Human Prohormone Convertase Research Articles

Reduced Stability and pH-Dependent Activity of a Common Obesity-Linked PCSK1 Polymorphism, N221D.

Common mutations in the human prohormone convertase (PC)1/3 gene (PCKSI) are linked to increased risk of obesity. Previous work has shown that the rs6232 single-nucleotide polymorphism (N221D) results in slightly decreased activity, although whether this decrease underlies obesity risk is not clear. We observed significantly decreased activity of the N221D PC1/3 enzyme at the pH of the trans-Golgi network; at this pH, the mutant enzyme was less stable than wild-type enzyme. Recombinant N221D PC1/3 also showed enhanced susceptibility to heat stress. Enhanced susceptibility to tunicamycin-induced endoplasmic reticulum stress was observed in AtT-20/PC2 cell clones in which murine PC1/3 was replaced by human N221D PC1/3, as compared with wild-type human PC1/3. However, N221D PC1/3-expressing AtT-20/PC2 clones processed proopiomelanocortin to α-MSH similarly to wild-type PC1/3. We also generated a CRISPR-edited mouse line expressing the N221D mutation in the PCKSI gene. When homozygous N221D mice were fed either a standard or a high-fat diet, we found no increase in body weight compared with their wild-type sibling controls. Sexual dimorphism was observed in pituitary ACTH for both genotypes, with females exhibiting lower levels of pituitary ACTH. In contrast, hypothalamic α-MSH content for both genotypes was higher in females compared with males. Hypothalamic corticotropin-like intermediate peptide content was higher in wild-type females compared with wild-type, but not N221D, males. Taken together, these data suggest that the increased obesity risk linked to the N221D allele in humans may be due in part to PC1/3-induced loss of resilience to stressors rather than strictly to decreased enzymatic activity on peptide precursors.

Production of bioactive peptides in an in vitro system

An in vitro system for the preparation of bioactive peptides is described. This system couples three different posttranslational modification enzymes, prohormone convertases (PCs), carboxypeptidase E, and peptidyl α-amidating enzyme, to transform recombinant precursors into bioactive peptides. Three different precursors, mouse proopiomelanocortin (mPOMC), rat proenkephalin (rPE), and human proghrelin, were used as model systems. The conversion of mPOMC and rPE to smaller peptide products was measured by radioimmunoassay. After optimization of the system, excellent efficiency was obtained: about 85% of starting mPOMC was converted to des-acetyl α-melanocyte-stimulating hormone (α-MSH). For proenkephalin, 75 and 96% yields were obtained for the opioid peptides Met-RGL and Met-enk, respectively. Cell-based assays demonstrated that in-vitro-generated des-acetyl α-MSH successfully activated the melanocortin 4 receptor. Proghrelin digestion was used to screen the specificity of PC cleavage and to confirm the cleavage site by mass spectroscopy. Mature ghrelin was produced by human furin, mouse prohormone convertase 1, and human prohormone convertase 7 but not by mouse prohormone convertase 2. These results demonstrate that our in vitro system (1) can produce peptides in quantities sufficient to carry out functional analyses, (2) can be used to determine the specificity of proprotein convertases on recombinant precursors, and (3) has the potential to identify novel peptide functions on both known and orphan G-protein-coupled receptors.

Gene organization and alternative splicing of human prohormone convertase PC8.

The mammalian Ca2+-dependent serine protease prohormone convertase PC8 is expressed ubiquitously, being transcribed as 3.5, 4.3 and 6.0 kb mRNA isoforms in various tissues. To determine the origin of these various mRNA isoforms we report the characterization of the human PC8 gene, which has been previously localized to chromosome 11q23-24. Consisting of 16 exons, the human PC8 gene spans approx. 27 kb. A comparison of the position of intron-exon junctions of the human PC8 gene with the gene structures of previously reported prohormone convertase genes demonstrated a divergence of the human PC8 from the highly conserved nature of the gene organization of this enzyme family. The nucleotide sequence of the 5'-flanking region of the human PC8 is reported and possesses putative promoter elements characteristic of a GC-rich promoter. Further supporting the potential role of a GC-rich promoter element, multiple transcriptional initiation sites within a 200 bp region were demonstrated. We propose that the various mRNA isoforms of PC8 result from the inclusion of intronic sequences within transcripts.

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.

Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.

Human obesity has an inherited component, but in contrast to rodent obesity, precise genetic defects have yet to be defined. A mutation of carboxypeptidase E (CPE), an enzyme active in the processing and sorting of prohormones, causes obesity in the fat/fat mouse. We have previously described a women with extreme childhood obesity (Fig. 1), abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism and elevated plasma proinsulin and pro-opiomelanocortin (POMC) concentrations but a very low insulin level, suggestive of a defective prohormone processing by the endopeptidase, prohormone convertase 1 (PC1; ref. 4). We now report this proband to be a compound heterozygote for mutations in PC1. Gly-->Arg483 prevents processing of proPC1 and leads to its retention in the endoplasmic reticulum (ER). A-->C+4 of the intro-5 donor splice site causes skipping of exon 5 leading to loss of 26 residues, a frameshift and creation of a premature stop codon within the catalytic domain. PC1 acts proximally to CPE in the pathway of post-translational processing of prohormones and neuropeptides. In view of the similarity between the proband and the fat/fat mouse phenotype, we infer that molecular defects in prohormone conversion may represent a generic mechanism for obesity, common to humans and rodents.

Cell Type-specific Protein-DNA Interactions at the cAMP Response Elements of the Prohormone Convertase 1 Promoter: EVIDENCE FOR ADDITIONAL TRANSACTIVATORS DISTINCT FROM CREB/ATF FAMILY MEMBERS

The proximal promoter region of the neuroendocrine-specific human prohormone convertase 1 (PC1) gene contains two distinct cAMP response elements (CRE-1 and CRE-2). Both elements are essential in directing the cAMP-mediated hormonal regulation of PC1 gene transcription. In this study, we have demonstrated that CRE-1 binds several trans-acting factors. In electrophoretic mobility shift assay experiments with nuclear extracts prepared from neuroendocrine AtT-20 and beta-TC3 cells and non-neuroendocrine COS-1 cells, three specific protein-DNA complexes (I-III) were detected. Complexes II and III were shown to contain CREB-1 and ATF-1, respectively. The most slowly migrating complex I was only detected with the neuroendocrine cell lines and appeared to comprise a c-Jun-containing heterodimer. In addition, CRE-2 was shown to bind a protein that was only detected in nuclear extracts derived from the neuroendocrine cell lines. Antibody supershift experiments indicated that both the c-Jun-interacting protein in CRE-1 complex I and the CRE-2-interacting protein are distinct from known members of the basic domain, leucine zipper family of transcription factors. UV cross-linking experiments demonstrated that these potential novel proteins are approximately 100 and 60 kDa in size, respectively. Site-specific mutagenesis experiments demonstrated that the formation of both CRE-1 and CRE-2 complexes is correlated with the transcriptional activity of the proximal PC1 promoter as has been shown in transient transfections with wild-type and mutant promoter constructs. In addition, it was shown that both CREB-1 and ATF-1 transactivate the human PC1 promoter in transient transfection experiments.

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.

Human prohormone convertase 3 gene: exon-intron organization and molecular scanning for mutations in Japanese subjects with NIDDM

Human prohormone convertase 3 gene: exon-intron organization and molecular scanning for mutations in Japanese subjects with NIDDM

Fluorescent Peptidyl Substrates as an Aid in Studying the Substrate Specificity of Human Prohormone Convertase PC1 and Human Furin and Designing a Potent Irreversible Inhibitor

The substrate specificities of two human prohormone convertases, furin and PC1, were examined with a series of 7-amino-4-methylcoumarinamide (MCA) containing peptidyl substrates. Using acetyl-Arg-Ser-Lys-Arg-MCA as model, P4 Arg substitution by Lys or Orn resulted for furin in a 538- and a 280-fold lower kcat/Km value, but only in a 14- and 18-fold decrease for PC1. Substitution of P3 Ser by either Pro, Glu, or Lys does not modify significantly the kcat/Km value for PC1, whereas furin activity is seriously impaired by the Glu substitution. Elongating the peptidyl sequence up to the P8 position decreases the kcat/Km value for furin but not for PC1. In both the P3 or P5 Glu substitution, the decrease of kcat/Km was due primarily to lower kcat rather than higher Km, possibly because of the presence of a negatively charged side chain. Finally, an octapeptidyl chloromethane derivative proved to be a potent irreversible inhibitor of either PC1 and ruin. The 811-fold difference in the apparent Kapp/[I] (1.63 x 10(6) s-1 m-1), and kcat/Km determined with the corresponding peptidyl MCA substrate (2.01 x 10(3) s-1 m-1), supports the proposal that cleavage of the acylenzyme represents the rate-limiting step for PC1 and furin.

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.