Context (language use) ,Biology ,Kidney ,Monoclonal antibody ,Gene Expression Regulation, Enzymologic ,Species Specificity ,Western blot ,In vivo ,Prostate ,Testis ,medicine ,Glutamate carboxypeptidase II ,Animals ,Humans ,Amino Acid Sequence ,medicine.diagnostic_test ,Prostate-Specific Antigen ,Molecular biology ,Rats ,medicine.anatomical_structure ,Spinal Cord ,Oncology ,Rats, Inbred Lew ,Models, Animal ,Swine, Miniature ,Immunohistochemistry - Abstract
BACKGROUND Prostate specific membrane antigen (PSMA), also called glutamate carboxypeptidase II (GCPII), is a target enzyme for diagnosis and treatment of prostate cancer. Moreover, it is upregulated in the vasculature of most solid tumors and is therefore a potential target for the generation of novel antineoplastics. In this context, we analyze the possibility of using rat and pig as animal models for enzymologic and in vivo studies. METHODS We prepared the recombinant extracellular part of human, rat, and pig GCPII in S2 cell media and characterized the activity and inhibition profiles of the three orthologs by radioenzymatic assay. We performed Western blot analysis of GCPII expression in human, rat, and pig tissues using the monoclonal antibody GCP-04 and confirmed these findings by activity measurements and immunohistochemistry. RESULTS The three recombinant proteins show similar specific enzymatic activities and inhibition profiles. Tissue expression analysis revealed that most of the pig and human tissues show at least some GCPII-positivity, while the expression pattern in rat is more restricted. Moreover, tissues such as prostate and testes exhibit different GCPII expression levels among the species studied. CONCLUSIONS The rat and pig orthologs of GCPII seem to be suitable to approximate human GCPII in enzymologic studies. However, the diffuse expression pattern of GCPII in animal and human tissues could be a caveat for the potential utilization of GCPII-targeted anticancer drugs. Furthermore, variations in GCPII tissue distribution among the species studied should be considered when using rat or pig as models for antineoplastic drug discovery. Prostate 68: 171–182, 2008. © 2007 Wiley-Liss, Inc.
- Published
- 2007
7. Biochemical characterization of human glutamate carboxypeptidase III
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Cyril Barinka, Pavel Šácha, Jan Konvalinka, Klusak, Klára Hlouchová, Petra Mlčochová, Lubomír Rulíšek, and Pavel Majer
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chemistry.chemical_classification ,medicine.medical_specialty ,Metallopeptidase ,Biology ,Biochemistry ,Amino acid ,Cellular and Molecular Neuroscience ,Endocrinology ,Enzyme ,Glutamate carboxypeptidase ,chemistry ,Internal medicine ,medicine ,Carboxypeptidase A ,biology.protein ,Glutamate carboxypeptidase II ,Site-directed mutagenesis ,Peptide sequence - Abstract
Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N-acetyl-L-aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostate-specific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N-glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N-acetyl-L-aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes.
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- 2006
8. Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer
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Jan Konvalinka, Rolf Hilgenfeld, Barbara S. Slusher, Jeroen R. Mesters, Cyril Barinka, Weixing Li, Takashi Tsukamoto, and Pavel Majer
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Glutamate Carboxypeptidase II ,Male ,Models, Molecular ,Protein Folding ,Glycosylation ,Protein Conformation ,Molecular Sequence Data ,Glutamic Acid ,Biology ,Crystallography, X-Ray ,Neuroprotection ,Article ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Hydrolase ,Glutamate carboxypeptidase II ,Humans ,Amino Acid Sequence ,Enzyme Inhibitors ,Binding site ,Neurotransmitter ,Molecular Biology ,Neurons ,chemistry.chemical_classification ,Binding Sites ,General Immunology and Microbiology ,Hydrolysis ,General Neuroscience ,Glutamate receptor ,Prostatic Neoplasms ,Glutamic acid ,Recombinant Proteins ,Enzyme ,Biochemistry ,chemistry - Abstract
Membrane-bound glutamate carboxypeptidase II (GCPII) is a zinc metalloenzyme that catalyzes the hydrolysis of the neurotransmitter N-acetyl-L-aspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartate and L-glutamate (which is itself a neurotransmitter). Potent and selective GCPII inhibitors have been shown to decrease brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain. Here, we report crystal structures of the extracellular part of GCPII in complex with both potent and weak inhibitors and with glutamate, the product of the enzyme's hydrolysis reaction, at 2.0, 2.4, and 2.2 Å resolution, respectively. GCPII folds into three domains: protease-like, apical, and C-terminal. All three participate in substrate binding, with two of them directly involved in C-terminal glutamate recognition. One of the carbohydrate moieties of the enzyme is essential for homodimer formation of GCPII. The three-dimensional structures presented here reveal an induced-fit substrate-binding mode of this key enzyme and provide essential information for the design of GCPII inhibitors useful in the treatment of neuronal diseases and prostate cancer.
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- 2006
9. Poster sessions AP11: Neurotransmitters, Transporter and Enzymes
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Pavel Majer, Camilo Rojas, Markéta Rinnová, Pavel Šácha, Barbara S. Slusher, Jan Konvalinka, Cyril Barinka, and Petra Mlčochová
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chemistry.chemical_classification ,Dipeptidase ,Glycosylation ,biology ,Glutamate receptor ,Transporter ,Biochemistry ,Amino acid ,law.invention ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Enzyme ,chemistry ,law ,Glutamate carboxypeptidase II ,biology.protein ,Recombinant DNA - Abstract
Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as N-acetylated-α-linked-acidic dipeptidase, NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. We report the mapping of the entire coding region of GCPII and identification of the region sufficient and necessary for the production of active recombinant protein. Extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) was expressed in Drosophila Schneider's cells and purified to homogeneity. A novel assay for hydrolytic activity of GCPII, based on fluorimetric detection of released alpha-amino groups was established, and used for enzymological characterization of GCPII. The potential of this assay for high-throughput inhibitor testing was evaluated and pH dependence for the enzymatic activity have been analysed. Using a complete set of protected dipeptides, substrate specificity of recombinant GCPII was elucidated. Ac-Glu-Met, Ac-Asp-Met and surprisingly Ac-Ala-Met were identified as novel substrates for GCPII. The glycosylation has been found indispensable for the activity of the enzyme. A series of point mutants of the enzyme has been expressed and purified and the glycosylation sites critical for the proteolytic activity have been identified.
- Published
- 2008
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