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PubMed1995

Molecular cloning, cDNA sequence, and localization of a prohormone convertase (PC2) from the Aplysia atrial gland.

Neuropeptides and peptide hormones are synthesized as part of larger precursor proteins that are processed post-translationally by subtilisin-related calcium-dependent prohormone convertases (PCs), frequently at multiple basic sites, to generate biologically active peptides. The atrial gland of Aplysia californica produces large quantities of egg-laying hormone (ELH)-related peptides, providing a unique opportunity to study prohormone processing. We have screened an Aplysia atrial gland cDNA library using a Lymnaea stagnalis PC2 probe and have isolated an Aplysia PC2-related 4.6-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland PC2 nucleotide sequence was obtained by reverse transcription/polymerase chain reaction (RT-PCR). The composite cDNA structure (5.6 kb) was deduced from sequence analysis of the RT-PCR product combined with the sequence obtained from the cDNA clone. The deduced cDNA of Aplysia atrial gland PC2 encoded a putative preproendoprotease of 653 amino acids that was evolutionarily related to other eukaryotic PC2s, and showed the strongest sequence identity with recently reported Aplysia nervous tissue PC2 sequences. In situ hybridization demonstrated extensive expression of PC2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3'untranslated region (3'-UTR) of 3,632 nucleotides. Strikingly, the 3'-UTR also contained several major nucleotide repeat sequences including the microsatellite repeats, (CA)n and (TG)n, and a TA-rich region comprised largely of the triplet repeat (TTA)n. The characterized Aplysia PC2 is a candidate endoprotease that may play an important role in the processing of ELH-related precursors in the atrial gland and represents the first example of PC2 expression in exocrine tissue.

Amino Acid Sequence↗
PubMed2003

Impaired intestinal proglucagon processing in mice lacking prohormone convertase 1.

The neuroendocrine prohormone convertases 1 and 2 (PC1 and PC2) are expressed in endocrine intestinal L cells and pancreatic A cells, respectively, and colocalize with proglucagon in secretory granules. Mice lacking PC2 have multiple endocrinopathies and cannot process proglucagon to mature glucagon in the pancreas. Disruption of PC1 results in dwarfism and also multiple neuroendocrine peptide processing defects. This study compares the pancreatic and intestinal processing of proglucagon in mice lacking PC1 expression with that in age-matched wild-type controls. Because proglucagon was found to precipitate in acidic extracts, the intestinal processing profile was analyzed in both acidic and neutral extracts by gel filtration, HPLC, and RIA. Supporting a central role for PC2 in glucagon biosynthesis, we found normal processing of proglucagon to glucagon in the pancreas, whereas the intestinal proglucagon processing showed marked defects. Tissue proglucagon levels in null mice were elevated, and proglucagon processing to glicentin, oxyntomodulin, and glucagon-like peptide-1 and -2 (GLP-1 and GLP-2) was markedly decreased, indicating that PC1 is essential for the processing of all the intestinal proglucagon cleavage sites. This includes the monobasic site R(77) and, thereby, production of mature, biologically active GLP-1. We also found elevated glucagon levels, suggesting that factors other than PC1 that are capable of processing to mature glucagon are present in the secretory granules of the L cell. These findings strongly suggest that PC1 is essential for intestinal proglucagon processing in vivo and, thereby, plays an important role in production of the incretin hormone GLP-1 and the intestinotrophic hormone GLP-2.

Animals↗
PubMed2005

Regulation of regional expression in rat brain PC2 by thyroid hormone/characterization of novel negative thyroid hormone response elements in the PC2 promoter.

The prohormone convertases (PCs) PC1 and PC2 are involved in the tissue-specific endoproteolytic processing of neuropeptide precursors within the secretory pathway. We previously showed that changes in thyroid status altered pituitary PC2 mRNA and that this regulation was due to triiodothyronine-dependent interaction of the thyroid hormone receptor (TR) with negative thyroid hormone response elements (nTREs) contained in a large proximal region of the human PC2 promoter. In the current study, we examined the in vivo regulation of brain PC2 mRNA by thyroid status and found that 6-n-propyl-2-thiouracil-induced hypothyroidism stimulated, whereas thyroxine-induced hyperthyroidism suppressed, PC2 mRNA levels in the rat hypothalamus and cerebral cortex. To address the mechanism of T3 regulation of the PC2 gene, we used human PC2 (hPC2) promoter constructs transiently transfected into GH3 cells and found that triiodothyronine negatively and 9-cis-retinoic acid positively regulated hPC2 promoter activity. EMSAs, using purified TRalpha1 and retinoid X receptor-beta (RXRbeta) proteins demonstrated that TRalpha bound the distal putative nTRE-containing oligonucleotide in the PC2 promoter, and RXR bound to both nTRE-containing oligonucleotides. EMSAs with oligonucleotides containing deletion mutations of the nTREs demonstrated that the binding to TR and RXR separately is reduced, but specific binding to TR and RXR together persists even with deletion of each putative nTRE. We conclude that there are two novel TRE-like sequences in the hPC2 promoter and that these regions act in concert in a unique manner to facilitate the effects of thyroid hormone and 9-cis-retinoic acid on PC2.

Alitretinoin↗
PubMed1997

Mechanism of the facilitation of PC2 maturation by 7B2: involvement in ProPC2 transport and activation but not folding.

Among the members of the prohormone convertase (PC) family, PC2 has a unique maturation pattern: it is retained in the ER for a comparatively long time and its propeptide is cleaved in the TGN/ secretory granules rather than in the ER. It is also unique by its association with the neuroendocrine protein 7B2. This interaction results in the facilitation of proPC2 maturation and in the production of activatable proPC2 from CHO cells. In the present study, we have investigated the mechanism of this interaction. ProPC2 binds 7B2 in the ER, but exits this compartment much more slowly than 7B2. We found that proPC2 was also slow to acquire the capacity to bind 7B2, whereas 7B2 could bind proPC2 rapidly after synthesis. This indicated that proPC2 folding was the limiting step in the formation of the complex. Indeed, sensitivity of native proPC2 to N-glycanase F digestion and inhibition of proPC2 folding supported the notion that 7B2 is not involved in the early steps of proPC2 folding, and that proPC2 must fold before binding 7B2. Under experimental conditions that prevent propeptide cleavage, 7B2 expression increased proPC2 transport to the Golgi. This increase exhibited the same kinetics as the facilitation of the removal of the propeptide. Finally, proPC2 activation could be reconstituted in Golgi- enriched subcellular fractions. In vitro, 7B2 was required for proPC2 activation at an acidic pH. Taken together, our results demonstrate that rather than promoting proPC2 folding, 7B2 acts as a helper protein involved in proPC2 transport and is required in the proPC2 activation process. We propose, therefore, that 7B2 stabilizes proPC2 in a conformation already competent for these two events.

Animals↗
PubMed2005

Strain-dependent influences on the hypothalamo-pituitary-adrenal axis profoundly affect the 7B2 and PC2 null phenotypes.

Two null mouse models have previously been created to study the role of the prohormone convertase (PC2) and its helper protein 7B2; unexpectedly, the phenotypes of these two nulls differ profoundly, with the 7B2 but not the PC2 null dying at 5 wk. The genetic backgrounds of these two models differ, with the 7B2 null in a 129/SvEv (129) background and the PC2 null in a mixed C57BL/N6:129/SvEv (B6:129) background. Because background can contribute greatly to phenotype, we have here examined strain influence on the hypothalamo-pituitary-adrenal (HPA) axis and glucose levels in wild-type, 7B2 null, and PC2 null mice. Wild-type B6 and 129 mice differed in basal corticosterone and glucose levels. When 7B2 nulls were transferred onto the B6 background, they survived and showed greatly decreased circulating corticosterone and increased blood glucose levels, most likely due to the comparatively higher adrenal resistance of the B6 strain to ACTH stimulation. Circulating ACTH levels were increased over wild-type in the B6 7B2 null but did not reach levels as high as the 129 7B2 null. Conversely, when the mixed-strain PC2 nulls were bred into the 129 background at the N6 generation, they began to exhibit the Cushing's-like phenotype characteristic of 129 7B2 null mice and died before 6 wk of age. Taken together, these results indicate that background effects are critical because they increase the phenotypic differences between the 7B2 and PC2 nulls and play a life-or-death role in the ACTH hypersecretion syndrome present in both 129 nulls.

Adrenocorticotropic Hormone↗
PubMed2000

Interactions between the prohormone convertase 2 promoter and the thyroid hormone receptor.

The majority of prohormones are cleaved at paired basic residues to generate bioactive hormones by prohormone convertases (PCs). As PC1 and PC2, two neuroendocrine-specific PCs, appear to be the key enzymes capable of processing a variety of prohormones, alterations of PC2 and/or PC1 levels will probably have a profound effect on hormonal homeostasis. We investigated the regulation of PC2 messenger RNA (mRNA) by thyroid hormone using GH3 cells to demonstrate that T3 negatively regulated PC2 mRNA levels in a dose- and time-dependent fashion. Functional analysis of progressive 5'-deletions of the human (h) PC2 promoter luciferase constructs in GH3 cells demonstrated that the regulation probably occurs at the transcriptional level, and that putative negative thyroid hormone response elements were located within the region from -44 to + 137 bp relative to the transcriptional start site. Transient transfections in JEG-3 cells and COS-1 cells showed that the suppressive effect of T3 was equally mediated by the thyroid hormone receptor (TR) isoforms TRalpha1 and TRbeta1. Electrophoretic mobility shift assays using purified TRal and retinoid X receptor-beta protein as well as GH3 nuclear extracts showed that regions from +51 to +71 bp and from +118 to +137 bp of the hPC2 promoter bind to TRalpha1 as both a monomer and a homodimer and with TRalpha1/retinoid X receptor-beta as a heterodimer. Finally, the in vivo regulation of pituitary PC2 mRNA by thyroid status was demonstrated in rats. These results demonstrate that T3 negatively regulates PC2 expression at the transcriptional level and that functional negative thyroid hormone response elements exist in the hPC2 promoter. We postulate that the alterations of PC2 activity may mediate some of the pathophysiological consequences of hypo- or hyperthyroidism.

Animals↗
PubMed2003

Obliteration of alpha-melanocyte-stimulating hormone derived from POMC in pituitary and brains of PC2-deficient mice.

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuropeptide expressed in pituitary and brain that is known to regulate energy balance, appetite control, and neuroimmune functions. The biosynthesis of alpha-MSH requires proteolytic processing of the proopiomelanocortin (POMC) precursor. Therefore, this study investigated the in vivo role of the prohormone convertase 2 (PC2) processing enzyme for production of alpha-MSH in PC2-deficient mice. Specific detection of alpha-MSH utilized radioimmunoassay (RIA) that does not crossreact with the POMC precursor, and which does not crossreact with other adrenocorticotropin hormone (ACTH) and beta-endorphin peptide products derived from POMC. alpha-MSH in PC2-deficient mice was essentially obliterated in pituitary, hypothalamus, cortex, and other brain regions (collectively), compared to wild-type controls. These results demonstrate the critical requirement of PC2 for the production of alpha-MSH. The absence of alpha-MSH was accompanied by accumulation of ACTH, ACTH-containing imtermediates, and POMC precursor. ACTH was increased in pituitary and hypothalamus of PC2-deficient mice, evaluated by RIA and reversed-phase high pressure liquid chromatography (RP-HPLC). Accumulation of ACTH demonstrates its role as a PC2 substrate that can be converted for alpha-MSH production. Further analyses of POMC-derived intermediates in pituitary, conducted by denaturing western blot conditions, showed accumulation of ACTH-containing intermediates in pituitaries of PC2-deficient mice, which implicate participation of such intermediates as PC2 substrates. Moreover, accumulation of POMC was observed in PC2-deficient mice by western blots with anti-ACTH and anti-beta-endorphin. In addition, increased beta-endorphin1-31 was observed in pituitary and hypothalamus of PC2-deficient mice, suggesting beta-endorphin1-31 as a substrate for PC2 in these tissues. Overall, these studies demonstrated that the PC2 processing enzyme is critical for the in vivo production of alpha-MSH in pituitary and brain.

Adrenocorticotropic Hormone↗
PubMed1999

The role of the 7B2 CT peptide in the inhibition of prohormone convertase 2 in endocrine cell lines.

Prohormone convertase (PC) 2 plays an important role in the processing of neuropeptide precursors via the regulated secretory pathway in neuronal and endocrine tissues. PC2 interacts with 7B2, a neuroendocrine protein that is cleaved to a 21-kDa domain involved in proPC2 maturation and a carboxyl-terminal peptide (CT peptide) that represents a potent inhibitor of PC2 in vitro. A role for the CT peptide as an inhibitor in vivo has not yet been established. To study the involvement of the CT peptide in PC2-mediated cleavages in neuroendocrine cells, we constructed a mutant proenkephalin (PE) expression vector containing PE with its carboxyl-terminal peptide (peptide B) replaced with the 7B2 inhibitory CT peptide. This PECT chimera was stably transfected into two PC2-expressing cell lines, AtT-20/PC2 and Rin cells. Although recombinant PECT proved to be a potent (nM) inhibitor of PC2 in vitro, cellular PC2-mediated cleavages of PE were not inhibited by the PECT chimera, nor was proopiomelanocortin cleavage (as assessed by adrenocorticotropin cleavage to alpha-melanocyte-stimulating hormone) inhibited further than in control cells expressing only the competitive substrate PE. Tests of stimulated secretion showed that both the CT peptide and the PE portion of the chimera were stored in regulated secretory granules of transfected clones. In both AtT-20/PC2 and Rin cells expressing the chimera, the CT peptide was substantially internally hydrolyzed, potentially accounting for the observed lack of inhibition. Taken together, our data suggest that overexpressed CT peptide derived from PECT is unable to inhibit PC2 in mature secretory granules, most likely due to its inactivation by PC2 or by other enzyme(s).

Animals↗
PubMed1995

Enzymatic characterization of immunopurified prohormone convertase 2: potent inhibition by a 7B2 peptide fragment.

Prohormone convertase (PCs) are thought to mediate the controlled proteolysis of prohormones and neuropeptide precursors. While recombinant PC1 and furin are currently available, thus far it has not been possible to produce recombinant PC2. We have used conditioned medium obtained from the mouse insulinoma cell line beta TC3 to generate a working preparation of enzymatically active PC2 through immunopurification. Immunopurified PC2 cleaved the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC in a time- and calcium-dependent manner. It was half-maximally stimulated at 75 microM Ca2+, had an optimum pH of 5, and exhibited PCMS and EDTA sensitivity similar to that reported for furin and PC1. The tight-binding inhibitor 27 kDa 7B2 was used to calculate the Kd for this inhibitor and the active enzyme concentration. The Kd was 7.3 +/- 1.7 nM, and the turnover rate of PC2 was 5.2 molecules substrate per enzyme molecule per minute. The specific activity was 4.9 nmol/micrograms/h (assuming a molecular mass for PC2 of 64 kDa). The enzyme preparation was able to cleave recombinant proenkephalin at at least four of the expected paired basic sites in the absence, but not in the presence, of 27 kDa 7B2. Since 21 kDa 7B2 is functionally inactive as a proteinase inhibitor, we examined the inhibitory activity of the carboxy-terminal portion of 27 kDa 7B2 (7B2 CT-peptide). Synthetic peptides were used to demonstrate that the 7B2 CT-peptide (a) represents a potent inhibitor of PC2 (Ki = 57 nM), (b) can block the conversion of proPC2 to PC2, and (c) can block the PC2-mediated conversion of proenkephalin to smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Inactivation of the 7B2 inhibitory CT peptide depends on a functional furin cleavage site.

The eukaryotic subtilisin prohormone convertase 2 (PC2) is known to require in vivo exposure to the neuroendocrine protein 7B2 in order to produce an enzymatically active species capable of proteolytic action on prohormone substrates. In the present study, we examined the role of the pentabasic site within 27-kDa 7B2 in this process. We prepared two His-tagged recombinant 7B2s by overexpression in bacteria: 7B2-Ser-Ser (SS), with an inactivating mutation in the CT peptide from Lys171-Lys172 (KK) to SS, rendering the CT peptide non-inhibitory; blockade-SS, a double mutant of both the CT peptide as well as of the pentabasic furin cleavage site. These purified proteins were used in a cell-free proPC2 activation assay. Both 7B2-SS as well as blockade-SS were able to facilitate the activation of proPC2 (as judged by efficient production of enzyme activity), suggesting that cleavage at the furin site is not required for 7B2s lacking inhibitory CT peptides. Plasmids encoding proPC2 and various 7B2s were transiently transfected into human embryonic kidney (HEK293) cells and PC2 enzymatic activity and CT forms in each overnight conditioned medium were measured. Cells transfected with proPC2 and wild-type 7B2 secreted CT peptide cleavage products, but cells transfected with proPC2 and the blockade mutant overwhelmingly secreted intact, 27-kDa, blockaded 7B2. Medium obtained from HEK293 cells transfected with proPC2 and either wild-type 7B2, 7B2-SS, or blockade-SS exhibited PC2 activity, but medium from cells expressing the 7B2 blockade mutant did not. We conclude that cleavage at the 7B2 furin consensus site is required to produce PC2 capable of efficient proteolytic inactivation of the CT peptide.

Animals↗
PubMed1997

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.

Activating Transcription Factor 1↗
PubMed2003

Abrogation of protein convertase 2 activity results in delayed islet cell differentiation and maturation, increased alpha-cell proliferation, and islet neogenesis.

To date, the role of pancreatic hormones in pancreatic islet growth and differentiation is poorly understood. To address this issue, we examined mice with a disruption in the gene encoding prohormone convertase 2 (PC2). These mice are unable to process proglucagon, prosomatostatin, and other neuroendocrine precursors into mature hormones. Initiation of insulin (IN) expression during development was delayed in PC2 mutant mice. Cells containing IN were first detected in knockout embryos on d 15 of development, 5 d later than in wild-type littermates. However, the IN(+) cells of d 15 PC2 mutant mice coexpressed glucagon, as did the first appearing beta-cells of controls. In addition, lack of PC2 perturbed the pattern of expression of transcription factors presumed to be involved in the determination of the mature alpha-cell phenotype. Thus, in contrast to controls, alpha-cells of mutant mice had protracted expression of Nkx 6.1 and Pdx-1, but did not express Brn-4. Islets of adult mutant mice also contained cells coexpressing insulin and somatostatin, an immature cell type found only in islets of the wild-type strain during development. In addition to the effects on islet cell differentiation, the absence of PC2 activity resulted in a 3-fold increase in the rate of proliferation of proglucagon cells during the perinatal period. This increase contributed to the development of alpha-cell hyperplasia during postnatal life. Furthermore, the total beta-cell volume was increased 2-fold in adult mutants compared with controls. This increase was due to islet neogenesis, as the number of islets per section was significantly higher in knockout mice compared with wild-type mice, whereas both strains had similar rates of IN cell proliferation. These results indicate that hormones processed by PC2 affected processes that regulate islet cell differentiation and maturation in embryos and adults.

Age Factors↗
PubMed1996

Purification and enzymatic characterization of recombinant prohormone convertase 2: stabilization of activity by 21 kDa 7B2.

Although previous efforts to produce significant quantities of purified prohormone convertase 2 from either recombinant or natural sources have been unsuccessful, our recent finding that the neuroendocrine polypeptide 7B2 is necessary for the biosynthesis of enzymatically active prohormone convertase 2 (PC2) has enabled us to obtain active recombinant enzyme from the conditioned medium of PC2-producing CHO cells supertransfected with cDNA coding for 21 kDa 7B2. The recombinant enzyme was purified to apparent homogeneity, with a 40% recovery, in milligram quantities. Two protein bands of Mrs 71 and 75 kDa were observed after SDS-PAGE followed by either Coomassie staining or Western blotting with PC2 antiserum. Spontaneous conversion of the 71- and 75-kDa species to the 66-kDa form occurred during incubation at pH 5.0; the degree of conversion correlated with a dramatic increase in activity. Kms of 124 and 131 microM and Kcats of 0.49 and 0.81 s(-1) were obtained for the substrates Cbz-Arg-Ser-Lys-Arg-AMC and Pyr-Arg-Thr-Lys-Arg-AMC, respectively. The pH optimum was 5.0, and the enzyme was inhibited by h7B2(155-185') p-CMS, and EDTA but not by other inhibitors tested. Interestingly, 21 kDa 7B2 was observed to copurify with the enzyme in a molar ratio of about 1:100 (7B2:PC2). Prior addition of recombinant 21 kDa 7B2 to activated 66 kDa PC2 provided significant protection against thermal denaturation. When coassociated 7B2 was mostly removed from activated PC2 through gel filtration, subsequent addition of recombinant 7B2 exerted a significant stabilizing effect on enzyme activity. Millimolar Ca2+ and pHs between 5 and 6 were required to observe this effect. Since these conditions resemble those thought to occur within secretory granules, and since 21 kDa 7B2 represents a stored secretory granule protein, our data suggest a physiological role for 21 kDa 7B2 in the stabilization of PC2 activity.

Amino Acid Sequence↗
PubMed2002

Mortality in 7B2 null mice can be rescued by adrenalectomy: involvement of dopamine in ACTH hypersecretion.

The serine protease prohormone convertase 2 (PC2), principally involved in the processing of polypeptide hormone precursors in neuroendocrine tissues, requires interaction with the neuroendocrine protein 7B2 to generate an enzymatically active form. 7B2 null mice express no PC2 activity and release large quantities of uncleaved ACTH, resulting in a lethal endocrine condition that resembles pituitary Cushing's (Westphal, C. H., Muller, L., Zhou, A., Bonner-Weir, S., Schambelan, M., Steiner, D. F., Lindberg, I. & Leder, P. (1999) Cell 96, 689). Here, we have compared the 7B2 and PC2 null mouse models to determine why the 7B2 null, but not the PC2 null, exhibits a lethal disease state. Both 7B2 and PC2 nulls contained highly elevated pituitary adrenocorticotropic hormone (ACTH); the neurointermediate lobe content of ACTH in 7B2 nulls was 13-fold higher than in WT mice; that of the PC2 null was 65-fold higher. However, circulating ACTH levels were much higher in the 7B2 null than in the PC2 null. Because hypothalamic inhibitory dopaminergic control represents the major influence on intermediate lobe proopiomelanocortin-derived peptide secretion, dopamine levels were measured, and they revealed that 7B2 null pituitaries contained only one-fourth of WT pituitary dopamine. Adrenalectomized 7B2 null animals survived past the usual time of death at 5 weeks; a month after adrenalectomy, they exhibited normal levels of pituitary dopamine, circulating ACTH, and corticosterone. Elevated corticosterone, therefore, seems to play a central role in the lethal phenotype of the 7B2 null, whereas a 7B2-mediated dopaminergic deficiency state may be involved in the actual ACTH hypersecretion phenomenon. Interestingly, adrenalectomized 7B2 nulls also developed unexpectedly severe obesity.

Adrenal Glands↗
PubMed2003

Selective roles for the PC2 processing enzyme in the regulation of peptide neurotransmitter levels in brain and peripheral neuroendocrine tissues of PC2 deficient mice.

The prohormone convertase 2 (PC2) is hypothesized to convert multiple pro-neuropeptides into active peptides that function as neurotransmitters. To examine the in vivo role of PC2 in neuropeptide production, the tissue contents of six different neuropeptides in brain and peripheral nervous tissues were examined in PC2 deficient mice. Specific neuropeptide radioimmunoassays and RP-HPLC (reverse-phase HPLC) provided evaluation of processed, active neuropeptides in brain and neuroendocrine tissues of PC2 deficient mice. Results demonstrated three features with regard to the selective roles of PC2 in determining the production of NPY, somatostatin-28, enkephalin, VIP, galanin, and CRF in neuroendocrine tissues. Firstly, PC2 deficient mice showed changes in several neuropeptides, but not all neuropeptides examined. The absence of active PC2 resulted in altered cellular levels of NPY, somatostatin-28, and (Met)enkephalin; few changes in VIP or galanin occurred in the tissues examined. CRF content was not altered in brains of PC2 deficient mice. Secondly, comparison of a single neuropeptide among different tissues of PC2 deficient mice demonstrated tissue-selective roles for PC2 in production of the neuropeptide. For example, NPY levels were decreased in ileum of PC2 deficient mice, but NPY content was not altered in hypothalamus that is abundant in NPY. In addition, (Met)enkephalin levels in hypothalamus and cortex were decreased in PC2 deficient mice, but no changes were observed in adrenal or intestine. Thirdly, a single tissue region often showed selective alterations among different neuropeptides. For example, the neuropeptide-rich hypothalamus region showed decreased (Met)enkephalin in PC2 deficient mice, but NPY, VIP, galanin, and CRF were not altered. These results demonstrate the selective role of PC2 in neuropeptide production that provides active peptide neurotransmitter or hormones for biological functions in brain and neuroendocrine systems.

Animals↗
PubMed1998

The propeptide of prohormone convertase PC2 acts as a transferable aggregation and membrane-association signal.

Prohormone convertase 2 (PC2) is a subtilisin-like protease involved in the intracellular processing of prohormones and proneuropeptides. Like its substrates, it is synthesised as a prepropeptide which undergoes proteolysis during transit through the regulated secretory pathway. Previous studies have shown that aggregation and membrane association of proPC2 occurs in a calcium-dependent and pH-dependent manner and that the pro-region of PC2 may be involved in this process. These events may be involved in the sorting of proteins to the regulated secretory pathway. To investigate this further, we made a chimeric protein containing both the signal peptide and pro-region of PC2 and the N-terminal part of alpha1-antitrypsin, called pro2alpha1. PC2, alpha1-antitrypsin and pro2alpha1 were compared with regard to their membrane association and aggregation properties using, respectively, sucrose gradient centrifugation after expression in Xenopus oocytes, and an in vitro aggregation assay. The chimeric protein, pro2alpha1, underwent low-pH-dependent aggregation and membrane association similar to wild-type PC2. Membrane association occurred at pH 5.5 in the absence of calcium and at pH 6.0 in the presence of 10 mM calcium but not at pH 6.5 or 7.0. alpha1-antitrypsin, as expected of a constitutively secreted protein, did not aggregate at low pH, nor associate with membranes. Pro2alpha1 thus exhibits the membrane association and aggregation properties of PC2, confirming the role of the pro-region in these processes. A series of deletions were performed within the 84-residue propeptide in order to define the sequences involved. Deletion of amino acids 52-77 reduced aggregation but large deletions in the pro-region had only a minimal effect on membrane association. These data suggest that several regions within the propeptide are important in these events.

Animals↗
PubMed1994

Design and synthesis of novel inhibitors of prohormone convertases.

Prohormone convertase-1 (PC1) and furin are subtilisin-like endopeptidases involved in the biosynthesis of peptide hormones. Five decapeptides representing the junction between the pro-region and the catalytic region of PC1 were prepared. The core sequence corresponded to D-Tyr-Arg-Ser-Lys-Arg- Xaa-Val-Gln-Lys-Asp where D-Tyr replaces the native Glu residue and Xaa, representing the P1' position, corresponds to L-Ser, L-Leu or the unnatural amino acids, D-Ser, beta-Ala, gamma-Abu, beta-Cha or gamma-Hyp. Another analog incorporating an Orn residue in place of the Arg at the P1 site was also prepared. These peptides, synthesized by solid-phase Fmoc chemistry, were fully characterized by FAB-MS, 1H-NMR and amino acid composition. Except for Orn, gamma-Hyp, L/D-Ser and L-Leu containing analogs, the others were found to be moderate to potent competitive inhibitors of hPC1 activity in the following order: gamma-Abu > beta-Cha > beta-Ala, with Ki values ranging from 1 to 8.6 microM. Both L-Ser and L-Leu analogs were correctly cleaved at the acyl carbon COOH-terminal to the Lys-Arg pair by human PC1, whereas beta-Cha, gamma-Abu, beta-Ala and D-Ser analogs proved to be very poor substrates. The Orn and gamma-Hyp derivatives were not cleaved by the enzyme at all. The three analogs containing beta-Cha, gamma-Abu and beta-Ala also proved to be potent inhibitors of the human furin activity in the following order: beta-Ala > beta-Cha > gamma-Abu, with Ki ranging from 0.8 to 2.2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Neuroendocrine protein 7B2 can be inactivated by phosphorylation within the secretory pathway.

The prohormone convertases play important roles in the maturation of neuropeptides and peptide hormone precursors. Prohormone convertase-2 (PC2) is the only convertase that requires the expression of another neuroendocrine protein, 7B2, for expression of enzyme activity. In this study, we determined that 7B2 can be phosphorylated in Rin cells (a rat insulinoma cell line) and cultured chromaffin cells, but not in AtT-20 cells (derived from mouse anterior pituitary). Phosphoamino acid analysis of Rin cell 7B2 indicated the presence of phosphorylated serine and threonine. Phosphorylation of Ser115 (located within the minimally active 36-residue peptide) was confirmed by mutagenesis, although Ser115 did not represent the sole residue phosphorylated. Two independent assays were used to investigate the effect of phosphorylated 7B2 on PC2 activation: the ability of 7B2 to bind to pro-PC2 was assessed by co-immunoprecipitation, and activation of pro-PC2 was assessed in a cell-free assay. Phosphorylated 7B2 was unable to bind pro-PC2, and the phosphorylated 7B2 peptide (residues 86-121, known to be the minimally active peptide for pro-PC2 activation) was impaired in its ability to facilitate the generation of PC2 activity in membrane fractions containing pro-PC2. In vitro phosphorylation experiments using Golgi membrane fractions showed that 7B2 could be phosphorylated by endogenous Golgi kinases. Golgi kinase activity was strongly inhibited by the broad-range kinase inhibitor staurosporine and partially inhibited by the protein kinase C inhibitor bisindolylmaleimide I, but not by the other protein kinase A, Ca2+/calmodulin-dependent kinase II, myosin light chain kinase, and protein kinase G inhibitors tested. We conclude that phosphorylation of 7B2 functionally inactivates this protein and suggest that this may be analogous to the phosphorylating inactivation of BiP, which impairs its ability to bind substrate.

Adrenal Glands↗