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Iris Lindberg - One of the best experts on this subject based on the ideXlab platform.
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A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity
2016Co-Authors: Michael Wilschanski, Montaser Abbasi, Elias Blanco, Iris Lindberg, Michael Yourshaw, David Zangen, Itai Berger, Eyal Shteyer, Benjamin Bar-oz, Martin G. Martı́nAbstract:Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the Proprotein Convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine Convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate Proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that thi
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a novel familial mutation in the pcsk1 gene that alters the oxyanion hole residue of Proprotein Convertase 1 3 and impairs its enzymatic activity
PLOS ONE, 2014Co-Authors: Michael Wilschanski, Montaser Abbasi, Elias Blanco, Iris Lindberg, Michael Yourshaw, David Zangen, Itai Berger, Eyal Shteyer, Orit PappoAbstract:Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the Proprotein Convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine Convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate Proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.
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biochemical and cell biological properties of the human prohormone Convertase 1 3 ser357gly mutation a pc1 3 hypermorph
Endocrinology, 2014Co-Authors: Elias H Blanco, Martin G Martin, Juan R Peinado, Iris LindbergAbstract:Satiety and appetite signaling are accomplished by circulating peptide hormones. These peptide hormones require processing from larger precursors to become bioactive, often by the Proprotein Convertase 1/3 (PC1/3). Several subcellular maturation steps are necessary for PC1/3 to achieve its optimal enzymatic activity. Certain PC1/3 variants found in the general population slightly attenuate its enzymatic activity and are associated with obesity and diabetes. However, mutations that increase PC1/3 activity and/or affect its specificity could also have physiological consequences. We here present data showing that the known human Ser357Gly PC1/3 mutant (PC1/3S357G) represents a PC1/3 hypermorph. Conditioned media from human embryonic kidney-293 cells transfected with PC1/3WT and PC1/3S357G were collected and enzymatic activity characterized. PC1/3S357G exhibited a lower calcium dependence; a higher pH optimum (neutral); and a higher resistance to peptide inhibitors than the wild-type enzyme. PC1/3S357G exhibited increased cleavage to the C-terminally truncated form, and kinetic parameters of the full-length and truncated mutant enzymes were also altered. Lastly, the S357G mutation broadened the specificity of the enzyme; we detected PC2-like specificity on the substrate proCART, the precursor of the cocaine- and amphetamine regulated transcript neuropeptide known to be associated with obesity. The production of another anorexigenic peptide normally synthesized only by PC2, αMSH, was increased when proopiomelanocortin was coexpressed with PC1/3S357G. Considering the aberrant enzymatic profile of PC1/3S357G, we hypothesize that this enzyme possesses unusual processing activity that may significantly change the profile of circulating peptide hormones.
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congenital Proprotein Convertase 1 3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort
Gastroenterology, 2013Co-Authors: Martin G Martin, Iris Lindberg, Sergio Solorzano R Vargas, Jiafang Wang, Yaron Avitzur, Robert H J Bandsma, Christiane Sokollik, Sarah Lawrence, Lindsay A Pickett, Zijun ChenAbstract:Background & Aims Proprotein Convertase 1/3 (PC1/3) deficiency, an autosomal-recessive disorder caused by rare mutations in the Proprotein Convertase subtilisin/kexin type 1 ( PCSK1 ) gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 also have been associated with obesity in heterozygotes in several population-based studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency. Methods We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1 . We measured enzymatic activity of recombinant PC1/3 proteins. Results We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears. Conclusions In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1 , failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in the processing of one or more enteric hormones that are required for nutrient absorption.
Régis Coutant - One of the best experts on this subject based on the ideXlab platform.
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a new case of pcsk1 pathogenic variant with congenital Proprotein Convertase 1 3 deficiency and literature review
The Journal of Clinical Endocrinology and Metabolism, 2019Co-Authors: Lucie Pépin, Estelle Colin, Marine Tessarech, Stéphanie Rouleau, Dominique Bonneau, Natacha Bouhoursnouet, Régis CoutantAbstract:Issue To report a homozygous pathogenic variant in PCSK1 in a boy affected with Proprotein Convertase 1/3 (PC1/3) deficiency. Case description and literature review A male infant born to consanguineous Turkish parents presented in the first week of life with transient central diabetes insipidus, watery diarrhea, micropenis due to hypogonadotropic hypogonadism and GH deficiency, and transient asymptomatic hypoglycemia. Further endocrine defects gradually appeared, including central hypothyroidism and mild central hypocortisolism (at 1 year), central diabetes insipidus that reappeared progressively (at 2.5 years), and obesity (at 2 years). Whole-exome sequencing revealed a homozygous nonsense pathogenic variant (NM_000439.4) c. 595 C>T in exon 5 of PCSK1, not yet reported in cases of PC1/3 deficiency. To date, 26 cases of PC1/3 deficiency have been reported in the literature. All individuals had early and severe malabsorptive diarrhea and 83% had polyuria-polydipsia syndrome (before 5 years). Most (79%) had early onset obesity. Various endocrine disorders were present, including GH deficiency (44%), mild central hypothyroidism (56%), central hypogonadism (44%), central hypocortisolism (57%), and postprandial hypoglycemia (52%). When described (n = 15), proinsulin levels were consistently high: between 8 and 154 times the upper limit of normal (mean 74). Conclusion We described a homozygous nonsense pathogenic variant (NM_000439.4) c. 595 C>T in exon 5 of PCSK1 in a boy with congenital PC1/3 deficiency. Elevated proinsulin could be useful in the diagnosis of this condition.
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A new case of pcsk1 pathogenic variant with congenital Proprotein Convertase 1/3 deficiency and literature review
The Journal of clinical endocrinology & metabolism, 2019Co-Authors: Lucie Pépin, Estelle Colin, Marine Tessarech, Stéphanie Rouleau, Natacha Bouhours-nouet, Dominique Bonneau, Régis CoutantAbstract:Issue: To report a homozygous pathogenic variant in PCSK1 in a boy affected with Proprotein Convertase 1/3 (PC1/3) deficiency. Case description and literature review: A male infant born to consanguineous Turkish parents presented in the first week of life with transient central diabetes insipidus, watery diarrhea, micropenis due to hypogonadotropic hypogonadism and GH deficiency, and transient asymptomatic hypoglycemia. Further endocrine defects gradually appeared, including central hypothyroidism and mild central hypocortisolism (at 1 yr), central diabetes insipidus that reappeared progressively (at 2.5 yr), and obesity (at 2 yr). Whole exome sequencing revealed a homozygous nonsense pathogenic variant (NM_000439.4) c. 595 C>T in exon 5 of PCSK1, not yet reported in cases of Proprotein Convertase 1/3 (PC1/3) deficiency. To date, 26 cases of PC1/3 deficiency have been reported in the literature. All individuals had early and severe malabsorptive diarrhea and 83% had polyuria-polydipsia syndrome (before 5 yr). Most (79%) had early-onset obesity. Various endocrine disorders were present, including growth hormone deficiency (44%), mild central hypothyroidism (56%), central hypogonadism (44%), central hypocortisolism (57%), and postprandial hypoglycemia (52%). When described (n=15), proinsulin levels were consistently high: between 8 and 154 times the upper limit of normal (mean 74). Conclusion: We described a homozygous nonsense pathogenic variant (NM_000439.4) c. 595 C>T in exon 5 of PCSK1 in a boy with congenital Proprotein Convertase 1/3 deficiency. Elevated proinsulin could be useful in the diagnosis of this condition.
Michel Chretien - One of the best experts on this subject based on the ideXlab platform.
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effects of rs6234 rs6235 and rs6232 rs6234 rs6235 pcsk1 single nucleotide polymorphism clusters on Proprotein Convertase 1 3 biosynthesis and activity
Molecular Genetics and Metabolism, 2011Co-Authors: Ajoy Basak, Michel Chretien, Majambu Mbikay, Francine Sirois, K K NkongoloAbstract:Abstract Background Proprotein Convertase 1/3 (PC1/3) is one of the endoproteases initiating the proteolytic activation of prohormones and proneuropeptides in the secretory pathway. It is produced as a zymogen that is subsequently modified by activity-determining cleavages at the amino and the carboxyl termini. In human, it is encoded by the PCSK1 locus on chromosome 5. Spontaneous inactivating mutations in its gene have been linked to obesity. Minor alleles of the common non-synonymous single-nucleotide polymorphisms (SNPs) rs6232 (T > C, N221D), rs6234 (G > C, Q665E) and rs6235 (C > G, S690T) have been associated with increased risk of obesity. We have shown that the variations associated with these SNPs are linked on minor PCSK1 alleles. Goal In this study, we examined the impact of amino acid substitutions specified by the minor PCSK1 alleles on PC1/3 biosynthesis and prohormone processing activity in cultured cells. Methods The common and variant isoforms of PC1/3 were expressed in transfected rat pituitary GH4C1 cells with or without proopiomelanocortin (POMC) as a substrate. Secreted PC1/3- or POMC-related proteins and peptides were analyzed by immunoblotting and immunoprecipitation. Results When expressed in GH4C1 cells, the triple-variant PC1/3 underwent significantly more proteolytic processing at the amino and carboxyl termini than the common and double-variant isoforms. However, there was no detectable difference among these isoforms in their ability to process POMC in the transfected cells. Conclusions Since truncation of PC1/3 in its C-terminal region reportedly renders the enzyme unstable, we speculate that the accentuated processing of the triple variant in this region may, in vivo , create a subtle deficit of PC1/3 enzymatic activity in endocrine and neuroendocrine cells, causing impaired processing of prohormones and proneuropeptides to their bioactive forms.
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expression and transient nuclear translocation of Proprotein Convertase 1 pc1 during mouse preimplantation embryonic development
Molecular Reproduction and Development, 2005Co-Authors: Carly St Germain, Michel Chretien, Gilles Croissandeau, Janice Mayne, Jay M Baltz, Majambu MbikayAbstract:Preimplantation embryos express a number of hormones, neuropeptides, and membrane receptors known to derive from proteolytic activation of their precursors by the seven-member family of subtilisin-like, calcium-dependent serine proteinases known as Proprotein Convertases (PCs). The goal of this study was to determine the pattern of PC expression in mouse preimplantation embryos. Transcripts for all PCs, except PC2, were detected by reverse transcription-polymerase chain reaction (RT-PCR) in unfertilized and fertilized eggs. Furin, PACE4, PC1, and PC7 transcripts remained present at subsequent stages of preimplantation embryonic development, whereas the levels of transcripts for PC4 and PC5 gradually disappeared after the 2-cell stage. Proprotein Convertase 1 (PC1) expression was further examined at the protein level. Immunoblotting revealed the presence of the zymogen and mature forms of this enzyme in eggs and embryos. Immunofluorescence laser confocal microscopy showed PC1-specific staining throughout the cytoplasm of unfertilized eggs. After fertilization, surprisingly, the staining was concentrated in pronuclei. It relocated to the cytoplasm at postzygotic stages and was particularly strong at junctions between blastomeres. The nuclear translocation of PC1 in fertilized eggs is probably mediated by its prodomain. Indeed, when transduced in human colon carcinoma LoVo cells, a mutant proPC1 incapable of cleaving off its prodomain was shown to accumulate in the nucleus. Furthermore, when N-terminally fused to green fluorescent protein, this domain was able to direct the reporter protein to the nucleus of these cells. Collectively, these data establish that eggs and preimplantation embryos express various PCs necessary for proteolytic activation of precursors of hormones and growth factors. They also raise the possibility of a nuclear function for PC1 during zygote formation. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.
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inhibitory specificity and potency of prosaas derived peptides toward Proprotein Convertase 1
Journal of Biological Chemistry, 2001Co-Authors: Ajoy Basak, Michel Chretien, Peter Koch, Marcel Dupelle, Lloyd D Fricker, Lakshmi A Devi, Nabil G SeidahAbstract:Prohormone Convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.
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a highly selective cell permeable peptide inhibitor of Proprotein Convertase 1 design synthesis and biological evaluation in cellular pc1 mediated proteolysis
ChemInform, 2001Co-Authors: Ajoy Basak, Michel Chretien, Nabil G Seidah, Francine Sirois, Peter Koch, Marcel Dupelle, Majambu MbikayAbstract:Hormonal peptides, neuropeptides, growth factors, receptors, bacterial toxins, viral glycoproteins and many biologically active proteins are generated as their precursors that must undergo proteolytic cleavages in order to be active. The delicate balance between these cleaved functional proteins and their precursors is the key element for normal growth, function, metabolism, development, and in pathophysiologic conditions [1,2]. These cleavage sites are generally composed of a pair of basic amino acids (aa) within the consensus sequence R/K/H-(X) n -R↓ (n = 0, 2, 4 or 6, X = any aa except Cys). These cleavages are mostly performed by a family of Ca+2-dependent serine proteases called Proprotein Convertases (PCs) related to bacterial subtilisin and yeast kexin [1]. PC1 is the first member of this family that mediates the processing of neural and endocrine precursor proteins, such as proopiomelanocortin (POMC) found in the secretary pathway [1,2]. PC1 activity was selectively regulated by grannin neuroendocrine protein, proSAAS [2]. The PC1-inhibitory property of proSAAS is confined within a short peptide segment near the C-terminus that contains the critical KR244 motif [2–4], In this article, we present kinetic evaluation of the specificity and potency of PC1-inhibition by a 12-mer h(human) proSAAS peptide and its mutants. The specificity and potency of these peptides were also tested against other PCs and compared with that of r (rat) proSAAS221–254, known as PenLen, the major processing form of proSAAS in rat brain [4].
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the rgd motif and the c terminal segment of Proprotein Convertase 1 are critical for its cellular trafficking but not for its intracellular binding to integrin α5β1
Journal of Biological Chemistry, 1999Co-Authors: Carole Rovere, Jose Luis, Jeanclaude Lissitzky, Ajoy Basak, Jacques Marvaldi, Michel Chretien, Nabil G SeidahAbstract:Cellular trafficking of subtilisin/kexin-like precursor Convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin α5β1 associates primarily with the zymogens proPC1, proPC1-ΔC (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as α5β1.
Ajoy Basak - One of the best experts on this subject based on the ideXlab platform.
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effects of rs6234 rs6235 and rs6232 rs6234 rs6235 pcsk1 single nucleotide polymorphism clusters on Proprotein Convertase 1 3 biosynthesis and activity
Molecular Genetics and Metabolism, 2011Co-Authors: Ajoy Basak, Michel Chretien, Majambu Mbikay, Francine Sirois, K K NkongoloAbstract:Abstract Background Proprotein Convertase 1/3 (PC1/3) is one of the endoproteases initiating the proteolytic activation of prohormones and proneuropeptides in the secretory pathway. It is produced as a zymogen that is subsequently modified by activity-determining cleavages at the amino and the carboxyl termini. In human, it is encoded by the PCSK1 locus on chromosome 5. Spontaneous inactivating mutations in its gene have been linked to obesity. Minor alleles of the common non-synonymous single-nucleotide polymorphisms (SNPs) rs6232 (T > C, N221D), rs6234 (G > C, Q665E) and rs6235 (C > G, S690T) have been associated with increased risk of obesity. We have shown that the variations associated with these SNPs are linked on minor PCSK1 alleles. Goal In this study, we examined the impact of amino acid substitutions specified by the minor PCSK1 alleles on PC1/3 biosynthesis and prohormone processing activity in cultured cells. Methods The common and variant isoforms of PC1/3 were expressed in transfected rat pituitary GH4C1 cells with or without proopiomelanocortin (POMC) as a substrate. Secreted PC1/3- or POMC-related proteins and peptides were analyzed by immunoblotting and immunoprecipitation. Results When expressed in GH4C1 cells, the triple-variant PC1/3 underwent significantly more proteolytic processing at the amino and carboxyl termini than the common and double-variant isoforms. However, there was no detectable difference among these isoforms in their ability to process POMC in the transfected cells. Conclusions Since truncation of PC1/3 in its C-terminal region reportedly renders the enzyme unstable, we speculate that the accentuated processing of the triple variant in this region may, in vivo , create a subtle deficit of PC1/3 enzymatic activity in endocrine and neuroendocrine cells, causing impaired processing of prohormones and proneuropeptides to their bioactive forms.
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synthetic peptides derived from the prosegments of Proprotein Convertase 1 3 and furin are potent inhibitors of both enzymes
Biochemical Journal, 2003Co-Authors: Ajoy Basak, Claude LazureAbstract:Proprotein Convertases (PCs) are Ca(2+)-dependent serine proteases of the subtilisin/kexin family which are known specifically to cleave propeptide and Proprotein substrates at the C-terminal of R-X-(K/R)-R/ to generate the relevant biologically active peptides. PCs are initially synthesized as enzymically inactive proenzyme forms where the prosegments play an important inhibitory role to the respective enzymes. Here we investigated whether synthetic peptides derived from the pro-region could also represent specific and potent inhibitors. Based upon sequence alignment, secondary structure analysis and hydrophilicity plot, a number of peptides ranging from 8 to 33 residues were selected. These included segments encompassing residues 55-62, 50-62, 39-62, 50-83, 55-83, 64-83 and 74-83 in the pro-mouse PC1/3 sequence and residues 54-62, 48-62 and 39-62 of the pro-human furin sequence. All peptides were prepared by solid-phase FastMoc chemistry, purified by reversed-phase HPLC and characterized by MS and amino acid analysis. These peptides were tested in vitro for inhibitory activity towards recombinant mouse PC1/3 and human furin. Progress-curve and end-time kinetic analysis demonstrated that a number of these peptides, particularly those containing both the primary and the secondary processing sites, displayed strong inhibition of both enzymes with inhibition constants (K (i)) in the high nanomolar range. Unlike the whole propeptide, these small synthetic peptide inhibitors exhibited either true competitive or mixed competitive inhibition, depending on the sequence. Our data revealed further the critical role of the last two basic amino acid residues (e.g. Lys(82)-Arg(83) for the mouse PC1/3 sequence) of the prodomain in imparting a strong anti-Convertase activity. The study also establishes the inhibitory potential of certain regions contained within the prosegment of the two Convertases.
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inhibitory specificity and potency of prosaas derived peptides toward Proprotein Convertase 1
Journal of Biological Chemistry, 2001Co-Authors: Ajoy Basak, Michel Chretien, Peter Koch, Marcel Dupelle, Lloyd D Fricker, Lakshmi A Devi, Nabil G SeidahAbstract:Prohormone Convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.
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a highly selective cell permeable peptide inhibitor of Proprotein Convertase 1 design synthesis and biological evaluation in cellular pc1 mediated proteolysis
ChemInform, 2001Co-Authors: Ajoy Basak, Michel Chretien, Nabil G Seidah, Francine Sirois, Peter Koch, Marcel Dupelle, Majambu MbikayAbstract:Hormonal peptides, neuropeptides, growth factors, receptors, bacterial toxins, viral glycoproteins and many biologically active proteins are generated as their precursors that must undergo proteolytic cleavages in order to be active. The delicate balance between these cleaved functional proteins and their precursors is the key element for normal growth, function, metabolism, development, and in pathophysiologic conditions [1,2]. These cleavage sites are generally composed of a pair of basic amino acids (aa) within the consensus sequence R/K/H-(X) n -R↓ (n = 0, 2, 4 or 6, X = any aa except Cys). These cleavages are mostly performed by a family of Ca+2-dependent serine proteases called Proprotein Convertases (PCs) related to bacterial subtilisin and yeast kexin [1]. PC1 is the first member of this family that mediates the processing of neural and endocrine precursor proteins, such as proopiomelanocortin (POMC) found in the secretary pathway [1,2]. PC1 activity was selectively regulated by grannin neuroendocrine protein, proSAAS [2]. The PC1-inhibitory property of proSAAS is confined within a short peptide segment near the C-terminus that contains the critical KR244 motif [2–4], In this article, we present kinetic evaluation of the specificity and potency of PC1-inhibition by a 12-mer h(human) proSAAS peptide and its mutants. The specificity and potency of these peptides were also tested against other PCs and compared with that of r (rat) proSAAS221–254, known as PenLen, the major processing form of proSAAS in rat brain [4].
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the rgd motif and the c terminal segment of Proprotein Convertase 1 are critical for its cellular trafficking but not for its intracellular binding to integrin α5β1
Journal of Biological Chemistry, 1999Co-Authors: Carole Rovere, Jose Luis, Jeanclaude Lissitzky, Ajoy Basak, Jacques Marvaldi, Michel Chretien, Nabil G SeidahAbstract:Cellular trafficking of subtilisin/kexin-like precursor Convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin α5β1 associates primarily with the zymogens proPC1, proPC1-ΔC (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as α5β1.
Ujwal Shinde - One of the best experts on this subject based on the ideXlab platform.
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mechanism of fine tuning ph sensors in Proprotein Convertases identification of a ph sensing histidine pair in the propeptide of Proprotein Convertase 1 3
Journal of Biological Chemistry, 2015Co-Authors: Danielle M Williamson, Johannes Elferich, Ujwal ShindeAbstract:The propeptides of Proprotein Convertases (PCs) regulate activation of cognate protease domains by sensing pH of their organellar compartments as they transit the secretory pathway. Earlier experimental work identified a conserved histidine-encoded pH sensor within the propeptide of the canonical PC, furin. To date, whether protonation of this conserved histidine is solely responsible for PC activation has remained unclear because of the observation that various PC paralogues are activated at different organellar pH values. To ascertain additional determinants of PC activation, we analyzed PC1/3, a paralogue of furin that is activated at a pH of ∼5.4. Using biophysical, biochemical, and cell-based methods, we mimicked the protonation status of various histidines within the propeptide of PC1/3 and examined how such alterations can modulate pH-dependent protease activation. Our results indicate that whereas the conserved histidine plays a crucial role in pH sensing and activation of this protease an additional histidine acts as a "gatekeeper" that fine-tunes the sensitivity of the PC1/3 propeptide to facilitate the release inhibition at higher proton concentrations when compared with furin. Coupled with earlier analyses that highlighted the enrichment of the amino acid histidine within propeptides of secreted eukaryotic proteases, our work elucidates how secreted proteases have evolved to exploit the pH of the secretory pathway by altering the spatial juxtaposition of titratable groups to regulate their activity in a spatiotemporal fashion.
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determination of histidine pka values in the propeptides of furin and Proprotein Convertase 1 3 using histidine hydrogen deuterium exchange mass spectrometry
Analytical Chemistry, 2015Co-Authors: Johannes Elferich, Danielle M Williamson, Larry L David, Ujwal ShindeAbstract:Propeptides of Proprotein Convertases regulate activation of their protease domains by sensing the organellar pH within the secretory pathway. Earlier experimental work highlighted the importance of a conserved histidine residue within the propeptide of a widely studied member, furin. A subsequent evolutionary analysis found an increase in histidine content within propeptides of secreted eukaryotic proteases compared with their prokaryotic orthologs. However, furin activates in the trans-golgi network at a pH of 6.5 while a paralog, Proprotein Convertase 1/3, activates in secretory vesicles at a pH of 5.5. It is unclear how a conserved histidine can mediate activation at two different pH values. In this manuscript, we measured the pKa values of histidines within the propeptides of furin and Proprotein Convertase 1/3 using a histidine hydrogen-deuterium exchange mass spectrometry approach. The high density of histidine residues combined with an abundance of basic residues provided challenges for generation of peptide ions with unique histidine residues, which were overcome by employing ETD fragmentation. During this analysis, we found slow hydrogen-deuterium exchange in residues other than histidine at basic pH. Finally, we demonstrate that the pKa of the conserved histidine in Proprotein Convertase 1/3 is acid-shifted compared with furin and is consistent with its lower pH of activation.
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propeptides are sufficient to regulate organelle specific ph dependent activation of furin and Proprotein Convertase 1 3
Journal of Molecular Biology, 2012Co-Authors: Stephanie L Dillon, Johannes Elferich, Danielle M Williamson, David Radler, Rajendra Joshi, Gary Thomas, Ujwal ShindeAbstract:Abstract The Proprotein Convertases (PCs) furin and Proprotein Convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO FUR ), which regulates furin activation at pH ~ 6.5 within the trans ‐Golgi network. Although this residue is conserved in the PC1 propeptide (PRO PC1 ), PC1 nonetheless activates at pH ~ 5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO FUR regulates furin activation and examine why PRO FUR and PRO PC1 differ in their pH-dependent activation. Sequence analyses establish that while both PRO FUR and PRO PC1 are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO FUR is ~ 2-fold greater than that in PRO PC1 , which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO FUR when compared to PRO PC1 to enhance autoproteolysis. We further demonstrate that PRO FUR and PRO PC1 are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles.
