37 results on '"Rholam, M"'
Search Results
2. CD and NMR studies on model peptides reproducing the proocytocin/neurophysin processing site
- Author
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Scatturin, A., primary, Vertuani, G., additional, D’Auria, G., additional, Gargiulo, M., additional, Paolillo, L., additional, Trivellone, E., additional, Brakch, N., additional, Rholam, M., additional, Cohen, P., additional, and Di Bello, C., additional
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- 1991
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3. Structural modeling of the pro-ocytocin-neurophysin precursor
- Author
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Velikson, B., primary, Cohen, P., additional, Rholam, M., additional, Rose, J. P., additional, Wang, B. C., additional, and Smith, J. C., additional
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- 1998
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4. Structural and functional properties of Bos taurus tryptase: a search for a possible propeptide processing role
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Fiorucci, L, primary, Pallaoro, M, additional, Erba, F, additional, Colombo, A.P, additional, Rholam, M, additional, Cohen, P, additional, and Ascoli, F, additional
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- 1998
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5. ChemInform Abstract: Peptides as Tools for Studying Propeptide and Proprotein Processing
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RHOLAM, M., primary and COHEN, P., additional
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- 1998
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6. Evidence for the presence of a secondary structure at the dibasic processing site of prohormone: the pro-ocytocin model.
- Author
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Paolillo, L., primary, Simonetti, M., additional, Brakch, N., additional, D'Auria, G., additional, Saviano, M., additional, Dettin, M., additional, Rholam, M., additional, Scatturin, A., additional, Di Bello, C., additional, and Cohen, P., additional
- Published
- 1992
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7. Site‐specific mutagenesis identifies amino acid residues critical in prohormone processing.
- Author
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Gomez, S., Boileau, G., Zollinger, L., Nault, C., Rholam, M., and Cohen, P.
- Abstract
Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site‐directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta‐turn structures in the immediate vicinity of the somatostatin‐28 (S‐28) and somatostatin‐14 (S‐14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S‐28 and S‐14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta‐turns had various effects on prosomatostatin processing: substitution of Ala for Pro‐5 drastically decreased prosomatostatin processing and replacement of Pro‐9 by Ala led to the accumulation of the intermediate maturation product [Arg‐2Lys‐1]‐S‐14. In contrast, substitution of Ala for Asn‐12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.
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- 1989
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8. Processing Endoprotease Recognizes a Structural Feature at the Cleavage Site of Peptide Prohormones
- Author
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Brakch, N, Boussetta, H, Rholam, M, and Cohen, P
- Abstract
Pro-ocytocin/neurophysin convertase is a divalent cation-dependent endoprotease isolated from both bovine corpus luteum and neurohypophyseal secretory granules. The putative pro-ocytocin/neurophysin converting enzyme cleaves the Arg12-Ala13bonds of both pro-ocytocin/neurophysin (1 → 20) and pro-ocytocin/neurophysin obtained by hemisynthesis. The minimal efficient substrate structure allowing recognition by this processing endoprotease was defined by measuring its cleavage efficiency and the inhibitory properties of a set of 34 selectively modified derivatives of the (1 → 20) NH2-terminal domain of the ocytocin/neurophysin precursor. The data demonstrate that: (i) the basic Lys11-Arg12doublet, although necessary, is not sufficient; (ii) a minimal substrate length of nine amino acids (residues 7–15 or 8–16) is essential; (iii) those amino acids around the Lys-Arg doublet which contribute to the formation of a possible β-turn-α-helix secondary structure are critical; (iv) substrate recognition by the enzyme may involve several subsites in which structural determinants, situated on both sides of the basic doublet, participate; (v) the NH2-terminal sequence of neurophysin plays a critical role in the correct reading of the cleavage sequence by the processing endoprotease.
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- 1989
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9. Peptides as tools for studying propeptide and proprotein processing
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Rholam, M. and Cohen, P.
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- 1997
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10. ChemInform Abstract: CATION BINDING TO BIOMOLECULES. VI. SCF AB INITIO (PSEUDOPOTENTIAL) COMPUTATIONS ON THE INTERACTION OF ZINC(2+) WITH THE PURINE AND PYRIMIDINE BASES OF THE NUCLEIC ACIDS
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PULLMAN, A., primary, EBBESEN, T., additional, and RHOLAM, M., additional
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- 1979
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11. Investigating the effects of different natural molecules on the structure and oligomerization propensity of hen egg-white lysozyme.
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Chaari A, Fahy C, Chevillot-Biraud A, and Rholam M
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- Dose-Response Relationship, Drug, Kinetics, Protein Aggregates drug effects, Biological Products pharmacology, Muramidase chemistry, Protein Multimerization drug effects
- Abstract
The formation of amyloid aggregates is the hallmark of systemic and neurodegenerative diseases, also known as amyloidosis. Many proteins have been found to aggregate into amyloid-like fibrils and thus process is recognized as general tendency of polypeptides. Inhibition of protein aggregation and fibril formation is thus one of the important strategies in the prevention and treatment of such disease. There is a growing interest of identification of small molecules mainly natural compounds that can prevent or delay amyloid fibril formation. In this work, we report the effect of various compounds from different groups on the amyloid fibrillation of hen egg white lysozyme, a model protein for amyloid formation. Herein, a range of biophysical techniques have been employed in order to establish a systematic approach to study the effect of candidate inhibitors on amyloid aggregation. Results demonstrated that the strategy used show that the different techniques are complimentary in order to elucidate a complete in vitro picture of the effect of the used compounds on HEWL aggregation. Moreover, compared to the data obtained by other groups for the inhibition of lysozyme fibril formation, this work provides new insights into the structural changes (local, secondary, oligomeric, fibrillar structures) undergone by HEWL during aggregation in the presence and absence of inhibitors., (Copyright © 2019 Elsevier B.V. All rights reserved.)
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- 2019
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12. Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods.
- Author
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Chaari A, Fahy C, Chevillot-Biraud A, and Rholam M
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- Acrylamide chemistry, Amyloid metabolism, Animals, Benzothiazoles, Chickens, Chromatography, High Pressure Liquid, Hot Temperature, Hydrogen-Ion Concentration, Kinetics, Light, Microscopy, Atomic Force, Protein Denaturation, Protein Folding, Protein Structure, Secondary, Scattering, Radiation, Spectrophotometry, Spectroscopy, Fourier Transform Infrared, Thiazoles chemistry, Time Factors, Tryptophan chemistry, Muramidase chemistry
- Abstract
Protein misfolding and amyloid formation are an underlying pathological hallmark in a number of prevalent diseases of protein aggregation ranging from Alzheimer's and Parkinson's diseases to systemic lysozyme amyloidosis. In this context, we have used complementary spectroscopic methods to undertake a systematic study of the self-assembly of hen egg-white lysozyme under agitation during a prolonged heating in acidic pH. The kinetics of lysozyme aggregation, monitored by Thioflavin T fluorescence, dynamic light scattering and the quenching of tryptophan fluorescence by acrylamide, is described by a sigmoid curve typical of a nucleation-dependent polymerization process. Nevertheless, we observe significant differences between the values deduced for the kinetic parameters (lag time and aggregation rate). The fibrillation process of lysozyme, as assessed by the attenuated total reflection-Fourier transform infrared spectroscopy, is accompanied by an increase in the β-sheet conformation at the expense of the α-helical conformation but the time-dependent variation of the content of these secondary structures does not evolve as a gradual transition. Moreover, the tryptophan fluorescence-monitored kinetics of lysozyme aggregation is described by three phases in which the temporal decrease of the tryptophan fluorescence quantum yield is of quasilinear nature. Finally, the generated lysozyme fibrils exhibit a typical amyloid morphology with various lengths (observed by atomic force microscopy) and contain exclusively the full-length protein (analyzed by highly performance liquid chromatography). Compared to the data obtained by other groups for the formation of lysozyme fibrils in acidic pH without agitation, this work provides new insights into the structural changes (local, secondary, oligomeric/fibrillar structures) undergone by the lysozyme during the agitation-induced formation of fibrils.
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- 2015
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13. Clearance of genetic variants of amyloid β peptide by neuronal and non-neuronal cells.
- Author
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Panchal M, El Abida B, Lazar N, Fahy C, Dubost L, Friguet B, and Rholam M
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- Amino Acid Sequence, Amyloid beta-Peptides genetics, Animals, CHO Cells, COS Cells, Chlorocebus aethiops, Circular Dichroism, Cricetinae, Cricetulus, Culture Media, Conditioned, Humans, K562 Cells, Kinetics, Molecular Sequence Data, Mutation, Peptide Fragments genetics, Protein Conformation, Rats, Sequence Alignment, Amyloid beta-Peptides chemistry, Amyloid beta-Peptides metabolism, Neurons chemistry, Neurons metabolism, Peptide Fragments chemistry, Peptide Fragments metabolism
- Abstract
The presence of senile plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). The biogenesis and clearance of the amyloid β peptide (A β ), the main component of the lesions, lie at the center of the pathogenesis of AD. In sporadic AD, the increase of A β levels seems to be indicative of failure of clearance mechanisms. We previously showed that the clearance of the wild type A β40 peptide by various neuronal and non-neuronal cells occurs through a same proteolytic process and that A β degradation was primarily dictated by its conformational state (Panchal et al., 2007). To gain further insights on the role of the peptide conformation in the clearance mechanism of A β , two A β40 peptides, known to be associated with amyloid angiopathy (Dutch and Flemish mutations), and the rodent A β40 peptide were catabolized by several cells by using the same experimental approach. The peptide fragments, generated by proteolytic cleavage of substrates in cell supernatants, were identified by LC-MS and the cleavage sites of proteases were deduced. In parallel, conformational states of wild type A β 40 peptide and of the three A β 40 variants were characterized by circular dichroism spectroscopy. We provide data suggesting that discrete conformational changes of A β 40 peptide regulate its clearance rate by neuronal and non-neuronal cells.
- Published
- 2013
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14. Acetonic extract of Buxus sempervirens induces cell cycle arrest, apoptosis and autophagy in breast cancer cells.
- Author
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Ait-Mohamed O, Battisti V, Joliot V, Fritsch L, Pontis J, Medjkane S, Redeuilh C, Lamouri A, Fahy C, Rholam M, Atmani D, and Ait-Si-Ali S
- Subjects
- Apoptosis Regulatory Proteins metabolism, Beclin-1, Blotting, Western, Cell Line, Tumor, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Female, Fluorescent Antibody Technique, Humans, Inhibitor of Apoptosis Proteins metabolism, Membrane Proteins metabolism, Survivin, Acetone chemistry, Apoptosis drug effects, Autophagy drug effects, Breast Neoplasms metabolism, Buxus chemistry, Cell Cycle drug effects, Plant Extracts chemistry, Plant Extracts pharmacology
- Abstract
Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC(50) ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC(50) of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC(50) did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer.
- Published
- 2011
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15. Processing of peptide and hormone precursors at the dibasic cleavage sites.
- Author
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Rholam M and Fahy C
- Subjects
- Amino Acid Sequence, Animals, Hormones chemistry, Hormones genetics, Humans, Models, Molecular, Molecular Sequence Data, Neuropeptides chemistry, Neuropeptides genetics, Proprotein Convertases genetics, Proprotein Convertases metabolism, Protein Precursors chemistry, Protein Precursors genetics, Protein Processing, Post-Translational, Protein Structure, Secondary, Hormones metabolism, Neuropeptides metabolism, Protein Precursors metabolism
- Abstract
Many functionally important cellular peptides and proteins, including hormones, neuropeptides, and growth factors, are synthesized as inactive precursor polypeptides, which require post-translational proteolytic processing to become biologically active polypeptides. This is achieved by the action of a relatively small number of proteases that belong to a family of seven subtilisin-like proprotein convertases (PCs) including furin. In view of this, this review focuses on the importance of privileged secondary structures and of given amino acid residues around basic cleavage sites in substrate recognition by these endoproteases. In addition to their participation in normal cell functions, PCs are crucial for the initiation and progress of many important diseases. Hence, these proteases constitute potential drug targets in medicine. Accordingly, this review also discusses the approaches used to shed light on the cleavage preference and the substrate specificity of the PCs, a prerequisite to select which PCs are promising drug targets in each disease.
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- 2009
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16. Clearance of amyloid-beta peptide by neuronal and non-neuronal cells: proteolytic degradation by secreted and membrane associated proteases.
- Author
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Panchal M, Lazar N, Munoz N, Fahy C, Clamagirand C, Brouard JP, Dubost L, Cohen P, Brakch N, and Rholam M
- Subjects
- Amino Acid Sequence, Amyloid beta-Peptides chemistry, Cell Line, Cell Membrane enzymology, Cell Survival, Chromatography, High Pressure Liquid, Culture Media, Enzyme-Linked Immunosorbent Assay, Extracellular Space metabolism, Intracellular Space metabolism, Mass Spectrometry, Molecular Sequence Data, Neurons chemistry, Neurons enzymology, Neuropeptide Y metabolism, Peptide Fragments chemistry, Spectrophotometry, Ultraviolet, Amyloid beta-Peptides metabolism, Neurons metabolism, Peptide Fragments metabolism, Peptide Hydrolases metabolism
- Abstract
Deposition of amyloid-beta peptide (Abeta) in the brain is an early and invariant feature of all forms of Alzheimer's disease (AD). As for all proteins or peptides, the steady-state level of Abeta peptide is determined not only by its production, but also by its degradation. So, overactive proteases involved in generating Abeta from amyloid precursor protein or underactive Abeta-degrading enzymes could lead to abnormal Abeta deposition in the brain. Since in the sporadic forms of AD (90% of all AD cases) an impaired clearance of Abeta appears to be at the origin of its aggregation and tissue deposition, we have investigated its proteolytic degradation by several neuronal and non-neuronal cells. In this report, we show that these cell types exhibit a similar profile of Abeta-degradation by cell-surface and secreted proteases which were respectively characterized as metallo- and serine proteases. By using a combination of the liquid chromatography/on-line mass spectrometry, we demonstrate that: (i)-the membrane associated protease(s) hydrolizes Abeta40 essentially at Lys(28) Gly(29), Phe(19) Phe(20) and Val(18) Phe(19) bonds; and (ii)-the secreted protease(s) cleaves the generating fragments Abeta (1-28), Abeta (1-19), Abeta (1-18) at His(14) Gln(15) bond and also Abeta (1-28) at Phe(20) Ala(21) and Asp(23) Val(24) sites. This is the first time our results define a proteolytic degradation process of Abeta40 that appears to be independent of the cell type and may represent a general pattern of its enzymatic clearance.
- Published
- 2007
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17. Reactivity of basic amino acid pairs in prohormone processing: model of pro-ocytocin/neurophysin processing domain.
- Author
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Lazar N, Brakch N, Panchal M, Fahy C, and Rholam M
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- Amino Acids, Basic analysis, Circular Dichroism, Kinetics, Neurophysins metabolism, Oxytocin metabolism, Proprotein Convertases metabolism, Protein Precursors metabolism, Protein Structure, Secondary, Protein Structure, Tertiary, Substrate Specificity, Amino Acids, Basic chemistry, Neurophysins chemistry, Oxytocin chemistry, Protein Precursors chemistry
- Abstract
Statistical analysis of several potential dibasic cleavage sites reveals differences in the distribution of basic doublets when the in vivo cleaved sites were compared to those which are not cleaved. Analysis of the substrate specificity of protease Kex2 towards the pro-ocytocin/neurophysin processing domain (pro-OT/Np(7-15) with altered basic pairs shows a cleavage efficiency order in accord with the statistical data. Structural analysis of these substrates indicates that each basic pair is associated with a local and specific conformational change. Thus, the in vivo cleavage hierarchy of dibasic sites is encoded by both the nature of basic pairs and the plasticity of proteolytic processing domains.
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- 2007
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18. Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate.
- Author
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Clamagirand C, El Abida B, Der Garabedian PA, Hanquez C, Dubost L, Marie A, Rholam M, Friguet B, and Cohen P
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- Amino Acid Sequence, Amyloid beta-Protein Precursor immunology, Animals, Chromatography, Gel methods, Chromatography, High Pressure Liquid methods, Humans, Mass Spectrometry methods, Molecular Sequence Data, Peptide Fragments isolation & purification, Peptide Fragments metabolism, Proprotein Convertases metabolism, Sequence Analysis, Protein, Xenopus Proteins immunology, Xenopus Proteins metabolism, Amyloid beta-Protein Precursor isolation & purification, Amyloid beta-Protein Precursor metabolism, Exudates and Transudates chemistry, Skin chemistry, Xenopus Proteins isolation & purification, Xenopus laevis
- Abstract
Using a monoclonal antibody against the entire C-terminal end of human APP(695) (643-695 sequence) and a monoclonal antibody directed against human beta[1-40] amyloid peptide (betaA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of betaA. According to the nature of N- and C-terminal amino acids we identified endogenous beta-, gamma-, epsilon-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the beta-amyloid sequence at same sites as those observed in human betaA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study betaA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human betaA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.
- Published
- 2007
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19. Abnormalities of peptide metabolism in Alzheimer disease.
- Author
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Panchal M, Rholam M, and Brakch N
- Subjects
- Amyloid Precursor Protein Secretases, Amyloid beta-Peptides metabolism, Amyloid beta-Protein Precursor metabolism, Animals, Aspartic Acid Endopeptidases classification, Aspartic Acid Endopeptidases metabolism, Endopeptidases, Endothelin-1 pharmacology, Humans, Models, Biological, Peptide Hydrolases metabolism, Alzheimer Disease metabolism, Peptides metabolism
- Abstract
The steady-state level of peptide hormones represents a balance between their biosynthesis and proteolytic processing by convertases and their catabolism by proteolytic enzymes. Low levels of neuropeptide Y, somatostatin and corticotropin-releasing factor, described in Alzheimer disease (AD), were related to a defect in proteolytic processing of their protein precursors. In contrast the abundance of beta-amyloid peptides, the major protein constituents of senile plaques is likely related to inefficient catabolism. Therefore, attention is mainly focused on convertases that generate active peptides and counter-regulatory proteases that are involved in their catabolism. Some well-described proteases such as NEP are thought to be involved in beta-amyloid catabolism. The search of other possible candidates represents a primary effort in the field. A variety of vascular risk factors such as diabetes, hypertension and arteriosclerosis suggest that the functional vascular defect contributes to AD pathology. It has also been described that beta-amyloid peptides potentiate endothelin-1 induced vasoconstriction. In this review, we will critically evaluate evidence relating proteases implicated in amyloid protein precursor proteolytic processing and beta-amyloid catabolism.
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- 2004
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20. Specific cleavage of beta-amyloid peptides by a metallopeptidase from Xenopus laevis skin secretions.
- Author
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Clamagirand C, Joulie C, Panchal M, Sekhri R, Hanquez C, Cohen P, and Rholam M
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- Amyloid beta-Peptides genetics, Animals, Humans, Metalloendopeptidases isolation & purification, Peptide Fragments genetics, Peptides genetics, Peptides metabolism, Protease Inhibitors metabolism, Skin enzymology, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Xenopus Proteins isolation & purification, Zinc metabolism, Amyloid beta-Peptides metabolism, Bodily Secretions enzymology, Metalloendopeptidases metabolism, Peptide Fragments metabolism, Skin metabolism, Xenopus Proteins metabolism, Xenopus laevis metabolism
- Abstract
Dactylysin (EC 3.5.24.60) is a metalloendopeptidase first isolated from the skin granular gland secretions of Xenopus laevis. This peptidase hydrolyzes bonds on the amino-terminus of singlets and between doublets of hydrophobic amino acids and was considered to play a role in the in vivo inactivation of biologically active regulatory peptides. Here, we show that dactylysin has also the ability to cleave human beta[1-40]-amyloid peptide and related peptides. Cleavage of the wild type beta[1-40]-amyloid peptide form, and to a lesser extent Flemish and Dutch mutants, occurred predominantly at the His14-Glu15 bond. We demonstrate that frog skin exudate contains a full-length amyloid protein precursor detected by immunochemical cross-reactivity with monoclonal antibody against C-terminal human amyloid protein precursor. The possibility that dactylysin, might be involved in normal catabolism of beta amyloid peptide of Xenopus laevis is discussed., (Copyright 2002 Elsevier Science Inc.)
- Published
- 2002
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21. Kinetics of precursor cleavage at the dibasic sites. Involvement of peptide dynamics.
- Author
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Glandieres JM, Hertzog M, Lazar N, Brakch N, Cohen P, Alpert B, and Rholam M
- Subjects
- Amino Acid Sequence, Arginine Vasopressin chemistry, Arginine Vasopressin metabolism, Binding Sites, Circular Dichroism, In Vitro Techniques, Kinetics, Neurophysins chemistry, Neurophysins metabolism, Oxytocin chemistry, Oxytocin metabolism, Protein Processing, Post-Translational, Spectrometry, Fluorescence, Oxytocin analogs & derivatives, Peptides chemistry, Peptides metabolism, Protein Precursors chemistry, Protein Precursors metabolism
- Abstract
The presence in the P'1 position relative to the LysArg doublet of either Phe, Tyr or Trp residues affects only pro-OT/Np(7-15) flexibility. This has a measurable effect on the dynamics of the peptide. Since the same modifications have a major influence on the K(m) and V(max) values of the peptide cleavage, these kinetic parameters should depend on the peptide substrate motions. Therefore, the primary kinetic contribution of substrate cleavage should arise from substrate dynamics rather than from the enzyme.
- Published
- 2002
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22. The somatostatin-28(1-12)-NPAMAP sequence: an essential helical-promoting motif governing prosomatostatin processing at mono- and dibasic sites.
- Author
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Brakch N, Lazar N, Panchal M, Allemandou F, Boileau G, Cohen P, and Rholam M
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- Amino Acid Motifs genetics, Amino Acid Sequence, Amino Acid Substitution genetics, Animals, Arginine metabolism, Circular Dichroism, Fishes, Humans, Hydrolysis, Lysine metabolism, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligopeptides genetics, Protein Conformation, Protein Precursors genetics, Protein Structure, Secondary genetics, Protein Structure, Tertiary genetics, Sequence Deletion, Somatostatin genetics, Somatostatin-28, Spectroscopy, Fourier Transform Infrared, Tumor Cells, Cultured, Oligopeptides chemistry, Oligopeptides metabolism, Protein Precursors chemistry, Protein Precursors metabolism, Protein Processing, Post-Translational genetics, Somatostatin chemistry, Somatostatin metabolism
- Abstract
Proline residues, known to have special structural properties, induce particular conformations which participate in some biological functions. Two prolines (Pro(-9), Pro(-5)) located near the processing sites (Arg(-15) and Arg(-2)Lys(-)(1)) of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin-28 (S-28) and somatostatin-14 (S-14) [Gomez et al. (1989) EMBO J. 8, 2911-2916]. In this study, the importance of the pentapeptide P-A-M-A-P sequence (P-(X)(3)-P pattern), located in the S-28(1-12) segment connecting the mono- and dibasic cleavage sites, was investigated by using site-directed mutagenesis. Analysis of prosomatostatin-derived peptides produced by expression of mutated cDNA species in Neuro2A cells indicated that (i) deletion of PAMAP decreased S-14 production, (ii) deletion of the two Pro residues almost abolished the cleavage at the dibasic site, and (iii) Pro displacement generating the AMAPP motif resulted in a decrease of S-28 production. Moreover, both theoretical and spectroscopic studies of synthetic peptides reproducing the S-28(1-12) sequence bearing critical mutations showed that this sequence can organize as an alpha helical structure. These observations demonstrate that NPAMAP constitutes an accurate alpha-helix nucleation motif allowing for the generation of equal amounts of S-28 and S-14 from their common precursor in Neuro2A cells. Moreover, they emphasize the importance of the S-28(1-12) segment joining Arg(-15) and Arg(-2)Lys(-1) cleavage sites whose conformational organization is essential for controlling their accessibility to the appropriate processing proteases.
- Published
- 2002
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23. Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3.
- Author
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Brakch N, Rholam M, Simonetti M, and Cohen P
- Subjects
- Amino Acid Sequence, Animals, Catalysis, Dimethyl Sulfoxide pharmacology, Kinetics, Molecular Sequence Data, Oxytocin chemistry, Oxytocin metabolism, Peptide Fragments metabolism, Proprotein Convertases, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Fusion Proteins metabolism, Structure-Activity Relationship, Substrate Specificity, Aspartic Acid Endopeptidases metabolism, Endopeptidases metabolism, Oxytocin analogs & derivatives, Oxytocin biosynthesis
- Abstract
Previous studies using selectively modified pro-ocytocin/neurophysin substrate analogues and the purified metalloprotease, pro-ocytocin/neurophysin convertase (magnolysin; EC 3.4 24.62), have shown that dibasic cleavage site processing is associated with a prohormone sequence organized in a beta-turn structure. We have used various peptide analogues of the pro-ocytocin-neurophysin processing domain, and recombinant prohormone convertase 1/3, to test the validity of this property towards this member of the family of prohormone convertases (PCs). The enzymatic cleavage analysis and kinetics showed that: (a) with methyl amide (N-Met) modification, a secondary structure beta-turn breaker, the enzyme substrate interaction was abolished; (b) cleavage was favoured when the dibasic substrate side-chains were oriented in opposite directions; (c) the amino acid present at the P'1 position is important in the enzyme-substrate interaction; (d) the flexibility of the peptide substrate is necessary for the interaction; (e) Addition of dimethylsulfoxide to the cleavage assay favoured the cleavage of the pro-ocytocin/neurophysin large substrate over that of the smaller one pGlu-Arg-Thr-Lys-Arg-methyl coumarin amide. These data allowed us to conclude that proteolytic processing of pro-ocytocin-related peptide substrates by PC1/3 as well as by the metalloenzyme, magnolysin, involves selective recognition of precise cleavage site local secondary structure by the processing enzyme. It is hypothesized that this may represent a general property of peptide precursor proteolytic processing systems.
- Published
- 2000
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24. Role of amino acid sequences flanking dibasic cleavage sites in precursor proteolytic processing. The importance of the first residue C-terminal of the cleavage site.
- Author
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Rholam M, Brakch N, Germain D, Thomas DY, Fahy C, Boussetta H, Boileau G, and Cohen P
- Subjects
- Amino Acid Sequence, Binding Sites, Databases, Factual, Endopeptidases metabolism, Hormones chemistry, Hormones metabolism, Models, Biological, Molecular Sequence Data, Oligopeptides chemistry, Oligopeptides metabolism, Substrate Specificity, Subtilisins metabolism, Peptide Hydrolases metabolism, Proprotein Convertases, Protein Precursors chemistry, Protein Precursors metabolism, Protein Processing, Post-Translational, Saccharomyces cerevisiae Proteins
- Abstract
The amino acid sequences flanking 352 dibasic moieties contained in 83 prohormones and pro-proteins listed in a database were examined. Frequency calculations on the occurrence of given residues at positions P6 to P'4 allowed us to delineate a number of features which might be in part responsible for the in vivo discrimination between cleaved and uncleaved dibasic sites. These include the following: amino acids at these positions were characterized by a large variability in composition and properties; no major contribution of a given precursor subsite to endoprotease specificity was observed; some amino acid residues appeared to occupy preferentially certain precursor subsites (for instance, Met in P6 and P3, Asp and Ala in P'1, Pro in P6, Gly in P3 and P'2 etc.) whereas some others appeared to be excluded. Most amino acid residues occupying the P'1 position in these precursor cleavage sites were tolerated. But the beta-carbon branched side chain residues (Thr, Val, Leu, Ile) and Pro, Cys, Met and Trp were either totally excluded or poorly represented, suggesting that they might be unfavourable to cleavage. The biological relevance of these observations to the efficacy of dibasic cleavage by model propeptide convertases was in vitro tested using both pro-ocytocin convertase and Kex2 protease action on a series of pro-ocytocin related synthetic substrates reproducing the Pro7-->Leu15 sequence of the precursor in which the Ala13 residue (P'1 in the LysArg-Ala motif) was replaced by various amino acid residues. A good correlation was obtained on this model system indicating that P'1 residue of precursor dibasic processing sites is an important feature and may play the role of anchoring motif to S'1 convertase subsite. We tentatively propose that the present database, and the corresponding model, may be used for further investigation of dibasic endoproteolytic processing of propeptides and pro-proteins.
- Published
- 1995
- Full Text
- View/download PDF
25. Prosomatostatin processing in Neuro2A cells. Role of beta-turn structure in the vicinity of the Arg-Lys cleavage site.
- Author
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Brakch N, Boileau G, Simonetti M, Nault C, Joseph-Bravo P, Rholam M, and Cohen P
- Subjects
- Amino Acid Sequence, Animals, Arginine metabolism, Circular Dichroism, DNA, Fourier Analysis, Humans, Lysine metabolism, Mice, Molecular Sequence Data, Mutagenesis, Mutagenesis, Site-Directed, Proline metabolism, Protein Conformation, Protein Precursors genetics, Protein Precursors metabolism, Protein Structure, Secondary, Somatostatin genetics, Somatostatin metabolism, Tumor Cells, Cultured, Arginine chemistry, Lysine chemistry, Proline chemistry, Protein Precursors chemistry, Protein Processing, Post-Translational, Somatostatin chemistry
- Abstract
Proline residues located near the processing sites of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin 28 and somatostatin 14 [Gomez, S., Boileau, G., Zollinger, L., Nault, C., Rholam, M. & Cohen, P. (1989) EMBO J. 8, 2911-2916]. In this study, site-directed and regional mutagenesis of the human prosomatostatin cDNA coupled with analysis by circular-dichroism and Fourier-transform-infrared spectroscopies of the native and mutated peptide sequences were used to elucidate the role of proline in proteolytic processing. Glycine was substituted for proline a position -5 and the beta-turn-promoting sequence Pro-Arg-Glu-Arg, located near the somatostatin-14 cleavage site and predicted to form a beta-turn structure, was replaced by Ser-Ser-Asn-Arg or Tyr-Lys-Gly-Arg, which have been shown by X-ray diffraction to form beta turns in other proteins. Analysis of the prosomatostatin-derived peptides produced by expression of the mutated cDNA species in Neuro2A cells indicated that while Pro-5-->Ala abolished cleavage at the dibasic site, the formation of mutants [Gly-5] prosomatostatin, [Ser-5, Ser-4, Arg-3] prosomatostatin and [Tyr-5, Lys-4, Gly-3] prosomatostatin did not affect cleavage at the dibasic site but produced modifications in both the relative proportions of the generated hormones and in precursor processing efficiency. Moreover, spectroscopical analysis showed that whereas these substitutions did not modify the presence of a beta turn structure in the corresponding peptide sequences, replacement of Pro-5-->Ala resulted in a dramatic increase in alpha-helix accompanied by the significant decrease of other structures including beta turn. The data support the hypothesis that the proline residue near the processing site for somatostatin-14 production is an important structural feature for conferring on the cleavage domain the adequate conformation for accessibility to processing enzymes and permitting production of equivalent amounts of both hormones.
- Published
- 1993
- Full Text
- View/download PDF
26. Role of beta-turn in proteolytic processing of peptide hormone precursors at dibasic sites.
- Author
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Brakch N, Rholam M, Boussetta H, and Cohen P
- Subjects
- Amino Acid Sequence, Circular Dichroism, Hormones metabolism, Molecular Sequence Data, Structure-Activity Relationship, Substrate Specificity, Endopeptidases metabolism, Protein Precursors metabolism, Protein Processing, Post-Translational, Protein Sorting Signals metabolism, Protein Structure, Secondary
- Abstract
Proteolytic activation of prohormones and proproteins occurs most frequently at the level of basic amino acids arranged in doublets. Previous predictions by Rholam et al. [Rholam, M., Nicolas, P., & Cohen, P. (1986) FEBS Lett. 207. 1-6] have indicated, on the basis of 20 prohormone sequences containing 53 dibasic potential processing sites, that dibasic sites situated in, or next to, beta-turns were cleaved in vivo, whereas sites included in ordered structures like beta-sheets or alpha-helices were not. We have used peptide analogs of the proocytocin/neurophysin processing domain and a purified preparation of the putative proocytocin convertase from bovine tissues as a model to demonstrate that (1) processing at dibasic sites is associated with a prohormone sequence organized in a beta-turn structure; (2) the beta-turn is an interchangeable motif since the original sequence could be replaced by an heterologous one possessing the ability to organize as a beta-turn; and (3) this particular secondary structure participates in the catalytic reaction, most likely by favoring the interactions of the substrate with the processing endoprotease. It is concluded that, in addition to the dibasic and other amino acids around the cleavage loci, the beta-turn constitutes a key feature in the proteolytic processing reaction in participating as the favorable conformation for optimal substrate-enzyme active site recognition.
- Published
- 1993
- Full Text
- View/download PDF
27. Differential processing of hormone precursor. Independent production of somatostatins 14 and 28 in transfected neuroblastoma 2A cells.
- Author
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Brakch N, Rholam M, Nault C, Boileau G, and Cohen P
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, DNA Mutational Analysis, In Vitro Techniques, Molecular Sequence Data, Molecular Weight, Protein Processing, Post-Translational, Recombinant Proteins, Transfection, Protein Precursors metabolism, Somatostatin metabolism
- Abstract
Neuro 2A cells infected with a retroviral vector carrying human prosomatostatin cDNA expressed and processed correctly the precursor into somatostatins-14 and -28 [(1989) EMBO J. 8, 2911-2916]. In order to study the mechanisms by which the active hormone sequences arise, site directed mutagenesis was performed on either the dibasic (ArgLys) or monobasic (Arg) cleavage sites involved in the production of somatostatins-14 and -28, respectively. Radioimmunochemical analysis of the somatostatin-related products indicated that replacement of either Arg-2-Lys-1 by Asn-2-Asn-1 or of Arg-15 by Asn-15 resulted in the exclusive production of either somatostatin-28 or -14, respectively. Moreover only prosomatostatin[1-76] was detected and no somatostatin-28[1-12] could be measured in cell extracts. Selective suppression of either somatostatin-14 or somatostatin-28 release by mutation did not affect the level of production of the other hormone but resulted in a correlative increase of unprocessed prosomatostatin. It is concluded that in this cell type (i) somatostatin-14 is exclusively generated by dibasic cleavage at the Arg-2-Lys-1 site of the intact precursor with concomitant production of prosomatostatin[1-76], and (ii) no direct interactions between the monobasic and dibasic processing domains occur.
- Published
- 1991
- Full Text
- View/download PDF
28. Evidence for beta-turn structure in model peptides reproducing pro-ocytocin/neurophysin proteolytic processing site.
- Author
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Rholam M, Cohen P, Brakch N, Paolillo L, Scatturin A, and Di Bello C
- Subjects
- Amino Acid Sequence, Circular Dichroism, Magnetic Resonance Spectroscopy, Models, Chemical, Molecular Sequence Data, Oligopeptides metabolism, Oxytocin metabolism, Protein Conformation, Neurophysins metabolism, Oligopeptides chemical synthesis, Oxytocin analogs & derivatives, Peptide Hydrolases metabolism
- Abstract
The structural organization of small peptides reproducing the amino acid sequence of the common ocytocin/neurophysin precursor around the LysArg cleavage locus was investigated by a combination of spectroscopical techniques. In water both circular dichroism and [1H] NMR spectra indicated that these peptides adopted a random conformation. Evidence for folded structures was obtained when these compounds were placed in a membrane-like environment i.e. 40 mM SDS in phosphate buffer or trifluoroethanol. Whereas the CD spectra indicated the formation of various types of beta-turn in rapid equilibrium, measurements of NH temperature coefficients and Nuclear Overhauser Effects by 400 and 500 MHz NMR revealed the existence of contacts and of a folded conformation. These observations are discussed in relation with previous hypothesis made on the secondary structure organization of the proteolytic processing site of polypeptide hormone precursors.
- Published
- 1990
- Full Text
- View/download PDF
29. Salt-dependent structural changes of neurohormones: lithium ions induce conformational rearrangements of ocytocin to a vasopressin-like structure.
- Author
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Rholam M, Nicolas P, and Cohen P
- Subjects
- Amino Acid Sequence, Lithium Chloride, Neurophysins metabolism, Peptides, Cyclic metabolism, Protein Conformation, Structure-Activity Relationship, Chlorides pharmacology, Lithium pharmacology, Oxytocin metabolism, Vasopressins metabolism
- Abstract
The preferred average conformation and structural subdomain interactions of the nonapeptide hormones vasopressin and ocytocin have been analyzed through the determination of their hydrodynamic volume and the thermal coefficient of the frictional resistance to rotation of their tyrosine residue. A spherical gross shape and an ellipsoidal gross shape were assessed respectively for ocytocin and vasopressin by fluorescence polarization analysis. Investigation of the thermal coefficient of viscosity and the critical temperature of both hormones and analogues indicated that strong interactions hold together the two structural subdomains of ocytocin (the flexible six-membered ring and the COOH-terminal tripeptide tail). An opposite situation was found in the case of vasopressin where such interactions could not be detected between the rigid ring and the flexible COOH-terminal tail. Lithium ions were shown to promote ocytocin binding to specific neurophysin sites restricted, under standard conditions, to vasopressin. In the presence of lithium, the gross conformational shape of ocytocin becomes similar to that of vasopressin but in the absence of salt. In addition, the ocytocin ring becomes more rigid in the presence of lithium while decreasing interactions between the ring and the COOH-terminal tail were detected. It is proposed that lithium ions induce specific conformational rearrangements of ocytocin toward a vasopressin-like structure, allowing recognition of this hormonal ligand by a specific vasopressin binding domain of neurophysins.
- Published
- 1985
- Full Text
- View/download PDF
30. Synthetic peptide substrates as models to study a pro-ocytocin/neurophysin converting enzyme.
- Author
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Créminon C, Rholam M, Boussetta H, Marrakchi N, and Cohen P
- Subjects
- Animals, Brain Chemistry, Camelus, Cattle, Chromatography, High Pressure Liquid, Mass Spectrometry, Protein Conformation, Structure-Activity Relationship, Endopeptidases metabolism, Neurophysins metabolism, Oxytocin metabolism, Protein Precursors metabolism
- Abstract
The selectivity and mechanism of processing at paired basic amino acids in hormone precursors was studied on several analogues of the (1-20)-aminoterminal domain of the ocytocin/neurophysin precursor in a cleavage assay by an endoprotease partially purified from bovine pituitary secretory granules. Peptide analogues with amino acid substitutions in, and around, the basic doublet were synthesized and used as substrates. The data obtained demonstrate the strict requirement of the processing enzyme for basic amino acids in tandem within a possibly preferred conformation which may be highly conserved in the aminoterminal domain of this hormone precursor.
- Published
- 1988
- Full Text
- View/download PDF
31. Side-by-side dimerization of neurophysin: sedimentation velocity, viscometry, and fluorescence polarization studies.
- Author
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Rholam M and Nicolas P
- Subjects
- Animals, Cattle, Chemical Phenomena, Chemistry, Physical, Fluorescence Polarization, Macromolecular Substances, Mathematics, Molecular Weight, Ultracentrifugation, Viscosity, Neurophysins
- Published
- 1981
- Full Text
- View/download PDF
32. Proocytocin/neurophysin convertase from bovine neurohypophysis and corpus luteum secretory granules: complete purification, structure-function relationships, and competitive inhibitor.
- Author
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Plevrakis I, Clamagirand C, Créminon C, Brakch N, Rholam M, and Cohen P
- Subjects
- Amino Acid Sequence, Animals, Cattle, Chromatography, Affinity, Chromatography, Gel, Endopeptidases metabolism, Female, Kinetics, Molecular Sequence Data, Molecular Weight, Oligopeptides chemical synthesis, Peptides chemical synthesis, Protease Inhibitors, Substrate Specificity, Corpus Luteum enzymology, Cytoplasmic Granules enzymology, Endopeptidases isolation & purification, Pituitary Gland, Posterior enzymology
- Abstract
Structure-function relationship studies were conducted on the proocytocin/neurophysin endoprotease previously characterized in both bovine neurohypophyseal and corpus luteum granules, using as a reference substrate a synthetic peptide reproducing the entire (1-20) NH2-terminal domain of the precursor. The [D-Arg12] derivative of proocytocin/neurophysin (1-20) was found to be a good competitive inhibitor of the enzyme (Ki = 30 microM), while the [D-Lys11] derivative was not. This allowed the complete purification of two isoforms of the endoprotease (Mr 58,000 and 52,000, respectively) by affinity chromatography using covalently immobilized [D-Arg12] proocytocin/neurophysin (1-20) as the affinity adsorbent. The use of selectively modified or truncated forms of the reference substrate or of the [D-Arg12] competitive inhibitor of the endoprotease established clearly that this basic pair specific convertase is sensitive to modification of the substrate structure either at the basic residues of the cleavage locus or at amino acids around this site (i.e., Pro7 and Gly9). It is concluded that longer distance interactions between amino acids situated on both the NH2 and COOH sides of the basic doublet Lys11Arg12 may contribute to the stabilization of a preferred substrate conformation allowing recognition by the enzyme subsites.
- Published
- 1989
- Full Text
- View/download PDF
33. Precursors for peptide hormones share common secondary structures forming features at the proteolytic processing sites.
- Author
-
Rholam M, Nicolas P, and Cohen P
- Subjects
- Amino Acid Sequence, Probability, Protein Conformation, Hormones biosynthesis, Peptide Hydrolases physiology, Protein Precursors metabolism
- Abstract
We have analyzed the amino acid sequences situated around the putative proteolytic cleavage sites in twenty different biosynthetic precursors of peptide hormones by processing enzymes. The prediction of the probability for forming secondary structures around the basic amino acids, constituting the cleavage sites, was made using the modified method of Chou and Fasman. The results indicate that the processing sequences which are cleaved in vivo, are in all cases located inside regions with high beta-turn formation probability or else immediately adjacent to these structures. The beta-turn forming region at the cleavage locus, is flanked on both sides by amino acid sequences with a high probability for forming highly ordered structures, either beta-sheet or alpha-helix. These conformational features are not found in precursors around dibasic pairs, i.e. putative cleavage loci, but which are not cleaved in vivo and appear to be conserved. We hypothesize that beta-turns including the basic amino acids doublets, flanked by highly ordered secondary structures (either beta-sheet or alpha-helix) may constitute a minimal requirement for the recognition by the endoproteases involved in the processing of these precursors.
- Published
- 1986
- Full Text
- View/download PDF
34. Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets.
- Author
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Nicolas P, Delfour A, Boussetta H, Morel A, Rholam M, and Cohen P
- Subjects
- Amino Acids analysis, Animals, Biological Assay, Chromatography, High Pressure Liquid, In Vitro Techniques, Mass Spectrometry, Pituitary Gland, Anterior metabolism, Rats, Somatostatin chemical synthesis, Somatostatin-28 analogs & derivatives, Somatostatin analogs & derivatives
- Abstract
Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells.
- Published
- 1986
- Full Text
- View/download PDF
35. Binding of neurohypophyseal peptides to neurophysin dimer promotes formation of compact and spherical complexes.
- Author
-
Rholam M, Nicolas P, and Cohen P
- Subjects
- Binding Sites, Ligands, Macromolecular Substances, Models, Chemical, Oligopeptides, Peptide Fragments, Protein Conformation, Viscosity, Neurophysins, Oxytocin, Vasopressins
- Abstract
Previous hydrodynamic studies [Rholam, M., & Nicolas, P. (1981) Biochemistry 20, 5837-5843] have demonstrated that the dimerization of a neurophysin monomer (prolate ellipsoid with an axial ratio, due to asymmetry, of 5.2) results in a decreased asymmetry (axial ratio, due to asymmetry, of 3.6) as the consequence of a side-by-side association process. By a combination of hydrodynamic measurements, including the use of sedimentation velocity, viscometry, and fluorescence polarization spectroscopy, the influence of hormone binding on the shape and asymmetry properties of the neurophysin dimer was evaluated. The binding of ocytocin, vasopressin, and the tripeptide analogue of the N-terminal sequence of ocytocin, Cys(S-Me)-Tyr-Ile-NH2, results in an increase of S020,W and a decrease in both the reduced viscosity and rotational relaxation time of the bis-liganded dimeric species vs. the nonliganded form. The axial ratio (a/b) due to asymmetry of the ligand-bound dimers was found in each case to be equal to, or slightly greater than, 1.0, indicating a compact spherical shape (Stokes radius 21 A). The profound alteration on molecular dimensions observed upon ligand binding is shown to be the consequence of a ligand-induced conformational change and might explain the intradimeric binding sites positive cooperativity. It is tentatively proposed that the pseudospherical shape of the neurophysin-hormone complexes may enhance the stability of neurophysin and contribute to the prevention of leakage of neuropeptides through the membrane of neurosecretory granules. The data provide a remarkable example of a small protein with a high content in disulfide links and that undergoes conspicuous changes in conformation under the influence of nonapeptide, or tripeptide, ligands.
- Published
- 1982
- Full Text
- View/download PDF
36. Conformational flexibility of neurophysin as investigated by local motions of fluorophores. Relationships with neurohypophyseal hormone binding.
- Author
-
Rholam M and Nicolas P
- Subjects
- Animals, Dansyl Compounds, Fluorescence Polarization, Kinetics, Protein Binding, Protein Conformation, Thermodynamics, Tyrosine, Neurophysins metabolism, Pituitary Hormones, Posterior metabolism
- Abstract
Flexibility of various structural domains of neurophysin and neurophysin-neurohypophyseal hormone complexes has been investigated through the fast rotational motion of fluorophores in highly viscous medium. Despite seven intrachain disulfide links, it is shown that some domains of neurophysin remain highly flexible. Dimerization of neurophysin does not affect the structural integrity of the individual subunits, each subdomain being conformationally equivalent within each protomer of the unliganded dimer. The absence of heterogeneous fluorescence anisotropy precludes the existence of a dimer tautomerization equilibrium. Binding of the hormonal ligands to neurophysin dimer promotes a large conformational change over the whole protein structure as assessed by differential alterations of the flexibility-rigidity and intrasegmental interaction properties of domains that do not participate directly to the dimerization/binding areas. The order of free-energy coupling between ligand binding and protein subunit association has been evaluated. Data are consistent with a model in which the first mole of bound ligand stabilizes the dimer by increasing the intersubunit contacts while the second mole of ligand induces most of the described conformational change. Accordingly, the positive cooperativity between the two dimeric binding sites is linked mainly to the binding of the second ligand. The induced structural change is perceived differently by each subunit as assessed by opposite local motions of Tyr49 in each liganded protomer and leads to the formation of a dimeric complex with a global pseudospherical symmetry although containing domains of local asymmetry.
- Published
- 1985
- Full Text
- View/download PDF
37. Bovine neurophysin dimerization and neurohypophyseal hormone binding.
- Author
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Nicolas P, Batelier G, Rholam M, and Cohen P
- Subjects
- Animals, Cattle, Hydrogen-Ion Concentration, Kinetics, Ligands, Macromolecular Substances, Mathematics, Protein Binding, Temperature, Thermodynamics, Lypressin, Neurophysins metabolism, Oxytocin chemical synthesis
- Published
- 1980
- Full Text
- View/download PDF
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