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PubMed1993

Release of the prohormone convertase PC1 from AtT-20 cells.

AtT-20 cells are known to synthesize two molecular weight forms of the prohormone converting enzyme PC1 with molecular masses of 87 and 66 kDa. In this study we have analyzed basal and stimulated secretion of these proteins. Western blot results show that basal secretion medium of cultured AtT-20 cells contained low concentrations of both the 87 and 66 kDa forms of PC1 with the former protein predominant. During the stimulation period with CRF, cAMP and cAMP + BaCl2, increased release of both proteins was observed, but the 66 kDa protein predominated. Secretion medium obtained from stimulated and unstimulated cells was enzymatically active against the Cbz-Arg-Ser-Lys-Arg-AMC fluorogenic substrate as well as against 35S-proenkephalin. This activity was Ca+2 dependent and was inhibited by the chelating agent EDTA. The activity was insensitive to acid and thiol proteinase inhibitors as well as to N-alpha-p-tosyl-L-Lys-chloromethyl ketone; it was slightly sensitive to phenylmethyl sulfonyl fluoride and was strongly inhibited by D-Tyr-Ala-Lys-Arg-chloromethyl ketone. This inhibitor profile exhibits strong similarities to furin and kexin. After partial purification of medium by gel filtration chromatography, a portion of the enzymatic activity and immunoreactivity for both 87 kDa and 66 kDa proteins eluted with an apparent molecular weight of 400 kDa (suggesting aggregation); however the highest activity appeared in the elution position of the 66 kDa monomer. When the 87 kDa protein was removed from the medium by means of an affinity column containing an antibody against the carboxyl terminal portion of PC1, the column flow-through, which included the 66 kDa protein, still remained enzymatically active. These data support the notion that the 66 kDa protein, which is the most concentrated PC1 product stored in AtT-20 cells and is released during stimulation, is enzymatically active.

Amino Acid Sequence↗
PubMed2003

The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein.

The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

Adrenocorticotropic Hormone↗
PubMed1994

Prohormone convertases PC2 and PC3 in rat neutrophils and macrophages. Parallel changes with proenkephalin-derived peptides induced by LPS in vivo.

Prohormone- or proneuropeptide-converting enzymes PC2 and PC3 have been observed exclusively in nervous and endocrine tissues. In this work the presence of these enzymes in cells of the immune system was demonstrated. PC2 was detected in peripheral and liver-infiltrating polymorphonuclear leukocytes (PMN) but not in alveolar macrophages (AM) or spleen mononuclear cells (SMC). PC2 proteins corresponded to 75, 71 and 56 kDa forms. PC3 appeared in AM and SMC but not in PMN, and a 66 kDa protein was the only PC3 form detected. Proenkephalin-derived peptides (PENKp) were observed in PMN and AM, showing peptides of 35, 28, 21, 18 and 14 kDa in the former cells and a doublet of 35 and 32 kDa in the latter. PC2 proteins and PENKp decreased in liver PMN and peripheral PMN 90 min after intravenous (i.v.) infusion of LPS, suggesting an increased release. However, in vitro assays showed that the chemotactic peptide FMLP but not LPS increased the basal secretion of PC2 proteins and PENKp in PMN. These results indicate that PC2 proteins are released from PMN, together with PENKp, and suggest that LPS in vivo may act through an indirect mechanism. Low levels of PC3 and PENK were detected in the AM of rats treated for 90 min with SAL or LPS. However, a significant increase of PC3 and PENKp appeared 30 h after LPS infusion. These results show for the first time that PC2 and PC3 are differentially expressed in PMN and AM, respectively, which were paralleled by the presence of different post-translational products of PENK. In addition, the in vivo effect of LPS on PC2, PC3 and PENKp levels in PMN and AM resembles the effect of LPS on prohormone levels in endocrine tissues, suggesting that similar mechanisms may control the turnover of PENK in endocrine and in these immune cells.

Animals↗
PubMed1993

Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells.

Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.

Amino Acid Sequence↗
PubMed2006

A novel loss of function mutation of PCSK9 gene in white subjects with low-plasma low-density lipoprotein cholesterol.

OBJECTIVES: The PCSK9 gene, encoding a pro-protein convertase involved in posttranslational degradation of low-density lipoprotein receptor, has emerged as a key regulator of plasma low-density lipoprotein cholesterol. In African-Americans two nonsense mutations resulting in loss of function of PCSK9 are associated with a 30% to 40% reduction of plasma low-density lipoprotein cholesterol. The aim of this study was to assess whether loss of function mutations of PCSK9 were a cause of familial hypobetalipoproteinemia and a determinant of low-plasma low-density lipoprotein cholesterol in whites. METHODS AND RESULTS: We sequenced PCSK9 gene in 18 familial hypobetalipoproteinemia subjects and in 102 hypocholesterolemic blood donors who were negative for APOB gene mutations known to cause familial hypobetalipoproteinemia. The PCSK9 gene variants found in these 2 groups were screened in 42 subjects in the lowest (<5th) percentile, 44 in the highest (>95th) percentile, and 100 with the average plasma cholesterol derived from general population. In one familial hypobetalipoproteinemia kindred and in 2 hypocholesterolemic blood donors we found a novel PCSK9 mutation in exon 1 (c.202delG) resulting in a truncated peptide (Ala68fsLeu82X). Two familial hypobetalipoproteinemia subjects and 4 hypocholesterolemic blood donors were carriers of the R46L substitution previously reported to be associated with reduced low-density lipoprotein cholesterol as well as other rare amino acid changes (T77I, V114A, A522T and P616L) not found in the other groups examined. CONCLUSIONS: We discovered a novel inactivating mutation as well as some rare nonconservative amino acid substitutions of PCSK9 in white hypocholesterolemic individuals.

Adult↗
PubMed2006

Melanocortins in human melanocytes.

Human epidermal keratinocytes and melanocytes express proopiomelanocortins (POMC) and all of the enzymes for POMC processing, i.e. prohormone convertases PC-1 and PC-2 including the regulatory protein 7B2. In melanocytes POMC processing also occurs in the melanosome, a lysosome-derived organelle that specializes in the biosynthesis of melanin. Consequently, the autocrine synthesis and release of the key hormones ACTH, alpha and beta-MSH and beta-endorphin takes also place in melanocytes. All four hormones have been reported to promote the biosynthesis of eumelanin in melanocytes. ACTH and alpha-MSH bind to the melanocortin-1 receptor (MC-1-R) on the plasma membrane and activate the signalling pathway predominantly coupled to production of cAMP, and in some cell lines raising intracellular calcium levels. In the melanocyte this signalling is redundant due to the high expression of alpha1 and beta2-adrenoceptors. Downstream events increase melanocyte this signalling is redundant due to the high expression of a tyrosinase expression / activity to stimulate eumelanogenesis. Studies with rMC-1-R transfected COS cells showed that both ACTH and alpha-MSH bind to the receptor with similar or different affinity depending on the species (human vs mice). We have modelled the MC-1-R based on the X-ray crystal structure of a homologous 7 receptor rhodopsin. Docking studies with ACTH1-39, ACTH1-17 and ACTH11-17 and alpha-MSH1-13 revealed that all 3 ACTH peptides yield thermodynamically stable (key ACTH1-13 in-lock) complexes. Interestingly, alpha-MSH is predicted to only have a kinetic effect on the MC-1-R and beta-MSH has even a weaker affinity for the MC-1-R than alpha-MSH. Based on these results the relative importance of ACTH versus alpha-MSH in the human epidermis has been re-evaluated.

Adrenocorticotropic Hormone↗
PubMed2006

Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor.

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.

Adenoviridae↗
PubMed2001

Can melatonin regulate the expression of prohormone convertase 1 and 2 genes via monomeric and dimeric forms of RZR/ROR nuclear receptor, and can melatonin influence the processes of embryogenesis or carcinogenesis by disturbing the proportion of cAMP and cGMP concentrations? Theoretic model of controlled apoptosis.

The presented model of controlled apoptosis has been based on the assumption that correct information exchange between an organism as a whole, and each of its cells is conditioned by mutual proportions of cAMP and cGMP concentrations (CcAMP, CcGMP), according to the formula CcAMP x CcGMP = 'a' (constant). The regulation of balance of these 'second messengers' in a cell and an extracellular space would depend on the mutual proportions of concentrations of Melatonin and monomers of Melanin. These indoloderived compounds could be the activators of the transcription factors i.e. RZR and NFkappa-B, regulating the expression of Prohormone Convertase (PC) gen and Nitric Oxide Synthase (NOS) gen, respectively. Additionally, maternal Melatonin and Nitric Oxide (NO), being able to pass through trophoblast or placenta freely, would play decisive role in the synchronization of embryogenesis and intrauterine development of the fetus. In case of an embryo or a fetus, the result of CcAMP and CcGMP multiplication, different from the proper constant 'a'-value, would mean occurrence of disorders in the structure and functioning of the cellular tensegrity system and, in consequence, disturbances in the intercellular information exchange. It would lead to deviation in cellular metabolism, oriented cell movement, uncontrolled apoptosis, and as a consequence, would lead to the development of fetal defects. In case of a child or an adult, a sudden occurrence and prolongation of such disturbances in CcAMP-CcGMP proportions would induce a process of apoptosis of normal cells and an initiation of a cancerogenesis. On the other hand, the recovery of equilibrium in the information exchange system would initiate apoptosis of neoplastic cells, and simultaneously, proliferation of connective tissue cells. According to the presented hypothesis, a decrease in CcAMP and destabilization of the CcAMP-CcGMP balance in an embryo or a fetus would result from relatively excessive amounts of maternal Melatonin (monomers) in fetal circulation, while a decrease of CcAMP and destabilization of the CcAMP-CcGMP balance in a child or an adult would be a consequence of relatively insufficient amounts of Melatonin (dimers) in an organism. It seems possible, that determination of both CcAMP and CcGMP would enable an early detection of high risk of developmental defects occurrence in an embryo or a fetus and neoplastic processes in a child or an adult. This method might also be considerably useful in monitoring a safe substitutional hormonotherapy.

Apoptosis↗
PubMed2003

Precursor-protein convertase 1 gene expression in the mouse hypothalamus: differential regulation by ob gene mutation, energy deficit and administration of leptin, and coexpression with prepro-orexin.

The expression of precursor-protein convertase (PC)1, PC2 and paired basic amino acid cleaving enzyme four mRNA was studied by in situ hybridisation in regions of the hypothalamus involved in energy regulation in relation to obese (ob) gene mutation and energy deficit. PC1 gene was differentially expressed in hypothalamic nuclei of mice from different genetic backgrounds or energetic status, whereas no differences in expression were observed for either the PC2 or paired basic amino acid cleaving enzyme four genes. In obese ob/ob mice, PC1 mRNA levels were increased in the paraventricular nucleus, decreased in the lateral hypothalamus and unchanged in the ventromedial nucleus and arcuate nucleus relative to lean controls. In response to intraperitoneal injection of murine leptin, PC1 mRNA levels in obese ob/ob mice decreased in the arcuate nucleus, increased in the lateral hypothalamus and were unchanged in both the paraventricular nucleus and ventromedial nucleus. In mice deprived of food for 24 h, PC1 mRNA levels were reduced in the ventromedial nucleus, increased in the lateral hypothalamus and unchanged in the paraventricular nucleus and arcuate nucleus relative to ad libitum-fed controls. Overall, whilst the data show effects related to leptin and energetic status, they do not support a strong and consistent link between PC1 gene expression and energy balance. This suggests that if PC1 is important to the control of energy balance then protein expression and activity, rather than gene expression may be the more critical parameters of regulation. The relationship between PC1 and candidate energy balance-related genes in the lateral hypothalamus was investigated by dual in situ hybridisation. PC1 mRNA was localised in prepro-orexin mRNA expressing neurons in the lateral hypothalamus, which suggests a functional relationship.

Animals↗
PubMed2001

Coregulation of glucagon-like peptide-1 synthesis with proglucagon and prohormone convertase 1 gene expression in enteroendocrine GLUTag cells.

The insulinotropic hormone glucagon-like peptide-1 (GLP-1) is synthesized in the intestinal L cell by prohormone convertase 1 (PC1)-mediated posttranslational processing of proglucagon. Previous studies have demonstrated that proglucagon gene transcription in the L cell is stimulated by the protein kinase A (PKA) pathway through a cAMP response element (CRE). Because the PC1 gene contains two functional CREs, the present studies were conducted to investigate whether the PC1 and proglucagon genes are coregulated by PKA, and to elucidate the temporal relationship(s) of PC1 and proglucagon gene expression with production of GLP-1, in the intestinal cell. The GLUTag enteroendocrine cell line, which is known to express the proglucagon gene and to synthesize and secrete GLP-1, was used as a model. Proglucagon and PC1 messenger RNA transcript levels were both increased after 12 h (but not 24 h) of treatment of GLUTag cells with forskolin/isobutylmethylxanthine (IBMX), by 2.7 +/- 0.3- and 2.4 +/- 0.3-fold, respectively, compared with controls (P < 0.01-0.001). Activation of PKA resulted in a 2.1 +/- 0.1-fold increase in PC1 reporter construct expression (P < 0.001) at 12 h, which was dependent on the presence of the CRE, and a 13- to 24-fold increment in PC1 protein levels (P < 0.01) at 12 and 24 h. Similarly, forskolin/IBMX increased secretion of GLP-1, by 1.8 +/- 0.2- and 2.2 +/- 0.6-fold at 12 and 24 h, respectively (P < 0.05-0.01). Although the cell content of GLP-1 was diminished after 12 h of treatment (P < 0.001), GLP-1 levels increased back to control values after 24 h of forskolin/IBMX treatment (P < 0.01 vs. 12-h levels). Thus, PKA-induced secretion of GLP-1 from the L cell is followed by restoration of the cellular peptide levels through a PKA-mediated, CRE-dependent up-regulation of proglucagon and PC1 gene expression.

1-Methyl-3-isobutylxanthine↗
PubMedDate not supplied

Peptidyl substrates containing unnatural amino acid at the P'1 position are potent inhibitors of prohormone convertases.

In order to study further the importance of the P'1 residue upon the activity of human PC1 and human furin, two important members of subtilisin/kexin family of enzymes, we have prepared by solid-phase Fmoc or recently introduced FastMoc chemistry a series of 10 peptidyl substrate analogs. The structures of these analogs are based upon the core sequence of pro-mPC1(83-93) namely, D-Tyr-Lys-Glu-Arg-Ser-Lys-Arg-Xaa-Val-Gln-Lys-Asp, where D-Tyr replaces the native L-Tyr residue and Xaa, representing the P'1 position, corresponds to L-Ser or to nonproteinacous amino acids such as Tle, Sarc, MLeu, Aib, D-Tic or L-Tic. Two more analogs with L-Tic at P'1 position but with one amino acid less, namely P5 Glu or P'3 Gln, and one with a Cit residue in place of Arg at P1 site of the dodecapeptide were also obtained. These peptides were all fully characterized by a combination of MS, 1H-NMR and amino acid analysis. In contrast to the Ser analog, which is an excellent substrate for both hPC1 and hfurin, these analogs displayed moderate to strong inhibition of both hPC1 and hfurin activity in a reversible competitive manner. They all exhibited higher potency for hfurin than for hPC1, with an inhibition constant (Ki) ranging from 0.8 to 10 microM and from 1.0 to 170 microM, respectively. Incorporation of L-Tic yielded an analog with a two to four-fold increased inhibition of either enzymes when compared to its D-Tic counterpart, the effect being more pronounced for hPC1 than for hfurin. Comparison of these data with those for the corresponding N-terminal Fmoc protected peptides revealed that the highly hydrophobic N-terminal Fmoc function occupying the P8 position can contribute positively or negatively towards proteinase inhibition depending on the nature of the unnatural amino acid at P'1 and the enzyme used. Finally, none of the analogs was significantly cleaved by either enzyme. FTIR data on these analogs revealed some important structural differences between the substrate and inhibitor analogs, as there appears to be a conformational shift from a more beta-sheet-like structure for the substrates to a more alpha-helical-like structure for the inhibitors.

Amino Acid Sequence↗
PubMed1994

Expression patterns of messenger ribonucleic acids encoding prohormone convertases (PC2 and PC3) in human pituitary adenomas.

OBJECTIVE: Prohormone convertases (PC2 and PC3) have been reported to play an important role for prohormone processing in rodent pituitaries. However, expression of mRNAs encoding these enzymes has not been characterized in human pituitaries. In addition, altered or insufficient prohormone processing has been reported in some human pituitary adenomas. Thus, the expression patterns of these mRNAs in non-tumorous and tumorous human pituitaries should be examined. DESIGN: Total RNAs were extracted from non-tumorous or tumorous human pituitaries to analyse PC2 and PC3 mRNA expression. SAMPLES: Five ACTH producing adenomas, 11 GH producing adenomas, one PRL producing adenoma and five non-functioning adenomas were obtained at surgery. Two non-tumorous pituitaries were also included in this study. MEASUREMENTS: The contents were quantitatively measured by Northern blot analysis using rat PC3 cDNA or human PC2 cDNA as a probe. The method was also developed for the detection of PC2 mRNA by Southern blot analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products. RESULTS: PC2 and PC3 mRNAs in non-tumorous samples were detected by Northern blot analysis whereas their contents in tumorous samples varied from high levels to undetectable. Marked variation of PC3 mRNA levels was observed among GH producing adenomas. ACTH producing adenomas were observed to express PC3 mRNA. Northern blot analysis also revealed that PC2 mRNA levels in ACTH producing adenomas were low except for one sample. PC2 mRNA expression in GH producing adenomas was confirmed by Southern blot analysis of RT-PCR products. This procedure also confirmed the various levels of PC2 mRNA expression among ACTH producing adenomas. CONCLUSION: The expression of PC2 and PC3 mRNA in human pituitaries has been confirmed. However, their expression has been observed to vary quantitatively and not to be restricted to certain types of pituitary cells.

Adenoma↗
PubMed1998

Functioning human insulinomas. An immunohistochemical analysis of intracellular insulin processing.

Sixty-seven insulinomas were investigated by immunohistochemistry using site-directed antibodies against insulin, proinsulin, chromogranin A, HISL-19, and four proteins directly or indirectly involved in the proteolytic processing of proinsulin: the prohormone convertases PC2 and PC3, carboxypeptidase H (CPH) and 7B2. Results were expressed in a six-grade score according to the frequency of immunoreactive tumour cells. Insulin was expressed by all tumours, appearing in either a diffuse or a polarized pattern and being detected in more than 30% of tumour cells in all cases but three. Proinsulin was also expressed in all tumours, with more than 50% of tumour cells immunoreactive in all cases but 5. It was consistently localized in the Golgi apparatus. In about half the cases, moreover, it also showed diffuse cytoplasmic staining, usually with a very sparse distribution. Trabecular and solid insulinomas did not present specific, homogeneous patterns of insulin immunostaining. However, insulin immunoreactivity was much more abundant in trabecular than in solid neoplasms, being present in virtually all tumour cells (score 6) in 50% and 8% of cases, respectively. Virtually all insulinomas expressed PC2, PC3, CPH and 7B2, usually in 30-100% of tumour cells, with a frequency significantly related to that of insulin. However, detection of PC2 and 7B2 was slightly less frequent than that of PC3 and CPH. In consecutive sections these proteins were found to be mostly co-localized with insulin and chromogranin A but not with proinsulin. They were heavily expressed in all 10 tumours with more than 10% of cells showing cytoplasmic proinsulin immunoreactivity, indicating that the leakage of proinsulin from the Golgi compartment is not associated with faulty expression of converting enzymes and possibly reflects a saturated processing capacity. HISL-19 immunoreactivity was found in both Golgi apparatus and insulin stores, indicating that the relevant antigen is different from all other proteins investigated. These results do not support a defect in expression or localization of proinsulin-processing enzymes in most insulinomas.

Adolescent↗
PubMed1995

Processing of prothyrotropin-releasing hormone (Pro-TRH) by bovine intermediate lobe secretory vesicle membrane PC1 and PC2 enzymes.

TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Expression of proopiomelanocortin and prohormone convertase-1 and -2 in the late gestation fetal sheep pituitary.

The biological activity of fetal plasma immunoreactive ACTH has been reported to increase during the final weeks of gestation in fetal sheep, indicative of enhanced processing of POMC to ACTH. The present study was aimed at examining the expression and localization of the prohormone convertases, PC1 and PC2, in the pituitary of fetal sheep during the final weeks of gestation. Pituitaries were obtained from fetal sheep during the final 50 days gestation (dGA) at 100-107 dGA (n = 6), 117-121 dGA (n = 6), 126-130 dGA (n = 7), and 144-147 dGA (n = 8; term = approximately 148 dGA). Pituitaries were cryosectioned and subjected to dual labeling in situ hybridization using 35S-labeled PC1 and/or PC2 complementary RNA probes with a digoxigenin-labeled POMC complementary RNA to localize and quantify PC1 and PC2 messenger RNA (mRNA) in POMC-hybridizing cells. Immunocytochemistry was also performed to assess coexpression of PC1 and PC2 with ACTH in the fetal pituitary. PC1 mRNA was heterogeneously distributed in the anterior pituitary (AP) at all gestational ages examined, with hybridization signals observed over POMC-expressing cells (corticotropes) as well as over noncorticotrope phenotypes. The inferior region of the AP contained an approximately 3-fold greater (P < 0.01) percentage of POMC cells containing PC1 transcripts compared with the superior region of the AP. The proportion of POMC cells containing PC1 was significantly higher (P < 0.01) in the 100-107 dGA and 144-147 dGA groups than in the 117-121 dGA and 126-130 dGA groups in both inferior and superior AP. The intensity of the PC1 hybridization signal over POMC-expressing cells was also about 2- to 4-fold greater (P < 0.01) in the inferior compared with the superior region of the fetal AP; the intensity of the PC1 hybridization signal associated with POMC cells remained constant within the AP region and did not change over the gestational ages examined. Hybridization for PC1 was highly variable over regions of AP not hybridizing for POMC, probably due to differences in the level of mRNA for PC1 between phenotypes. Similar to POMC cells, the average hybridization signal for PC1 over non-POMC-hybridizing regions was about 2-fold greater in the inferior vs. superior AP. A weak PC2 hybridization signal was observed over a small number of unidentified phenotypes in the fetal AP at all ages examined; no POMC cells were found to contain PC2 hybridization signal. In the neurointermediate lobe, POMC, PC1, and PC2 were ubiquitously expressed at all ages. Levels of PC1 and PC2 mRNA in the fetal neurointermediate lobe did not change over the period of gestation examined. Immunocytochemical analysis of PC1 and PC2 with ACTH confirmed the pattern of expression and the extent of coexpression observed with in situ hybridization methods. We conclude that both PC1 and PC2 are likely to contribute to POMC processing in the fetal pituitary during the final weeks of gestation.

Animals↗
PubMed1997

Comparative processing of bovine leukemia virus envelope glycoprotein gp72 by subtilisin/kexin-like mammalian convertases.

Intracellular proteolytic processing of bovine leukemia virus (BLV) envelope glycoprotein precursor (gp72) at the C-terminal end of the RVRR268 / site is an essential step for virus infectivity. Subtilisin/kexin-like convertases cleave proproteins at preferred RX(K/R)R / sites, including those commonly found in viral envelope glycoprotein precursors. We first demonstrated that gp72 is processed into gp51/gp30 in both CV1 cells and the furin-deficient LoVo cells, leading us to compare the ability of mammalian convertases to cleave BLV gp72 in vitro. In contrast to the inability of the neuroendocrine PC1 to cleave gp72, the convertases furin, PACE4, PC5-A and PC5-B, which process constitutively secreted precursors, can effectively cleave gp72 into gp51/gp30. N-terminal sequence analysis of the convertase-generated gp30 demonstrated that cleavage occurs at the in vivo-utilized RVRR / SPV site. Such furin-, PACE4- and PC5-mediated processing was completely inhibited by the alpha1-antitrypsin variant alpha1-PDX. Mutagenesis of the gp72 cleavage site into RVRG-TPV resulted in complete abrogation of gp72 processing by endogenous CV-1 cells and by convertases in vitro. Since our in vitro data suggest a redundancy in the ability of the convertases to cleave gp72, RT-PCR analysis was used to define the convertases expressed in B-lymphocytes, representing one of the major targets of BLV infection. Our data revealed that only furin and the newly discovered PC7 mRNAs are expressed in Raji, B-Jab and LG2 cell lines.

Amino Acid Sequence↗
PubMed2001

Furin and membrane type-1 metalloproteinase mRNA levels and activation of metalloproteinase-2 are associated with arterial remodeling.

Matrix metalloproteinase (MMP) activation is an essential feature of pathological and physiological arterial enlargement or shrinkage. Recently, furin-activated membrane type-1 MMP (MT1-MMP) was identified as the in vivo activator of MMP2 in mice. Although arterial enlargement and shrinkage are important in several pathological processes, this proprotein convertase-MT1-MMP axis has not been described during arterial remodeling. In rabbit femoral and carotid arteries, we report an increase in furin and MT1-MMP mRNA levels before and at the onset of arterial remodeling followed by an increase in activated MMP2. This reveals the presence of the proprotein convertase-MT1-MMP axis in flow-induced arterial remodeling and identifies furin as a possible target for local intervention in pathological arterial remodeling.

Animals↗
PubMed2006

Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain.

Bone morphogenetic protein-4 (BMP-4) is synthesized as a large precursor protein, which undergoes proprotein convertase-mediated proteolytic maturation along the secretory pathway to release the active ligand. Pro-BMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). This sequential cleavage liberates a small, evolutionarily conserved, prodomain fragment (the linker peptide) of unknown fate and function. Here we show that the linker domain is essential for proper folding, exit from the endoplasmic reticulum, and thus cleavage of the BMP-4 precursor when overexpressed in Xenopus oocytes and embryos but not in cultured mammalian cells. Mature BMP-4 synthesized from a precursor in which the S1 site is non-cleavable, such that the linker domain remains covalently attached to the ligand, has little or no activity in vivo. Finally, analysis of folding, cleavage, and bioactivity of chimeric precursors containing the BMP-7 prodomain and BMP-4 mature domain, or vice versa, with or without the BMP-4 linker domain revealed that the linker domain is only functional in the context of the BMP-4 prodomain, and that differential cleavage around this domain can regulate the activity of a heterologous ligand.

Amino Acid Sequence↗