48 results on '"Brakch, N."'
Search Results
2. Aortic remodelling in Fabry disease
- Author
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Barbey, F, Qanadli, S D, Juli, C, Brakch, N, Palacek, T, Rizzo, E, Jeanrenaud, X, Eckhardt, B, Linhart, A, Barbey, F, Qanadli, S D, Juli, C, Brakch, N, Palacek, T, Rizzo, E, Jeanrenaud, X, Eckhardt, B, and Linhart, A
- Abstract
Aims To evaluate thoracic aortic dilation in patients with Fabry disease (FD). Methods and results A cohort of 106 patients with FD (52 males; 54 females) from three European centres were studied. The diameter of the thoracic aorta was assessed at three levels (sinus of Valsalva, ascending aorta, and descending aorta) using echocardiograms and cardiovascular magnetic resonance imaging. Aortic dilation at the sinus of Valsalva was found in 32.7% of males and 5.6% of females; aneurysms were present in 9.6% of males and 1.9% of females. No aortic dilation was observed in the descending aorta. There was no correlation between aortic diameter at the sinus of Valsalva and cardiovascular risk factors. Conclusion Fabry disease should be considered as a cardiovascular disease that affects the heart and arterial vasculature, including the thoracic aorta. Thus, patients with FD should be closely monitored for the presence, and possible progression and complications of aortic dilation. Clinical Trial Registration: Protocol 101/01. Ethics committee, Faculty of Medicine, Lausanne
- Published
- 2010
3. Evidence for a role of sphingosine-1 phosphate in cardiovascular remodelling in Fabry disease
- Author
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Brakch, N, Dormond, O, Bekri, S, Golshayan, D, Correvon, M, Mazzolai, L, Steinmann, B, Barbey, F, Brakch, N, Dormond, O, Bekri, S, Golshayan, D, Correvon, M, Mazzolai, L, Steinmann, B, and Barbey, F
- Abstract
AIMS: A hallmark of Fabry disease is the concomitant development of left-ventricular hypertrophy and arterial intima-media thickening, the pathogenesis of which is thought to be related to the presence of a plasmatic circulating growth-promoting factor. We therefore characterized the plasma of patients with Fabry disease in order to identify this factor. METHODS AND RESULTS: Using a classical biochemical strategy, we isolated and identified sphingosine-1 phosphate (S1P) as a proliferative factor present in the plasma of patients with Fabry disease. Plasma S1P levels were significantly higher in 17 patients with Fabry disease compared with 17 healthy controls (225 +/- 40 vs. 164 +/- 17 ng/mL; P = 0.005). There was a positive correlation between plasma S1P levels and both common carotid artery intima-media thickness and left-ventricular mass index (r(2) = 0.47; P = 0.006 and r(2) = 0.53; P = 0.0007, respectively). In an experimental model, mice treated with S1P developed cardiovascular remodelling similar to that observed in patients with Fabry disease. CONCLUSION: Sphingosine-1 phosphate participates in cardiovascular remodelling in Fabry disease. Our findings have implications for the treatment of cardiovascular involvement in Fabry disease.
- Published
- 2010
4. Processo di maturazione della glicoproteina gp160: analisi conformazionale e studi cinetici su peptidi riproducenti il sito di clivaggio enzimatico di gp160
- Author
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Dettin, Monica, Scarinci, Claudia, Brakch, N., and DI BELLO, Carlo
- Published
- 1995
5. RENIN PROSEQUENCE INTERACTIONS WITH RENIN OPTIMIZE ENZYMATIC ACTIVITY AND ENHANCE CONSTITUTIVE SECRETION: PP.24.476
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Brakch, N, primary and Nussberger, J, additional
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- 2010
- Full Text
- View/download PDF
6. Aortic remodelling in Fabry disease
- Author
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Barbey, F., primary, Qanadli, S. D., additional, Juli, C., additional, Brakch, N., additional, Palacek, T., additional, Rizzo, E., additional, Jeanrenaud, X., additional, Eckhardt, B., additional, and Linhart, A., additional
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- 2009
- Full Text
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7. Renal endothelin receptor type b upregulation in rats with low or high renin hypertension
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BRAKCH, N, primary
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- 2004
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8. Predominant basolateral proteolytic processing of prosomatostatin into somatostatin-28 in polarized LLC-PK1 cells
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Brakch, N, primary, Yang, X.-F, additional, Crine, P, additional, Cohen, P, additional, and Boileau, G, additional
- Published
- 1997
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9. Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans
- Author
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Beauvais, A, primary, Monod, M, additional, Wyniger, J, additional, Debeaupuis, J P, additional, Grouzmann, E, additional, Brakch, N, additional, Svab, J, additional, Hovanessian, A G, additional, and Latgé, J P, additional
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- 1997
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10. Structural Investigation and Kinetic Characterization of Potential Cleavage Sites of HIV Gp160 by Human Furin and Pc1
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Brakch, N., primary, Dettin, M., additional, Scarinci, C., additional, Seidah, N.G., additional, and Dibello, C., additional
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- 1995
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11. Comparative proteolytic processing of rat prosomatostatin by the convertases PC1, PC2, furin, PACE4 and PC5 in constitutive and regulated secretory pathways
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Brakch, N., primary, Galanopoulou, A.S., additional, Patel, Y.C., additional, Boileau, G., additional, and Seidah, N.G., additional
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- 1995
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12. Processing of Human Prosomatostatin in AtT-20 Cells: S-28 and S-14 Are Generated in Different Secretory Pathways
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Brakch, N., primary, Cohen, P., additional, and Boileau, G., additional
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- 1994
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13. Evidence for the presence of a secondary structure at the dibasic processing site of prohormone: the pro-ocytocin model.
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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
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- 1992
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14. Dibasic cleavage site is required for sorting to the regulated secretory pathway for both pro- and neuropeptide Y.
- Author
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Brakch, N., Allemandou, F., Cavadas, C., Grouzmann, E., and Brunner, H.R.
- Subjects
- *
NEUROPEPTIDE Y , *PROTEIN kinases - Abstract
To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non-mutated proneuropeptide Y (ProNPY) in pituitary-derived AtT20 cells. The mutations were carried out on dibasic cleavage site and or ProNPY C-terminal sequence. Targeting to the regulated secretory pathway was studied using protein kinase A (8-BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase. The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive-like secretory pathways. ProNPY secretion behaviour was not Proteolytic cleavage efficiency-dependent. The dibasic cleavage was essential for ProNPY and NPY cAMP-dependent regulated secretion and may have function as a retention signal. [ABSTRACT FROM AUTHOR]
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- 2002
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15. 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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16. Blood pressure regulation in low renin subjects.
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Brakch N, Nussberger J, Brakch, Noureddine, and Nussberger, Juerg
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- 2003
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17. Predominant basolateral proteolytic processing of prosomatostatin into somatostatin-28 in polarized LLC-PK 1 cells
- Author
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Brakch, N, Yang, X.-F, Crine, P, Cohen, P, and Boileau, G
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- 1997
- Full Text
- View/download PDF
18. Proteolytic Processing of Pro-Neuropeptide Y In Neuroendocrine Cells.
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Brakch, N, Brunner, H R, and Grouzmann, E
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- 1998
19. Evidence for a role of sphingosine-1 phosphate in cardiovascular remodelling in Fabry disease
- Author
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Lucia Mazzolai, Soumeya Bekri, Beat Steinmann, Magali Correvon, Dela Golshayan, Frédéric Barbey, Noureddine Brakch, Olivier Dormond, University of Zurich, and Brakch, N
- Subjects
Adult ,Male ,medicine.medical_specialty ,Carotid Artery, Common ,610 Medicine & health ,Muscle, Smooth, Vascular ,2705 Cardiology and Cardiovascular Medicine ,Muscle hypertrophy ,Pathogenesis ,Mice ,chemistry.chemical_compound ,Sphingosine ,Internal medicine ,medicine.artery ,Animals ,Humans ,Medicine ,Common carotid artery ,Sphingosine-1-phosphate ,Rats, Wistar ,Aorta ,Cells, Cultured ,Cell Proliferation ,Ventricular Remodeling ,business.industry ,Middle Aged ,medicine.disease ,Fabry disease ,Rats ,Endocrinology ,chemistry ,Intima-media thickness ,10036 Medical Clinic ,Concomitant ,Fabry Disease ,Female ,Hypertrophy, Left Ventricular ,Endothelium, Vascular ,Lysophospholipids ,Tunica Intima ,Tunica Media ,Cardiology and Cardiovascular Medicine ,business ,Biomarkers - Abstract
AIMS: A hallmark of Fabry disease is the concomitant development of left-ventricular hypertrophy and arterial intima-media thickening, the pathogenesis of which is thought to be related to the presence of a plasmatic circulating growth-promoting factor. We therefore characterized the plasma of patients with Fabry disease in order to identify this factor. METHODS AND RESULTS: Using a classical biochemical strategy, we isolated and identified sphingosine-1 phosphate (S1P) as a proliferative factor present in the plasma of patients with Fabry disease. Plasma S1P levels were significantly higher in 17 patients with Fabry disease compared with 17 healthy controls (225 +/- 40 vs. 164 +/- 17 ng/mL; P = 0.005). There was a positive correlation between plasma S1P levels and both common carotid artery intima-media thickness and left-ventricular mass index (r(2) = 0.47; P = 0.006 and r(2) = 0.53; P = 0.0007, respectively). In an experimental model, mice treated with S1P developed cardiovascular remodelling similar to that observed in patients with Fabry disease. CONCLUSION: Sphingosine-1 phosphate participates in cardiovascular remodelling in Fabry disease. Our findings have implications for the treatment of cardiovascular involvement in Fabry disease.
- Published
- 2017
20. Lack of enantioselectivity in the SULT1A3-catalyzed sulfoconjugation of normetanephrine enantiomers: an in vitro and computational study.
- Author
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Grouzmann E, Gualtierotti JB, Gerber-Lemaire S, Abid K, Brakch N, Pedretti A, Testa B, and Vistoli G
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- Arylsulfotransferase chemistry, Catalysis, Humans, Kinetics, Models, Molecular, Molecular Dynamics Simulation, Normetanephrine chemical synthesis, Normetanephrine chemistry, Stereoisomerism, Arylsulfotransferase metabolism, Normetanephrine metabolism
- Abstract
(1R)-Normetanephrine is the natural stereoisomeric substrate for sulfotransferase 1A3 (SULT1A3)-catalyzed sulfonation. Nothing appears known on the enantioselectivity of the reaction despite its potential significance in the metabolism of adrenergic amines and in clinical biochemistry. We confronted the kinetic parameters of the sulfoconjugation of synthetic (1R)-normetanephrine and (1S)-normetanephrine by recombinant human SULT1A3 to a docking model of each normetanephrine enantiomer with SULT1A3 and the 3'-phosphoadenosine-5'-phosphosulfate cofactor on the basis of molecular modeling and molecular dynamics simulations of the stability of the complexes. The K(M), V(max), and k(cat) values for the sulfonation of (1R)-normetanephrine, (1S)-normetanephrine, and racemic normetanephrine were similar. In silico models were consistent with these findings as they showed that the binding modes of the two enantiomers were almost identical. In conclusion, SULT1A3 is not substrate-enantioselective toward normetanephrine, an unexpected finding explainable by a mutual adaptability between the ligands and SULT1A3 through an "induced-fit model" in the catalytic pocket., (Copyright © 2012 Wiley Periodicals, Inc.)
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- 2013
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21. Monoamine oxidase A down-regulation contributes to high metanephrine concentration in pheochromocytoma.
- Author
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Grouzmann E, Matter M, Bilz S, Herren A, Triponez F, Henzen C, Kim KS, Zulewski H, Buclin T, Brakch N, and Abid K
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- Adolescent, Adrenal Medulla metabolism, Adult, Aged, Catechol O-Methyltransferase analysis, Catechol O-Methyltransferase physiology, Catecholamines metabolism, Down-Regulation, Female, Gene Expression Regulation, Enzymologic, Humans, Male, Metanephrine analysis, Middle Aged, Monoamine Oxidase analysis, Monoamine Oxidase genetics, Adrenal Gland Neoplasms metabolism, Metanephrine metabolism, Monoamine Oxidase physiology, Pheochromocytoma metabolism
- Abstract
Context: The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes., Objective: The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT., Design: A multicenter comparative study was conducted., Study Participants: The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control., Main Outcome Measures: CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations., Results: At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor., Conclusion: High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.
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- 2012
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22. The renin prosequence enhances constitutive secretion of renin and optimizes renin activity.
- Author
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Brakch N, Allemandou F, Keller I, and Nussberger J
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Chlorocebus aethiops, Enzyme Activation physiology, Humans, Mice, Peptide Fragments metabolism, Protein Folding, Protein Precursors biosynthesis, Renin biosynthesis, Peptide Fragments chemistry, Protein Precursors chemistry, Protein Precursors metabolism, Renin chemistry, Renin metabolism
- Abstract
Renin is cleaved from its precursor prorenin into mature renin. We investigated the impact of the renin proregion on the generation and secretion of enzymatically active renin. We compared the effects of the following sequences of human prorenin with those of wild type prorenin[1-383]: prosequence [1-43], hinge sequence [1-62], Des[1-43]prorenin ("renin"), Des[1-62]prorenin and prorenin[N260]. These sequences were individually expressed in CV1 cells (constitutive pathway model) and AtT20 cells (regulated and constitutive pathways model), and Des[1-43]prorenin was also coexpressed together with the different prosequences. Renin concentration and activity were measured in cell extracts and culture media. Deletion of the prosequence reduces renin activity in both cell types, but it leaves (total) renin concentration unchanged. Coexpression of the prosequence with renin enhances renin secretion in both cell types: Constitutively secreted renin is enhanced by coexpression of renin together with any of the prosequence containing molecules [1-43], [1-62] or prorenin[N260]. Immunofluorescence in AtT20 cells shows lysosomal typical labeling of prorenin and Des[1-43]prorenin. In AtT20 cells expressing prorenin[1-383], stimulation of regulated secretion increases prorenin but not renin release. The renin prosequence [1-43] optimizes renin activity possibly through appropriate protein folding and it enhances the constitutive secretion of (pro)renin. The major part of generated renin may be targeted to lysosomes., (© 2011 Bentham Science Publishers Ltd.)
- Published
- 2011
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23. Autophagosome maturation is impaired in Fabry disease.
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Chévrier M, Brakch N, Céline L, Genty D, Ramdani Y, Moll S, Djavaheri-Mergny M, Brasse-Lagnel C, Annie Laquerrière AL, Barbey F, and Bekri S
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Adult, Biopsy, Blotting, Western, Enzyme Replacement Therapy, Fabry Disease drug therapy, Fibroblasts drug effects, Fibroblasts metabolism, Fibroblasts pathology, Gene Expression Regulation drug effects, Humans, Isoenzymes pharmacology, Isoenzymes therapeutic use, Kidney drug effects, Kidney pathology, Kidney ultrastructure, Male, Microscopy, Confocal, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, Middle Aged, Phagosomes drug effects, Phagosomes metabolism, Phagosomes ultrastructure, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Proteins, Sequestosome-1 Protein, Vacuoles drug effects, Vacuoles metabolism, Vacuoles ultrastructure, alpha-Galactosidase pharmacology, alpha-Galactosidase therapeutic use, Autophagy drug effects, Fabry Disease pathology, Phagosomes pathology
- Abstract
Fabry disease is a lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A. The disease is characterized by severe major organ involvement, but the pathologic mechanisms responsible have not been elucidated. Disruptions of autophagic processes have been reported for other LSDs, but have not yet been investigated in Fabry disease. Renal biopsies were obtained from 5 adult male Fabry disease patients before and after 3 years of enzyme replacement therapy (ERT) with agalsidase alfa. Vacuole accumulation was seen in renal biopsies from all patients compared with control biopsies. Decreases in the number of vacuoles were seen after 3 years of ERT primarily in renal endothelial cells and mesangial cells. Measurement of the levels of LC3, a specific autophagy marker, in cultured cells from Fabry patients revealed increased basal levels compared to cells from non-Fabry subjects and a larger increase in response to starvation than seen in non-Fabry cells. Starvation in the presence of protease inhibitors did not result in a significant increase in LC3 in Fabry cells, whereas a further increase in LC3 was observed in non-Fabry cells, an observation that is consistent with impaired autophagic flux in Fabry disease. Overexpression of LC3 mRNA in Fabry fibroblasts compared to control cells is consistent with an upregulation of autophagy. Furthermore, LC3 and p62/SQSTM1 (that binds to LC3) staining in renal tissues and in cultured fibroblasts from Fabry patients supports impairment of autophagic flux. These findings suggest that Fabry disease is linked to a deregulation of autophagy.
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- 2010
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24. Aortic remodelling in Fabry disease.
- Author
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Barbey F, Qanadli SD, Juli C, Brakch N, Palacek T, Rizzo E, Jeanrenaud X, Eckhardt B, and Linhart A
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- Adult, Aged, Aged, 80 and over, Echocardiography, Fabry Disease physiopathology, Female, Humans, Magnetic Resonance Angiography, Male, Middle Aged, Sinus of Valsalva pathology, Young Adult, Aortic Aneurysm, Thoracic pathology, Fabry Disease pathology
- Abstract
Aims: To evaluate thoracic aortic dilation in patients with Fabry disease (FD)., Methods and Results: A cohort of 106 patients with FD (52 males; 54 females) from three European centres were studied. The diameter of the thoracic aorta was assessed at three levels (sinus of Valsalva, ascending aorta, and descending aorta) using echocardiograms and cardiovascular magnetic resonance imaging. Aortic dilation at the sinus of Valsalva was found in 32.7% of males and 5.6% of females; aneurysms were present in 9.6% of males and 1.9% of females. No aortic dilation was observed in the descending aorta. There was no correlation between aortic diameter at the sinus of Valsalva and cardiovascular risk factors., Conclusion: Fabry disease should be considered as a cardiovascular disease that affects the heart and arterial vasculature, including the thoracic aorta. Thus, patients with FD should be closely monitored for the presence, and possible progression and complications of aortic dilation., Clinical Trial Registration: Protocol 101/01. Ethics committee, Faculty of Medicine, Lausanne.
- Published
- 2010
- Full Text
- View/download PDF
25. Evidence for a role of sphingosine-1 phosphate in cardiovascular remodelling in Fabry disease.
- Author
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Brakch N, Dormond O, Bekri S, Golshayan D, Correvon M, Mazzolai L, Steinmann B, and Barbey F
- Subjects
- Adult, Animals, Aorta pathology, Biomarkers blood, Carotid Artery, Common pathology, Cell Proliferation, Cells, Cultured, Endothelium, Vascular pathology, Fabry Disease complications, Fabry Disease pathology, Female, Humans, Hypertrophy, Left Ventricular etiology, Hypertrophy, Left Ventricular pathology, Lysophospholipids metabolism, Male, Mice, Middle Aged, Muscle, Smooth, Vascular pathology, Rats, Rats, Wistar, Sphingosine metabolism, Sphingosine physiology, Tunica Intima pathology, Tunica Media pathology, Fabry Disease blood, Lysophospholipids physiology, Sphingosine analogs & derivatives, Ventricular Remodeling physiology
- Abstract
Aims: A hallmark of Fabry disease is the concomitant development of left-ventricular hypertrophy and arterial intima-media thickening, the pathogenesis of which is thought to be related to the presence of a plasmatic circulating growth-promoting factor. We therefore characterized the plasma of patients with Fabry disease in order to identify this factor., Methods and Results: Using a classical biochemical strategy, we isolated and identified sphingosine-1 phosphate (S1P) as a proliferative factor present in the plasma of patients with Fabry disease. Plasma S1P levels were significantly higher in 17 patients with Fabry disease compared with 17 healthy controls (225 +/- 40 vs. 164 +/- 17 ng/mL; P = 0.005). There was a positive correlation between plasma S1P levels and both common carotid artery intima-media thickness and left-ventricular mass index (r(2) = 0.47; P = 0.006 and r(2) = 0.53; P = 0.0007, respectively). In an experimental model, mice treated with S1P developed cardiovascular remodelling similar to that observed in patients with Fabry disease., Conclusion: Sphingosine-1 phosphate participates in cardiovascular remodelling in Fabry disease. Our findings have implications for the treatment of cardiovascular involvement in Fabry disease.
- Published
- 2010
- Full Text
- View/download PDF
26. Kinetic study of neuropeptide Y (NPY) proteolysis in blood and identification of NPY3-35: a new peptide generated by plasma kallikrein.
- Author
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Abid K, Rochat B, Lassahn PG, Stöcklin R, Michalet S, Brakch N, Aubert JF, Vatansever B, Tella P, De Meester I, and Grouzmann E
- Subjects
- Adamantane analogs & derivatives, Adamantane pharmacology, Adult, Chromatography, Liquid methods, Female, Humans, Kinetics, Male, Mass Spectrometry methods, Neuropeptide Y chemistry, Nitriles pharmacology, Peptide Fragments metabolism, Peptides chemistry, Peptides pharmacology, Plasma Kallikrein chemistry, Protease Inhibitors pharmacology, Protein Structure, Tertiary, Pyrrolidines pharmacology, Substrate Specificity, Vildagliptin, Neuropeptide Y metabolism, Peptide Fragments chemistry, Plasma Kallikrein metabolism
- Abstract
There is little information on how neuropeptide Y (NPY) proteolysis by peptidases occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive forms and also because the factors affecting the expression of these enzymes have been poorly studied. In the present study, LC-MS/MS was used to identify and quantify NPY fragments resulting from peptidolytic cleavage of NPY(1-36) upon incubation with human serum. Kinetic studies indicated that NPY(1-36) is rapidly cleaved in serum into 3 main fragments with the following order of efficacy: NPY(3-36) >> NPY(3-35) > NPY(2-36). Trace amounts of additional NPY forms were identified by accurate mass spectrometry. Specific inhibitors of dipeptidyl peptidase IV, kallikrein, and aminopeptidase P prevented the production of NPY(3-36), NPY(3-35), and NPY(2-36), respectively. Plasma kallikrein at physiological concentrations converted NPY(3-36) into NPY(3-35). Receptor binding assays revealed that NPY(3-35) is unable to bind to NPY Y1, Y2, and Y5 receptors; thus NPY(3-35) may represent the major metabolic clearance product of the Y2/Y5 agonist, NPY(3-36).
- Published
- 2009
- Full Text
- View/download PDF
27. 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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- View/download PDF
28. 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
- Subjects
- 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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29. SLURP1 is a late marker of epidermal differentiation and is absent in Mal de Meleda.
- Author
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Favre B, Plantard L, Aeschbach L, Brakch N, Christen-Zaech S, de Viragh PA, Sergeant A, Huber M, and Hohl D
- Subjects
- Antigens, Ly metabolism, Biomarkers metabolism, Calcium metabolism, Cells, Cultured, Humans, Keratinocytes metabolism, Keratoderma, Palmoplantar metabolism, Mutation, Skin metabolism, Urokinase-Type Plasminogen Activator metabolism, Antigens, Ly genetics, Cell Differentiation, Epidermis pathology, Keratoderma, Palmoplantar genetics, Keratoderma, Palmoplantar pathology, Urokinase-Type Plasminogen Activator deficiency, Urokinase-Type Plasminogen Activator genetics
- Abstract
SLURP1 is a secreted member of the LY6/PLAUR protein family. Mutations in the SLURP1 gene are the cause of Mal de Meleda (MDM), a rare autosomal recessive genetic disease, characterized by inflammatory palmoplantar keratoderma. In this study, we have analyzed the expression of SLURP1 in normal and MDM skin. SLURP1 was found to be a marker of late differentiation, predominantly expressed in the granular layer of skin, notably the acrosyringium. Moreover, SLURP1 was also identified in several biological fluids such as sweat, saliva, tears, and urine from normal volunteers. In palmoplantar sections from MDM patients, as well as in their sweat, mutant SLURP1, including the new variant R71H-SLURP1, was either absent or barely detectable. Transfected human embryonic kidney 293T cells expressed the MDM mutant SLURP1 containing the single amino-acid substitution G86R but did not tolerate the MDM mutation W15R located in the signal peptide. Thus, most MDM mutations in SLURP1 affect either the expression, integrity, or stability of the protein, suggesting that a simple immunologic test could be used as a rapid screening procedure.
- Published
- 2007
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30. Increased carotid intima-media thickness in the absence of atherosclerotic plaques in an adult population with Fabry disease.
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Barbey F, Brakch N, Linhart A, Jeanrenaud X, Palecek T, Bultas J, Burnier M, and Hayoz D
- Subjects
- Adult, Carotid Artery Diseases diagnostic imaging, Carotid Artery, Common diagnostic imaging, Carotid Artery, Common pathology, Fabry Disease diagnostic imaging, Female, Humans, Male, Middle Aged, Ultrasonography, Carotid Artery Diseases pathology, Fabry Disease pathology, Tunica Intima pathology, Tunica Media pathology
- Abstract
Aim: Fabry disease is considered primarily as a progressive small vessel disease, with ischaemic degenerative lesions involving the kidneys, brain and heart. Macrovascular involvement in male patients includes an accelerated wall hypertrophy of the radial artery and a thickening of the intima-media of the common carotid artery. The aim of this study is to evaluate the prevalence and severity of carotid artery atherosclerosis in hemizygous and heterozygous patients with Fabry disease, compared with a matched control population., Methods: The common carotid artery intima-media thickness (IMT) of 53 patients with Fabry disease (24 men, 29 women) was measured by high-definition ultrasonography, and the presence or absence of atherosclerotic plaques reported. Results were compared with those of 120 age-matched healthy individuals (83 men, 37 women)., Results: The common carotid artery IMT was increased to the same extent in male and female patients with Fabry disease (706+/-211 microm and 749+/-395 microm, respectively) compared with that of the control population (614+/-113 microm). In the Fabry population, IMT did not correlate with either systolic blood pressure or with renal function (plasma creatinine). In the control population, only systolic blood pressure was positively and significantly correlated with IMT. Atherosclerotic plaques in the common carotid artery were not observed in any patient with Fabry disease, whereas 34% of the control population had carotid artery plaques, as evidenced by focal non-homogeneous intima-media thickening greater than 1.2 mm., Conclusion: This study presents evidence of a major increase in common carotid artery IMT, both in hemizygous and heterozygous patients with Fabry disease, in the absence of focal atherosclerotic plaques. These results suggest that the conduit arteries may be protected from atherosclerosis in Fabry disease.
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- 2006
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31. Cardiac and vascular hypertrophy in Fabry disease: evidence for a new mechanism independent of blood pressure and glycosphingolipid deposition.
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Barbey F, Brakch N, Linhart A, Rosenblatt-Velin N, Jeanrenaud X, Qanadli S, Steinmann B, Burnier M, Palecek T, Bultas J, and Hayoz D
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Animals, Blood Proteins pharmacology, Cell Proliferation drug effects, Cells, Cultured, Echocardiography, Fabry Disease blood, Fabry Disease etiology, Female, Humans, Hypertrophy, Left Ventricular etiology, Male, Mice, Middle Aged, Myocytes, Cardiac pathology, Rats, Tunica Intima pathology, Tunica Intima physiopathology, Tunica Media pathology, Tunica Media physiopathology, Blood Pressure, Carotid Artery, Common physiopathology, Fabry Disease physiopathology, Glycosphingolipids metabolism, Hypertrophy, Left Ventricular physiopathology
- Abstract
Objective: Fabry disease is an X-linked disorder resulting from alpha-galactosidase A deficiency. The cardiovascular findings include left ventricular hypertrophy (LVH) and increased intima-media thickness of the common carotid artery (CCA IMT). The current study examined the possible correlation between these parameters. To corroborate these clinical findings in vitro, plasma from Fabry patients was tested for possible proliferative effect on rat vascular smooth muscle cells (vascular smooth muscle cell [VSMC]) and mouse neonatal cardiomyocytes., Methods and Results: Thirty male and 38 female patients were enrolled. LVH was found in 60% of men and 39% of women. Increased CCA IMT was equally present in males and females. There was a strong positive correlation between LV mass and CCA IMT (r2=0.27; P<0.0001). VSMC and neonatal cardiomyocyte proliferative response in vitro correlated with CCA IMT (r2=0.39; P<0.0004) and LV mass index (r2=0.19; P=0.028), respectively., Conclusions: LVH and CCA IMT occur concomitantly in Fabry suggesting common pathogenesis. The underlying cause may be a circulating growth-promoting factor whose presence has been confirmed in vitro.
- Published
- 2006
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32. Abnormalities of peptide metabolism in Alzheimer disease.
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Panchal M, Rholam M, and Brakch N
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- 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.
- Published
- 2004
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33. Renal endothelin receptor type B upregulation in rats with low or high renin hypertension.
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Brakch N, Abdel-Sayed S, Allemandou F, Wang Q, Aubert JF, Brunner HR, and Nussberger J
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- Animals, Blood Pressure, Body Weight, Endothelin-1 metabolism, Endothelin-1 pharmacology, Heart Rate, Hypertension, Renal chemically induced, Iodine Radioisotopes, Myocardium metabolism, Radioligand Assay, Rats, Rats, Wistar, Receptor, Endothelin A metabolism, Sodium Chloride, Up-Regulation, Desoxycorticosterone analogs & derivatives, Hypertension, Renal metabolism, Kidney metabolism, Receptor, Endothelin B metabolism, Renin metabolism
- Abstract
Objectives: To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats., Methods: Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out., Results: MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h., Conclusions: Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.
- Published
- 2004
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34. Identification of SLURP-1 as an epidermal neuromodulator explains the clinical phenotype of Mal de Meleda.
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Chimienti F, Hogg RC, Plantard L, Lehmann C, Brakch N, Fischer J, Huber M, Bertrand D, and Hohl D
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- Acetylcholine metabolism, Amino Acid Sequence, Animals, Antigens, Ly chemistry, Antigens, Ly isolation & purification, Antigens, Ly pharmacology, Cell Line, Cell Nucleus metabolism, Clone Cells, DNA, Complementary administration & dosage, DNA, Complementary metabolism, Dose-Response Relationship, Drug, Female, Genes, Recessive, Humans, Keratoderma, Palmoplantar metabolism, Keratoderma, Palmoplantar pathology, Microinjections, Models, Molecular, Moths cytology, Mutation, Oocytes metabolism, Patch-Clamp Techniques, Peptides chemistry, Peptides genetics, Peptides metabolism, Phenotype, Protein Structure, Tertiary, Receptors, Cholinergic drug effects, Receptors, Cholinergic metabolism, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Urokinase-Type Plasminogen Activator chemistry, Urokinase-Type Plasminogen Activator isolation & purification, Urokinase-Type Plasminogen Activator pharmacology, Xenopus laevis physiology, Antigens, Ly genetics, Epidermis metabolism, Keratoderma, Palmoplantar genetics, Neurotransmitter Agents metabolism, Urokinase-Type Plasminogen Activator genetics
- Abstract
Mal de Meleda is an autosomal recessive inflammatory and keratotic palmoplantar skin disorder due to mutations in the ARS B gene, encoding for SLURP-1 (secreted mammalian Ly-6/uPAR-related protein 1). SLURP-1 belongs to the Ly-6/uPAR superfamily of receptor and secreted proteins, which participate in signal transduction, immune cell activation or cellular adhesion. The high degree of structural similarity between SLURP-1 and the three fingers motif of snake neurotoxins and Lynx1 suggests that this protein interacts with the neuronal acetylcholine receptors. We found that SLURP-1 potentiates the human alpha 7 nicotinic acetylcholine receptors that are present in keratinocytes. These results identify SLURP-1 as a secreted epidermal neuromodulator which is likely to be essential for both epidermal homeostasis and inhibition of TNF-alpha release by macrophages during wound healing. This explains both the hyperproliferative as well as the inflammatory clinical phenotype of Mal de Meleda.
- Published
- 2003
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35. Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects.
- Author
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Abdel-Sayed S, Nussberger J, Aubert JF, Gohlke P, Brunner HR, and Brakch N
- Subjects
- Animals, Enzyme-Linked Immunosorbent Assay, Humans, Male, Models, Animal, Rats, Rats, Wistar, Endothelin-1 blood, Hypertension blood
- Abstract
Background: Endothelin-1 is an endothelium-derived potent vasoconstrictor peptide of 21 amino acids. To establish reference values in different models of hypertension and in human subjects an assay for plasma immunoreactive endothelin-1 (ET-1) was optimized., Methods: ET-1 is extracted by acetone from 1 mL of plasma and subjected to a sensitive enzyme-linked immunosorbent assay., Results: The detection limit for plasma ET-1 is 0.05 fmol/mL. Mean recoveries of the 1, 2, 5, and 10 fmol of ET-1 added to 1 mL of plasma were 66%, 75%, 85%, and 92%, respectively. Within- and between-assay coefficients of variation were < or =12% and < or =10%, respectively. Assay accuracy was demonstrated by consistent recoveries of added ET-1 over the entire physiologic range of plasma concentrations and by the linearity of ET-1 concentrations measured in serially diluted plasma extracts (r = 0.99). No ET-1 was detected when albumin buffer was extracted instead of plasma. Using this method, we found increased ET-1 levels in plasma of three experimental rat models of hypertension: stroke prone spontaneously hypertensive rats (SP-SHR), deoxycorticosterone acetate-salt hypertensive rats, and one kidney-one clip hypertensive rats. In contrast, plasma ET-1 levels of SHR were half those of normotensive Wistar rats. In two kidney-one clip hypertensive rats, plasma ET-1 concentrations were not different from those found in sham-operated control rats. Plasma ET-1 concentrations of 37 healthy men were 0.85 +/- 0.26 fmol/ml (mean +/- SD)., Conclusions: The present assay reliably measures ET-1 levels in rat and human plasma. It allows to discriminate between different forms of hypertension with high or low circulating levels of ET-1.
- Published
- 2003
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36. Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template.
- Author
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Allemandou F, Nussberger J, Brunner HR, and Brakch N
- Abstract
We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed.
- Published
- 2003
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37. Loss of dipeptidylpeptidase IV activity in chronic rhinosinusitis contributes to the neurogenic inflammation induced by substance P in the nasal mucosa.
- Author
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Grouzmann E, Monod M, Landis B, Wilk S, Brakch N, Nicoucar K, Giger R, Malis D, Szalay-Quinodoz I, Cavadas C, Morel DR, and Lacroix JS
- Subjects
- Animals, Chronic Disease, Humans, Models, Biological, Nasal Mucosa blood supply, Nasal Mucosa drug effects, Rhinitis chemically induced, Rhinitis pathology, Substance P pharmacology, Swine, Vascular Resistance drug effects, Vasodilator Agents pharmacology, Dipeptidyl Peptidase 4 metabolism, Nasal Mucosa enzymology, Rhinitis enzymology
- Abstract
In this study, we have found that dipeptidylpeptidase IV (DPPIV) plays in vivo an active role in the modulation of the inflammatory response of chronic rhinosinusitis. Human nasal mucosa expresses DPPIV-like immunoreactivity in submucosal seromucus glands, leukocytes, and endothelial cells of blood vessels. DPPIV enzymatic activity in nasal tissue biopsies taken from patients suffering from chronic rhinosinusitis was correlated inversely with the density of inflammatory cells in the nasal mucosa, and the DPPIV activity rose when chronic rhinosinusitis was treated. By using a pig animal model, we have shown that the intranasal administration of recombinant DPPIV decreased the vasodilatation induced by exogenous substance P (SP), a proinflammatory peptide released by sensory nerves. In contrast, an inhibitor of DPPIV enhanced the vasodilatatory effect at low doses of SP. SP5-11 was 100- to 1000-fold less potent than SP as a vasodilator of the nasal mucosa. The vasodilatatory effect of SP was abolished by a NK1 receptor antagonist. In conclusion, these results suggest a new pathophysiological pathway for rhinitis based on clinical observations in humans, indicating the involvement of an enzyme to modulate non-adrenergic and non-cholinergic substrate that occurred during nasal dysfunctions.
- Published
- 2002
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38. 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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39. 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
- Subjects
- 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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40. 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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41. Role of prohormone convertases in pro-neuropeptide Y processing: coexpression and in vitro kinetic investigations.
- Author
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Brakch N, Rist B, Beck-Sickinger AG, Goenaga J, Wittek R, Bürger E, Brunner HR, and Grouzmann E
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Aspartic Acid Endopeptidases genetics, Furin, Gene Expression, Genetic Vectors, Kinetics, Mass Spectrometry, Mice, Molecular Sequence Data, Neuropeptide Y genetics, Peptide Fragments analysis, Proprotein Convertase 2, Proprotein Convertases, Protein Precursors genetics, Rats, Recombinant Proteins metabolism, Serine Endopeptidases genetics, Substrate Specificity, Subtilisins genetics, Tumor Cells, Cultured, Vaccinia virus genetics, Aspartic Acid Endopeptidases metabolism, Neuropeptide Y metabolism, Proprotein Convertase 1, Protein Precursors metabolism, Protein Processing, Post-Translational, Serine Endopeptidases metabolism, Subtilisins metabolism
- Abstract
Proneuropeptide Y (ProNPY) undergoes cleavage at a single dibasic site Lys38-Arg39 resulting in the formation of 1-39 amino acid NPY which is further processed successively by carboxypeptidase-like and peptidylglycine alpha-amidating monooxygenase enzymes. To investigate whether prohormone convertases are involved in ProNPY processing, a vaccinia virus derived expression system was used to coexpress recombinant ProNPY with each of the prohormone convertases PC1/3, PC2, furin, and PACE4 in Neuro2A and NIH 3T3 cell lines as regulated neuroendocrine and constitutive prototype cell lines, respectively. The analysis of processed products shows that only PC1/3 generates NPY in NIH 3T3 cells while both PC1/3 and PC2 are able to generate NPY in Neuro2A cells. The convertases furin and PACE4 are unable to process ProNPY in either cell line. Moreover, comparative in vitro cleavage of recombinant NPY precursor by the enzymes PC1/3, PC2 and furin shows that only PC1/3 and PC2 are involved in specific cleavage of the dibasic site. Kinetic studies demonstrate that PC1/3 cleaves ProNPY more efficiently than PC2. The main difference between the cleavage efficiency is observed in the Vmax values whereas no major difference is observed in Km values. In addition the cleavage by PC1/3 and PC2 of two peptides reproducing the dibasic cleavage site with different amino acid sequence lengths namely (20-49)-ProNPY and (28-43)-ProNPY was studied. These shortened ProNPY substrates, when recognized by the enzymes, are more efficiently cleaved than ProNPY itself. The shortest peptide is not cleaved by PC2 while it is by PC1/3. On the basis of these observations it is proposed, first, that the constitutive secreted NPY does not result from the cleavage carried out by ubiquitously expressed enzymes furin and PACE4; second, that PC1/3 and PC2 are not equipotent in the cleavage of ProNPY; and third, substrate peptide length might discriminate PC1/3 and PC2 processing activity.
- Published
- 1997
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42. 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
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43. 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
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44. 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
45. Differential processing of hormone precursor. Independent production of somatostatins 14 and 28 in transfected neuroblastoma 2A cells.
- Author
-
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
46. Synthesis and processing of pro-ocytocin in bovine corpus luteum and granulosa cells.
- Author
-
Camier M, Benveniste D, Barré N, Brakch N, and Cohen P
- Subjects
- Amino Acid Sequence, Animals, Blotting, Western, Cattle, Cells, Cultured, Chromatography, High Pressure Liquid, Female, Molecular Sequence Data, Neurophysins metabolism, Oxytocin biosynthesis, Protein Precursors biosynthesis, Corpus Luteum metabolism, Granulosa Cells metabolism, Oxytocin metabolism, Protein Precursors metabolism, Protein Processing, Post-Translational
- Abstract
Bovine corpus luteum is the site of intense production of pro-ocytocin-neurophysin mRNA at day 1 after estrus (Ivell et al. (1985) FEBS Lett. 190, 263-267) which is followed by apparent delayed production of ocytocin. Therefore it is a good model to study both the translational and post-translational production of this neuropeptide in non-hypothalamic tissues and its regulation. In order to assess if this mRNA is translated during the lag period we have analyzed the neurophysin-like species produced in this organ. As early as day 2 after estrus one neurophysin species (pI approximately 4.7) could be detected and was unequivocally identified as pro-ocytocin-neurophysin. In primary cultures of luteinizing granulosa cells, biosynthetic intermediates were characterized, i.e. ocytocin-Gly, ocytocin-Gly-Lys and ocytocin-Gly-Lys-Arg, whereas amidated, fully mature, ocytocin was undetectable. We conclude that translation of pro-ocytocin-neurophysin mRNA takes place soon after transcription and we propose that incomplete processing could be responsible for the low level of ocytocin in the early bovine corpus luteum.
- Published
- 1991
- Full Text
- View/download PDF
47. Evidence for beta-turn structure in model peptides reproducing pro-ocytocin/neurophysin proteolytic processing site.
- Author
-
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
48. 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
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