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PubMed1999

Endocytic delivery of intramolecularly quenched substrates and inhibitors to the intracellular yeast Kex2 protease1.

Kex2 in the yeast Saccharomyces cerevisiae is a transmembrane, Ca2+-dependent serine protease of the subtilisin-like pro-protein convertase (SPC) family with specificity for cleavage after paired basic amino acids. At steady state, Kex2 is predominantly localized in late Golgi compartments and initiates the proteolytic maturation of pro-protein precursors that transit the distal secretory pathway. However, Kex2 localization is not static, and its itinerary apparently involves transiting out of the late Golgi and cycling back from post-Golgi endosomal compartments during its lifetime. We tested whether the endocytic pathway could deliver small molecules to Kex2 from the extracellular medium. Here we report that intramolecularly quenched fluorogenic substrates taken up into intact yeast revealed fluorescence due to specific cleavage by Kex2 protease in endosomal compartments. Furthermore, the endocytic delivery of protease inhibitors interfered with Kex2 activity for precursor protein processing. These observations reveal that the endocytic pathway does intersect with the cycling itinerary of active Kex2 protease. This strategy of endocytic drug delivery has implications for modulating SPC protease activity needed for hormone, toxin and viral glycoprotein precursor processing in human cells.

Biological Transport↗
PubMed1998

Characterization of the promoter of the mouse prohormone convertase PC2 gene.

Prohormone convertase 2 (PC2) is a member of a family of mammalian subtilisin-like endoproteases that are involved in the processing of prohormones, neuropeptides and many other precursor derived proteins. The expression of PC2 is restricted to neuroendocrine tissues such as pancreatic islets, the pituitary and the brain. To understand the regulation of the PC2 gene, we cloned and characterized the promoter region of the mouse PC2 gene. The transcriptional start site of the mouse PC2 gene is identical to that of the human. There is 79% identity in the sequences of the promoter regions between the mouse and human PC2 genes. The mouse PC2 gene, like the human, does not have a TATA-like motif in the region just upstream of the start of the transcription. Studies with promoter-reporter gene, chloramphenicol acetyltransferase (CAT), constructs showed that the region from -400 to -170 bp was necessary for high level expression of the mouse PC2 gene in the betaTC-3 insulinoma cells.

Animals↗
PubMed2000

Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3.

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.

Amino Acid Sequence↗
PubMed1996

Prohormone convertase PC5 is a candidate processing enzyme for prorenin in the human adrenal cortex.

We isolated a cDNA clone encoding the human prohormone convertase PC5 from human adrenal gland mRNA. The deduced protein sequence would encode a 915 amino acid preproPC5 that shares a very high degree of homology with previously cloned rat and mouse homologues. PC5 mRNA was detected in multiple human tissues, including the brain, adrenal and thyroid glands, heart, placenta, lung, and testes. PC5 mRNA was undetectable in the liver and was present at lower levels in skeletal muscle, kidney, pancreas, small intestine, and stomach. Co-transfection of human PC5 and human prorenin expression vectors in cultured GH4C1 cells led to secretion of active renin. The activation of human prorenin by PC5 depended on a pair of basic amino acids at positions 42 and 43 of the prorenin prosegment and occurred only in cells containing dense core secretory granules. Human PC5 was colocalized with renin by immunohistochemistry in the zona glomerulosa of the adrenal gland, suggesting that it could participate in the activation of a local renin-angiotensin system in the human adrenal cortex.

Adrenal Cortex↗
PubMed1994

Autocatalytic maturation of the prohormone convertase PC2.

PC2 is a member of the eukaryotic family of subtilisin-like proteases, which is thought to participate in the processing of prohormones and proneuropeptides in neuroendocrine cells. PC2 is synthesized as a 69-kDa prepropolypeptide. The NH2-terminal signal sequence is removed during segregation within the endoplasmic reticulum, where glycosylation occurs to generate a 75-kDa propolypeptide. A combination of site-directed mutagenesis and a cell-free translation/translocation system from Xenopus eggs was used to investigate the processing of the pro-PC2 precursor. The 75-kDa polypeptide underwent slow cleavage after the sequence Arg-Lys-Lys-Arg84 to generate a 68-kDa mature enzyme. Cleavage was blocked when the tetrabasic sequence was deleted (PC2M3) or when the active site Asp142 was changed to Asn (PC2M4). This latter observation suggested that cleavage of the 75-kDa propolypeptide to the mature 68-kDa enzyme was autocatalytic. Incubation of the PC2M4 mutant with the wild type PC2 precursor resulted in cleavage of both the wild type polypeptide and the catalytically inactive PC2M4 mutant. This indicates that cleavage could occur through an intermolecular reaction. The results also demonstrate that the novel Xenopus egg extract translation/translocation system represents a powerful cell-free method for studying proteolytic processing of propolypeptides.

Amino Acid Sequence↗
PubMed2005

Proopiomelanocortin processing in the anterior pituitary of the ovine fetus after lesion of the hypothalamic paraventricular nucleus.

The hypothalamic-pituitary-adrenocortical axis plays an essential role in the maturation of fetal organs and, in sheep, birth. Lesioning the paraventricular nucleus (PVN) in fetal sheep prevents adrenocortical maturation and parturition without altering plasma immunoreactive ACTH concentrations. The purpose of this study was to determine the effect of PVN lesion on anterior pituitary processing of proopiomelanocortin (POMC) to ACTH, plasma concentrations of ACTH and ACTH precursors (POMC; 22-kDa proACTH), and expression of subtilisin-like prohormone convertase 3 (SPC3) in corticotropes in fetal sheep. PVN lesion did not affect anterior pituitary POMC and 22-kDa proACTH levels, whereas ACTH was significantly affected. The ACTH precursor (POMC plus 22-kDa proACTH) to ACTH ratio in the anterior pituitary was significantly increased after PVN lesion. Post-PVN lesion, fetal plasma ACTH(1-39), was below the limit of detection, whereas ACTH precursors (POMC plus 22-kDa proACTH) were not affected. In the inferior region of the anterior pituitary, 40-50% of corticotropes had detectable SPC3 hybridization signal, and PVN lesion did not change the extent of colocalization of POMC and SPC3, or SPC3 mRNA levels within corticotropes. Neither the percent of corticotropes in the superior region containing SPC3 hybridization (7-12%) or hybridization signal strength was altered in response to PVN lesion. In conclusion, the fetal PVN is necessary for sustaining adequate anterior pituitary processing of POMC to ACTH and ACTH release needed for maturing the adrenal cortex in the sheep fetus.

Adrenocorticotropic Hormone↗
PubMed1998

Residues unique to the pro-hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity.

The prohormone convertase PC2 is one of the major subtilisin/kexin-like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin-like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2-specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2-specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site-directed mutagenesis of Tyr194 and of the oxyanion hole Asp309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into beta-endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D-PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co-immunoprecipitations and Western blots, binding assays indicate that Tyr194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp309 is critical for the binding of proPC2 with pro7B2.

Animals↗
PubMed1997

Identification of a molluscan homologue of the neuroendocrine polypeptide 7B2.

In vertebrates, interaction of prohormone convertase 2 (PC2) with the highly conserved polypeptide 7B2 is essential for transport and maturation of proPC2 in the regulated secretory pathway. In vitro, 7B2 displays a strong inhibitory activity toward PC2. Here, we characterize a cDNA encoding the first invertebrate 7B2-related protein (L7B2) from the brain of the mollusc Lymnaea stagnalis. The overall amino acid sequence identity between L7B2 and its vertebrate counterparts is surprisingly low (29%) and is restricted to a few small stretches of amino acid residues. Of particular interest are a conserved proline-rich region in the middle portion of the L7B2 sequence and a repeated conserved region in the carboxyl-terminal domain. Synthetic peptides corresponding to the carboxyl-terminal regions inhibit Lymnaea PC2 enzyme activity in extracts of insulin-producing neurons, in which both L7B2 and Lymnaea PC2 are abundantly expressed. Moreover, the peptides inhibit mouse PC2 enzyme activity. Our cloning of invertebrate 7B2 helps to delineate residues that are important for 7B2-PC2 interaction.

Amino Acid Sequence↗
PubMed2003

Furin and prohormone convertase 1/3 are major convertases in the processing of mouse pro-growth hormone-releasing hormone.

We investigated the proteolytic processing of mouse pro-GHRH [84 amino acids (aa)] by furin, PC1/3, PC2, and PC5/6A. We created six point mutations in the N- and C-terminal cleavage sites, RXXR decreased and RXRXXR decreased, respectively. The following results were obtained after transient transfection/cotransfection and metabolic pulse-chase labeling studies in several neuroendocrine cells. 1) Furin was the most efficient convertase in cleaving the N-terminal RXXR/RXRR site to generate intermediate I, 12-84aa, whereas PC1/3 was the most potent in processing the C-terminal RXRXXR site to yield mature GHRH, 12-53aa. 2) Both PC1/3 and PC5/6A also processed the N-terminal site but less efficiently than furin. 3) PC2 was much weaker in cleaving the C-terminal site relative to PC1/3 to generate mature GHRH. 4) The Q10R mutant was significantly more susceptible to furin cleavage at the N-terminal site than the wild-type pro-GHRH. And 5) the N- and C-terminal P1 Arg residues, R11 and R54, respectively, were essential for mature GHRH production. We also showed localization of the GHRH immunoreactive peptides in Golgi and secretory granules in neuroendocrine cells by an immunofluorescence assay. We conclude that the efficient production of mature GHRH from pro-GHRH is a stepwise process mediated predominantly by furin at the N-terminal cleavage site followed by PC1/3 at the C terminus.

Animals↗
PubMed1997

Prohormone convertase 1 is necessary for the formation of cholecystokinin 8 in Rin5F and STC-1 cells.

Several immortalized cell lines serve as models for procholecystokinin (pro-CCK) processing. Rin5F cells, derived from a rat insulinoma, and STC-1 cells, derived from a murine intestinal tumor, process pro-CCK mainly to amidated CCK 8. Both also make significant quantities of amidated CCK 22, a slightly larger form found in the gut. Many modifications are necessary during pro-CCK processing including cleavages performed by endoproteases, the identities of which are unknown. A candidate endoprotease is prohormone convertase 1 (PC1) also known as PC3, a Ca2+-dependent serine endoprotease of the subtilisin family. Constitutive expression of antisense PC1 message in stably transfected Rin5F cells resulted in a significant reduction of the cellular content of CCK 8 as measured by radioimmunoassay. Several affected cell lines displayed about 80% reduction in CCK content in early passages after transfection. Expression of antisense PC1 message in these cell lines resulted in a selective depletion of CCK 8 and a comparative sparing of CCK 22. The induction of antisense PC1 message within a single subclone of Rin5F cells using the Lac Switch system also resulted in a significant inhibition of CCK content. Expression of antisense PC1 message in a stably transfected STC-1 cell line also resulted in a decrease in CCK content and in PC1 protein expression, and the specific depletion of CCK 8 with comparative sparing of CCK 22. These observations support the hypothesis that PC1 is necessary for pro-CCK processing in Rin5F and STC-1 cells and suggests a role for PC1 endoprotease in the biosynthesis of CCK 8 in vivo.

Animals↗
PubMed2006

Crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by Furin-like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein.

Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.

Cell Line↗
PubMed1996

PC8 [corrected], a new member of the convertase family.

A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23-11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8.

Amino Acid Sequence↗
PubMed1996

Expression of prohormone convertase, PC2, in adrenocorticotropin-producing thymic carcinoid with elevated plasma corticotropin-releasing hormone.

An autopsy case of ACTH-producing thymic carcinoid with Cushing's syndrome is reported. The patient was a 63-yr-old man with multiple bone metastases from an undetermined primary site. Hyperpigmentation was observed at the terminal stage. The plasma levels of ACTH, cortisol, chromogranin A, and urinary 17-hydroxy-corticosteroids were extremely high, and ectopic ACTH-producing neuroendocrine tumor was diagnosed. In addition, plasma CRH was high. Autopsy revealed that the patient had primary thymic carcinoid with extensive metastases. Remarkable hyperplasia of the adrenal cortexes and Crooke's hyaline degeneration of the pituitary gland were consistent with Cushing's syndrome by ectopic ACTH production. There were multiple CRH-producing cells without degenerative changes in the hypothalamus. The tumor cells were immunoreactive to ACTH, CRH, and the cleavage enzyme PC2. POMC messenger ribonucleic acid and PC2 messenger ribonucleic acid were detected in the tumor cells by an in situ hybridization method. Expression of PC2 was considered to induce hyperpigmentation by producing alpha MSH. Despite hypercortisolism and ectopic production of CRH by the tumor cells, hypothalamic CRH cells were not atrophic. This case is a good example to demonstrate the correlation between CRH and the hypothalamo-pituitary-adrenal axis as well as hyperpigmentation in Cushing's syndrome.

Adrenocorticotropic Hormone↗
PubMed2005

Embryonic gene expression and pro-protein processing of proSAAS during rodent development.

In vitro assays have demonstrated that peptides derived from the recently-identified proSAAS precursor inhibit prohormone convertase 1 (PC1) suggesting that this novel peptide may function as an endogenous inhibitor of PC1. To further understand the role of proSAAS in vivo, we have investigated the expression of proSAAS mRNA and processing of proSAAS during pre- and early postnatal rodent development. In situ hybridization showed that, by embryonic day 12.5 (e12.5) in the rat, proSAAS mRNA was present in essentially all differentiating neurons in the mantle layer of the myelencephalon, metencephalon, diencephalon, spinal cord and several sympathetic ganglia. During later stages of prenatal development, widespread proSAAS expression continues in post-mitotic neurons of both the CNS and PNS and begins in endocrine cells of the anterior and intermediate pituitary. Although proSAAS expression overlaps with PC1 in several regions, its overall expression pattern is significantly more extensive, suggesting that proSAAS may be multifunctional during development. Processed forms of proSAAS are present by at least mid-gestation with marked accumulation of two C-terminal forms, comprising the PC1 inhibitory fragment of proSAAS.

Animals↗
PubMed2000

Structure-function analysis of the 7B2 CT peptide.

Prohormone convertases play important roles in the proteolytic conversion of many protein precursors. The neuroendocrine protein 7B2 and its 31-residue carboxyl-terminal (CT) peptide potently and specifically inhibit prohormone convertase 2 (PC2). We have analyzed the residues contributing to inhibition using N-terminal truncation and alanine scanning. Removal of more than 3 residues from the amino-terminal end of CT1-18 resulted in a more than 190-fold drop in inhibitory activity, showing that most of the residues between 3 and 18 are required for inhibition. In agreement, an Ala scan indicated that only 4 residues could be replaced with Ala without losing mid-nanomolar inhibitory potency; in particular, Gln7, Gln9, and Asp12 could be Ala-substituted to yield peptides with a similar inhibitory potency to the starting peptide. The all-d-retro-inverso, all-l-inverso, and all-d analogues of CT peptide were completely inactive, indicating that amino acid side chains and the CT peptide main chain interact with PC2. CT peptide inhibition could not be competitively blocked by preincubation with truncated CT peptide forms, supporting an absolute requirement for the Lys-Lys pair in initial binding of the CT peptide to the active site.

Amino Acid Sequence↗
PubMed1997

Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2.

The prohormone convertase SPC2 (PC2) participates in the processing of proinsulin, proglucagon, and a variety of other neuroendocrine precursors, acting either alone or in conjunction with the structurally related dense-core granule convertase SPC3 (PC3/PC1). We have generated a strain of mice lacking active SPC2 by introducing the neomycin resistance gene (Neor) into the third exon of the mSPC2 gene. This gene insertion results in the synthesis of an exon 3-deleted form of SPC2 that does not undergo autoactivation and is not secreted. The homozygous mutant mice appear to be normal at birth. However, they exhibit a small decrease in rate of growth. They also have chronic fasting hypoglycemia and a reduced rise in blood glucose levels during an intraperitoneal glucose tolerance test, which is consistent with a deficiency of circulating glucagon. The processing of proglucagon, prosomatostatin, and proinsulin in the alpha, delta, and beta cells, respectively, of the pancreatic islets is severely impaired. The islets in mutant mice at 3 months of age show marked hyperplasia of alpha and delta cells and a relative diminution of beta cells. SPC2-defective mice offer many possibilities for further delineating neuroendocrine precursor processing mechanisms and for exploring more fully the physiological roles of many neuropeptides and peptide hormones.

Animals↗
PubMed2001

Biosynthesis and cellular trafficking of the convertase SKI-1/S1P: ectodomain shedding requires SKI-1 activity.

Subtilisin kexin isozyme-1 (SKI-1)/site 1 protease is a mammalian subtilase composed of distinct functional domains. Among the major substrates of SKI-1 are the sterol regulatory element-binding proteins, regulating cholesterol and fatty acid homeostasis. Other substrates include the stress response factor activating transcription factor-6, the brain-derived neurotrophic factor, and the surface glycoproteins of highly infectious viruses belonging to the family of Arenaviridae. Domain deletion and/or point mutants were used to gauge the role of the various domains of SKI-1. Biosynthesis, cellular trafficking, and sterol regulatory element-binding protein-2 cleavage activity were used as diagnostic tools. Results revealed that Arg(130) and Arg(134) are critical for the autocatalytic primary processing of the prosegment and for the subsequent efficient exit of SKI-1 from the endoplasmic reticulum. Functional mapping of the growth factor cytokine receptor motif suggested a folding role within the endoplasmic reticulum. Microsequencing of the remaining membrane-bound stub following ectodomain shedding of SKI-1 localized the shedding site to KHQKLL(953) downward arrow. Site-directed mutagenesis, in vitro cleavage of a synthetic peptide containing the shedding site, and inhibitor studies favor an autocatalytic event occurring at a non-canonical SKI-1 recognition sequence, with P2 and P1 Leu being very critical. In conclusion, multiple domains ensuring optimal functional characteristics control SKI-1 activity and cellular trafficking.

Amino Acid Sequence↗
PubMed2004

Prohormone convertase 1 (PC1) processing and sorting: effect of PC1 propeptide and proSAAS.

Prohormone convertase 1 (PC1) is a serine proteinase responsible for the proteolytic processing of many precursor proteins within the regulated secretory pathway. The activity of PC1 is potentially regulated by two endogenous inhibitors, the PC1 propeptide and proSAAS. Here we have investigated the effect of proSAAS and propeptide-containing constructs on PC1 carboxy-terminal processing and activity. In AtT-20 cells, proSAAS expression inhibited both C-terminal PC1 processing and proopiomelanocortin (POMC) processing under pulse/chase conditions. SAAS CT peptide-propeptide chimeric constructs had no effect on the cleavage of PC1 and POMC under pulse/chase conditions. However, a construct containing the propeptide alone reduced C-terminal PC1 processing under pulse/chase conditions and also inhibited POMC processing. In contrast, experiments using HEK293 cells transiently expressing PC1 plus the respective constructs demonstrated significant inhibition of zymogen processing and decreased C-terminal processing of PC1 by the SAAS CT peptide portion of the chimera. Our results suggest that the PC1 propeptide expressed in trans is able to act as an endogenous inhibitor of PC1, but that SAAS CT peptide-containing/propeptide constructs cannot function as effective inhibitors of precursor maturation in the regulated pathway.

Animals↗