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PubMed2006

Processing of cocaine- and amphetamine-regulated transcript (CART) precursor proteins by prohormone convertases (PCs) and its implications.

Cocaine- and amphetamine-regulated transcript (CART) peptides are expressed in several neuroendocrine tissues, including hypothalamus, pituitary, gut, adrenal and pancreas, and are involved in regulating important biological processes including feeding/appetite, drug reward and stress. CART is synthesized as larger, inactive peptide precursors (pro-CART) that require endoproteolytic processing to generate smaller, active forms. Prohormone/proprotein convertases (PCs), a family of calcium-dependent, serine endoproteases, have been shown to cleave many protein precursors in the regulated/constitutive secretory pathway to generate smaller fragments. In our previous studies, we have demonstrated the important roles of the two neuroendocrine-specific PCs, PC2 and PC1/3, in processing the two pro-CART isoforms, long (102aa) and short (89aa), to generate the bioactive CART peptides, I (55-102/42-89) and II (62-102/49-89) as well as the intermediate fragments, 10-89 and 33-102. Our subsequent studies have revealed the participation of another PC family member, PC5/6A (the soluble isoform of a widely expressed PC, PC5/6), in cleaving both precursor isoforms. We conclude that PC5/6A contributes to the normal efficient processing of pro-CART and is functionally more redundant with PC2 than PC1/3 in generating both CART I and II.

Animals↗
PubMed1992

Molluscan putative prohormone convertases: structural diversity in the central nervous system of Lymnaea stagnalis.

In the cerebral ganglia of the central nervous system of the freshwater snail Lymnaea stagnalis many neuropeptides are proteolytically processed from larger prohormones at sites marked by both single and multiple basic amino acids. In the present study we identified cloned cDNA and PCR products corresponding to three putative endoproteases that may be involved in prohormone processing. The cDNA encodes a protein of 653 residues with an overall sequence identity of 60%, 41%, 35%, 40%, and 27% with the recently characterized endoproteases PC2, PC1/3, PC4, furin and Kex2, respectively. The Lymnaea preproconvertase has approximately 80% homology with the catalytic domain, and approximately 40% and approximately 50% with the N- and C-terminal part, respectively, of the vertebrate PC2. Two cloned PCR products, Lfur 1 and Lfur 2, show highest sequence identity to furin. Expression of the LPC2 gene is exclusively in the central nervous system, where two LPC2 transcripts of 3.0 and 4.8 kb were detected.

Amino Acid Sequence↗
PubMed1997

Regulation of human prohormone convertase 2 promoter activity by the transcription factor EGR-1.

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 2 (PC2) gene and established characteristics of the PC2 promoter region. The proximal promoter region is very G+C-rich and does not contain a canonical TATA box or a CAAT box. Transient expression assays with a set of human PC2 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, we show that the transcription factor EGR-1 interacts with two distinct elements within the proximal human PC2 promoter region. Transfection experiments also demonstrate that EGR-1 is able to enhance PC2 promoter activity.

Animals↗
PubMed2006

Obesity, hyperphagia and increased metabolic efficiency in Pc1 mutant mice.

Prohormone convertase 1 (PC1) mutations lead to obesity in humans. However, Pc1 knockout mice do not become obese; in fact, they are runted due to a defect in growth-hormone releasing hormone processing, leading to the speculation that PC1 subserves different functions between mouse and human. Here, we report a novel allele of mouse Pc1 (N222D) that leads to obesity, abnormal proinsulin processing and multiple endocrinological defects. Increased energy intake and a more efficient metabolism contribute to the obesity in Pc1(N222D/N222D) mice. Defective proinsulin processing leads to glucose intolerance, but neither insulin resistance nor diabetes develop despite obesity. The obesity is associated with impaired autocatalytic activation of mature PC1 and reduced hypothalamic alpha-MSH. This is the first characterization of Pc1 mutation in a model organism that mimics human PC1 deficiency.

Amino Acid Sequence↗
PubMed1993

cDNA structure and in situ localization of the Aplysia californica pro-hormone convertase PC2.

The complete cDNA structure of the Aplysia californica pro-protein and pro-hormone convertase PC2 (aPC2) was obtained from a cDNA library of the nervous system. The deduced amino acid sequence revealed that aPC2 exhibits an 85%, 61% and 62% sequence identity to the Lymnaea stagnalis, Xenopus laevis and mouse PC2 homologues, respectively. The deduced stagnalis, Xenopus laevis and mouse PC2 homologues, respectively. The deduced primary sequence suggested a protein of 653 amino acids which includes a 27- and 88-amino acid signal peptide and pro-segment. The signal peptide and the C-terminal segments are the least conserved regions. On Northern blots of nervous system we detected a transcript of 6.8 kb. The in situ hybridization histochemistry on the abdominal ganglion revealed intense labeling of the bag cells. Large peptidergic cells and clusters of sensory and motor neurons also contained high levels of aPC2 mRNA.

Amino Acid Sequence↗
PubMed1998

Manipulation of disulfide bonds differentially affects the intracellular transport, sorting, and processing of neuroendocrine secretory proteins.

To investigate if the prevention of disulfide bond formation affects the intracellular transport, sorting, and processing of a distinct set of neuroendocrine proteins in the regulated secretory pathway, we have treated Xenopus intermediate pituitaries with the thiol-reducing agent dithiothreitol. Pulse-chase incubations in combination with immunoprecipitation analysis were used to monitor the fates of the prohormone proopiomelanocortin (POMC), prohormone convertase PC2 and its helper protein 7B2, as well as secretogranin III. Manipulation of the disulfide bonds in POMC and proPC2 blocked their transport to the trans-Golgi network and strongly inhibited their processing. Reduction of the single disulfide bond in 7B2 did not disturb its transport and cleavage, but caused its missorting to the constitutive secretory pathway. Moreover, the liaison between proPC2 and 7B2 was prevented. Dithiothreitol did not affect transport, sorting, and cleavage of secretogranin III, which lacks disulfide bonds. When the reducing agent was washed away, POMC processing, proPC2 maturation, and the association between proPC2 and 7B2 were reestablished. Collectively, our findings indicate that manipulation of disulfide bonds differentially affects the fates of neuroendocrine proteins during their transit through the secretory pathway.

Animals↗
PubMed1992

Structure and expression of Xenopus prohormone convertase PC2.

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including alpha-melanophore-stimulating hormone (alpha-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to alpha-MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85-87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28-36%) and the region amino-terminal to the catalytic domain (59-60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black- and white-adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to alpha-MSH.

Amino Acid Sequence↗
PubMed1997

Adenovirally encoded prohormone convertase-1 functions in atrial myocyte large dense core vesicles.

Bioactive peptides are usually synthesized as inactive precursor proteins that yield bioactive products only after specific biosynthetic processing events. Large dense core vesicles (LDCV) are usually the site of storage of mature peptides. Atrial myocyte LDCV are rather unique in their storage of intact prohormone, proatrial natriuretic factor (pro-ANF), with no storage of cleaved products. To investigate whether the lack of intracellular cleavage of pro-ANF is due to the absence of prohormone convertases (PCs) from the atrial granules or to other factors, we expressed PC1 in atrial myocyte cultures using a recombinant adenovirus vector. Pro-PC1 protein was processed to mature PC1 and to the COOH-terminally shortened neuroendocrine-specific form of PC1 and rapidly secreted. Integral membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) were processed by PC1, and two primary products were secreted: a monofunctional monooxygenase and a larger bifunctional form. The cleaved PAM products were stored in LDCV, as secretion of PAM-derived products was stimulatable. In addition, pro-ANF was processed to ANF within PC1-expressing cells. In primary atrial myocytes, virally encoded PC1 is active on three substrates; lack of cleavage of pro-ANF and PAM in atrial myocytes is not due to a fundamental inability of atrial LDCV to support endoproteolytic processing.

Adenoviridae↗
PubMed1997

Cleavage of CCK 33 by recombinant PC2 in vitro.

The specificity and kinetic properties of CCK 33 cleavage by recombinant prohormone convertase 2 (PC2) was investigated using a combination of Sephadex G-50 chromatography, HLPC and RIA methods. It is shown that CCK 33 can be cleaved effectively by PC2 to form CCK 8, the reaction of which has a K(m) of 104.8 microM. No CCK 22 or other products were detected and the reaction is completely inhibited by the PC2 inhibitor, p-CMS (p-chloromercuriphenylsulfonic acid), suggesting that the cleavage is PC2 specific. The time course of this reaction shows an initial lag phase followed by a rapid increase in velocity consistent with a previously reported spontaneous transformation from a 71 kDa relatively inactive form into a more active form of 62 kDa (1). PC2 did not cleave recombinant pro CCK or CCK 1-21. These results demonstrate that PC2, an enzyme usually associated with dibasic cleavages, can cleave easily at single basic residues.

4-Chloromercuribenzenesulfonate↗
PubMed1994

The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-1 SU) and gp41 (HIV-I TM).

Intracellular proteolytic processing of human immunodeficiency virus envelope glycoprotein precursor (gp160) is an essential step for virus infectivity. Northern blot analysis provided evidence that furin and PC1, but not PC2, are expressed in the CD4+ human lymphoblastoid H9 cell line, suggesting the possible participation of these convertases in human immunodeficiency virus (HIV) gp160 proteolytic processing. Purified PC1 and furin cleaved specifically in vitro gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). NH2-terminal sequence analysis of the produced gp41 (HIV-I TM) demonstrated that the cleavage occurred within the sequence Arg-Glu-Lys-Arg decreases Ala-Val-Gly-Ile, which is identical to the bond cleaved in vivo. Transition state analog peptides were designed and tested in vitro for their ability to inhibit the PC1- or furin-mediated gp160 cleavage. The best inhibitor was decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2.

Amino Acid Sequence↗
PubMed2005

XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFbeta proteins in Xenopus development.

XPACE4 is a member of the subtilisin/kexin family of pro-protein convertases. It cleaves many pro-proteins to release their active proteins, including members of the TGFbeta family of signaling molecules. Studies in mouse suggest it may have important roles in regulating embryonic tissue specification. Here, we examine the role of XPACE4 in Xenopus development and make three novel observations: first, XPACE4 is stored as maternal mRNA localized to the mitochondrial cloud and vegetal hemisphere of the oocyte; second, it is required for the endogenous mesoderm inducing activity of vegetal cells before gastrulation; and third, it has substrate-specific activity, cleaving Xnr1, Xnr2, Xnr3 and Vg1, but not Xnr5, Derriere or ActivinB pro-proteins. We conclude that maternal XPACE4 plays an important role in embryonic patterning by regulating the production of a subset of active mature TGFbeta proteins in specific sites.

Amino Acid Sequence↗
PubMed1994

[Analysis of the gene encoding human PC2, a prohormone processing enzyme].

Prohormone Convertase 2 (PC2) is a specific endoprotease responsible for the processing of proinsulin to insulin. PC2 is expressed in pancreatic islets, pituitary and brain but is very low or absent in most other tissues, such as liver, spleen and kidney. To evaluate the regulated expression of the human PC2 gene we have analyzed its structure and characterized its promoter. The gene spans > 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The promoter region of the PC2 gene is very G + C rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Analysis of the level of chrolamphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for the PC2 promoter activity.

Animals↗
PubMed2003

Prohormone convertase 2 enzymatic activity and its regulation in neuro-endocrine cells and tissues.

We used the fluorometric substrate, pGlu-Arg-Thr-Lys-Arg-MCA and the C-terminal peptide of human 7B2(155-185), a specific inhibitor of prohormone convertase 2 (PC2), to specifically measure the enzymatic activity of the prohormone convertases, PC2. Using lysates from the pancreatic alpha cell line, alphaTC1-6 cells, which contain moderate levels of PC2 enzymatic activity, we determined that the PC2 assay was linear with respect to time of incubation and protein added and had a pH optimum of 5.5 and a calcium optimum of 2.5 mM. Rat pituitary contained high levels of PC2 enzymatic activity, while the hypothalamus and other brain regions contained moderate levels. This enzyme assay was used to document that both mice null for PC2 as well as mice null for the PC2 cofactor, 7B2, had only trace levels of PC2 activity in various brain regions, while mice heterozygous for these alleles had approximately half of the PC2 activity in most brain regions. PC2 enzymatic activity and PC2 mRNA levels were somewhat discordant suggesting that PC2 mRNA levels do not always reflect PC2 enzymatic activity.

Animals↗
PubMed1999

Monogenic disorders of obesity and body fat distribution.

Recently, great progress has been made towards understanding the molecular basis of body fat regulation. Identification of mutations in several genes in spontaneous monogenic animal models of obesity and development of transgenic models have indicated the physiological roles of many genes in the regulation of body fat distribution. In humans, mutations in leptin, leptin receptor, prohormone convertase 1 (PC1), pro-opiomelanocortin (POMC), melanocortin 4-receptor (MC4-R), and peroxisome proliferator-activated receptor (PPAR) gamma2 genes have been described in patients with severe obesity. Most of these obesity disorders exhibit a distinct phenotype with varying degrees of hypothalamic and pituitary dysfunction and a recessive inheritance, whereas MC4-R mutation has a nonsyndromic phenotype with dominant inheritance. These mutations suggest the critical role of central signaling systems composed of leptin/leptin receptor and alpha-melanocyte stimulating hormone/MC4-R in human energy homeostasis. Although the genetic basis of monogenic disorders of body fat distribution, such as congenital generalized lipodystrophy and familial partial lipodystrophy, Dunnigan variety, is still unknown, the genes for these have recently been localized to chromosomes 9q34 and 1q21-22, respectively. The advances in our knowledge of the phenotypic manifestations and underlying molecular mechanisms of genetic body fat disorders may lead to better treatment and prevention of obesity and other disorders of adipose tissue in the future.

Adipose Tissue↗
PubMed1995

Neuroendocrine-specific expression of the human prohormone convertase 1 gene. Hormonal regulation of transcription through distinct cAMP response elements.

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 1 (PC1) gene and identified and characterized the PC1 promoter region. We found multiple transcription start sites located within a 15-base pair region, 205 base pairs upstream of the translation start codon. The promoter region is not G+C-rich and does not contain a canonical TATA box nor a CAAT box. Transient expression assays with a set of human PC1 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, the proximal promoter region confers both basal and hormone-regulated promoter activity. Site-specific mutagenesis experiments demonstrate that two closely spaced cAMP response elements within the proximal promoter region direct cAMP-mediated hormonal regulation of transcription of the PC1 gene.

Animals↗
PubMed2000

Immunohistochemical evidence for the implication of PC1 in the processing of proneurotensin in rat brain.

Biochemical studies have shown that prohormone convertases PC1, PC2 and PC5A all have the capacity to process, with different specificities, the neurotensin/neuromedin N precursor, pro-NT/NN. A previous study from our laboratory has demonstrated that in rat brain, both PC2 and PC5A may be co-expressed with NT, lending support to a physiological implication of these two enzymes in the endoproteolytic maturation of pro-NT/NN. In the present study, we sought to determine whether PC1 might also be involved in this process by comparing the immunohistochemical distribution of the enzyme with that of NT in both singly labeled and dually labeled serial brain sections. PC1 was found to co-localize extensively with NT throughout the rat neuraxis. However, there were important regional variations in the proportion of NT neurons co-expressing PC1. Furthermore, this proportion was negatively correlated with that of NT neurons expressing PC5, suggesting that PC1 may serve as an alternative to PC5A for processing pro-NT/NN in mammalian brain.

Animals↗
PubMed2005

Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells.

AIMS/HYPOTHESIS: Mutations in genes encoding HNF-4alpha, HNF-1alpha and IPF-1/Pdx-1 are associated with, respectively, MODY subtypes-1, -3 and -4. Impaired glucose-stimulated insulin secretion is the common primary defect of these monogenic forms of diabetes. A regulatory circuit between these three transcription factors has also been suggested. We aimed to explore how Pdx-1 regulates beta cell function and gene expression patterns. METHODS: We studied two previously established INS-1 stable cell lines permitting inducible expression of, respectively, Pdx-1 and its dominant-negative mutant. We used HPLC for insulin processing, adenovirally encoded aequorin for cytosolic [Ca2+], and transient transfection of human growth hormone or patch-clamp capacitance recordings to monitor exocytosis. RESULTS: Induction of DN-Pdx-1 resulted in defective glucose-stimulated and K+-depolarisation-induced insulin secretion in INS-1 cells, while overexpression of Pdx-1 had no effect. We found that DN-Pdx-1 caused down-regulation of fibroblast growth factor receptor 1 (FGFR1), and consequently prohormone convertases (PC-1/3 and -2). As a result, DN-Pdx-1 severely impaired proinsulin processing. In addition, induction of Pdx-1 suppressed the expression of glucagon-like peptide 1 receptor (GLP-1R), which resulted in marked reduction of both basal and GLP-1 agonist exendin-4-stimulated cellular cAMP levels. Induction of DN-Pdx-1 did not affect glucokinase activity, glycolysis, mitochondrial metabolism or ATP generation. The K+-induced cytosolic [Ca2+] rise and Ca2+-evoked exocytosis (membrane capacitance) were not abrogated. CONCLUSIONS/INTERPRETATION: The severely impaired proinsulin processing combined with decreased GLP-1R expression and cellular cAMP content, rather than metabolic defects or altered exocytosis, may contribute to the beta cell dysfunction induced by Pdx-1 deficiency.

Adenosine Triphosphate↗
PubMed1999

The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing's disease.

The neuroendocrine protein 7B2 has been implicated in activation of prohormone convertase 2 (PC2), an important neuroendocrine precursor processing endoprotease. To test this hypothesis, we created a null mutation in 7B2 employing a novel transposon-facilitated technique and compared the phenotypes of 7B2 and PC2 nulls. 7B2 null mice have no demonstrable PC2 activity, are deficient in processing islet hormones, and display hypoglycemia, hyperproinsulinemia, and hypoglucagonemia. In contrast to the PC2 null phenotype, these mice show markedly elevated circulating ACTH and corticosterone levels, with adrenocortical expansion. They die before 9 weeks of severe Cushing's syndrome arising from pituitary intermediate lobe ACTH hypersecretion. We conclude that 7B2 is indeed required for activation of PC2 in vivo but has additional important functions in regulating pituitary hormone secretion.

Adipose Tissue↗