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PubMed1998

Dynamics of proopiomelanocortin and prohormone convertase 2 gene expression in Xenopus melanotrope cells during long-term background adaptation.

The toad Xenopus laevis is able to adapt its skin color to background light intensity. In this neuroendocrine reflex, the proopiomelanocortin (POMC)-derived peptide alpha-melanophore-stimulating hormone (alphaMSH) is a key regulatory factor. In animals adapting to a black background, release of alphaMSH from the pituitary pars intermedia causes dispersal of melanin in skin melanophores. To investigate the long-term in vivo dynamics of alphaMSH production during black background adaptation, the biosynthetic rate of POMC and the contents of POMC, alphaMSH and the POMC processing enzyme precursor convertase 2 (PC2) have been studied in the pars intermedia using pulse-labeling, Western blot and radioimmunoassay. In control animals, adapted to a white background, the rate of POMC biosynthesis and the POMC content were low, while high alphaMSH and PC2 contents were found. After 1 week of adaptation to a black background, the rate of POMC biosynthesis and the POMC protein content had increased 19- and 3.7-fold respectively. These parameters attained a maximum level (28- and 5. 8-fold higher than control) after 3 weeks and remained at these elevated levels for at least 12 weeks. After 1 week, the pars intermedia content of alphaMSH was only 30% of the control level, but after 6 and 12 weeks, the alphaMSH level had increased to the control level. The PC2 content decreased to 52% of control after 1 week and stabilized after 3 weeks at a level slightly lower than the control value. The results show that during long-term background adaptation a steady-state situation is reached, with a balance between the biosynthesis, enzymatic processing and release of alphaMSH. The in vivo dynamics of the processing enzyme PC2 suggest a parallel storage and release of alphaMSH and mature PC2 in the Xenopus pituitary pars intermedia.

Adaptation, Physiological↗

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↗

PubMed2002

Glucagon replacement via micro-osmotic pump corrects hypoglycemia and alpha-cell hyperplasia in prohormone convertase 2 knockout mice.

Prohormone convertase 2 (PC2) plays an essential role in the processing of proglucagon to mature active glucagon in pancreatic alpha-cells (J Biol Chem 276:27197-27202, 2001). Mice lacking PC2 demonstrate multiple defects, including chronic mild hypoglycemia and dramatic hyperplasia of the pancreatic alpha-cells. To define the contribution of mature glucagon deficiency to the hypoglycemia and alpha-cell hyperplasia, we have attempted to correct the defects by delivery of exogenous glucagon by micro-osmotic pumps. Intraperitoneal delivery of 0.5 microg glucagon/h in PC2(-/-) mice resulted in the normalization of blood glucose concentrations. Islet remodeling through the loss of hyperplastic alpha-cells was evident by day 11 after pump implantation; by 25 days postimplantation, PC2(-/-) islets were indistinguishable from wild-type islets. These rapid changes were brought about by induction of apoptosis in the alpha-cell population. Morphological normalization of islets was also accompanied by marked downregulation of endogenous preproglucagon gene expression, but with little or no change in the level of preproinsulin gene expression. Exogenous glucagon delivery also normalized hepatic expression of the gluconeogenic enzyme PEPCK. These results demonstrate that the lack of mature glucagon in PC2(-/-) mice is responsible for the aberrant blood glucose levels, islet morphology, and gene expression, and they confirm the role of glucagon as a tonic insulin antagonist in regulating glycemia.

Animals↗

PubMed2004

Regulation of hypothalamic prohormone convertases 1 and 2 and effects on processing of prothyrotropin-releasing hormone.

Regulation of energy balance by leptin involves regulation of several neuropeptides, including thyrotropin-releasing hormone (TRH). Synthesized from a larger inactive precursor, its maturation requires proteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). Since this maturation in response to leptin requires prohormone processing, we hypothesized that leptin might regulate hypothalamic PC1 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Using hypothalamic neurons, we found that leptin stimulated PC1 and PC2 mRNA and protein expression and also increased PC1 and PC2 promoter activities in transfected 293T cells. Starvation of rats, leading to low serum leptin levels, decreased PC1 and PC2 gene and protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Exogenous administration of leptin to fasted animals restored PC1 levels in the median eminence (ME) and the PVN to approximately the level found in fed control animals. Consistent with this regulation of PCs in the PVN, concentrations of TRH in the PVN and ME were substantially reduced in the fasted animals relative to the fed animals, and leptin reversed this decrease. Further analysis showed that proteolytic cleavage of pro-thyrotropin-releasing hormone (proTRH) at known PC cleavage sites was reduced by fasting and increased in animals given leptin. Combined, these findings suggest that leptin-dependent stimulation of hypothalamic TRH expression involves both activation of trh transcription and stimulation of PC1 and PC2 expression, which lead to enhanced processing of proTRH into mature TRH.

Animals↗

PubMed2006

Short polybasic peptide sequences are potent inhibitors of PC5/6 and PC7: Use of positional scanning-synthetic peptide combinatorial libraries as a tool for the optimization of inhibitory sequences.

Positional scanning-synthetic peptide combinatorial libraries (PS-SPCLs) are powerful molecular tools to identify enzyme substrate and potent inhibitory sequences and also to provide crucial information about active site determinants. PS-SPCLs have been surveyed for furin, proprotein convertase (PC)2, PC1/3, and PACE4 and proven efficient to identify potent peptidyl inhibitors in the low nanomolar range for furin and PC1/3. We report herein the screenings of nonamidated and acetylated hexapeptide PS-SPCLs for PC5/6A and PC7. The L-configuration library surveys distinctively revealed that L-Arg, L-Lys, and sometimes L-His in all six positions would generate the most potent inhibitors for both enzymes. Based on this clear polybasic preference, L-poly-Arg peptides ranging from four to nine residues were assayed. Inhibitory potency of these polybasic peptides increased with chain length, making nona-l-Arg a potent nanomolar inhibitor of PC5/6A and PC7 (Ki of 150 and 120 nM). PC5/6 and PC7 inhibition by nona-l-arginine was equivalent to that of furin (Ki of 114 nM) (J Biol Chem 275: 36741-36749, 2000). Nona-d-arginine was a more potent inhibitor of PC5/6 and PC7 than its levorotatory version (Ki of 19 and 81 nM), reminiscent of furin (Ki of 1.3 nM) (J Biol Chem 279:36788-36794, 2004). Our data indicate that certain poly-arginine peptides represent potent inhibitors targeting PCs of the constitutive secretory pathway (furin, PC5/6, and PC7). We conclude that basic residues within PC peptide inhibitors might be responsible for targeting PCs in general and for inhibitory potency, but that select amino acid changes will be necessary to acquire true specificity toward a single PC.

Amino Acid Sequence↗

PubMed1996

Involvement of a polyproline helix-like structure in the interaction of 7B2 with prohormone convertase 2.

The neuroendocrine protein 7B2 is known to be involved in the biosynthesis and activity of prohormone convertase 2 (PC2). Previous studies have demonstrated that while the carboxyl-terminal portion of 7B2 (residues 155-186) regulates the enzymatic activity of PC2, the amino terminus of the molecule (residues 1-151) is required for maturation of proPC2. In this study we employed four different experimental approaches (co-immunoprecipitation with proPC2, facilitation of pro-PC2 maturation, acquisition of enzymatic activity, and thermal protection assays) to identify structural elements of 7B2 important for bioactivity. Inspection of the sequence of 7B2 indicated potential involvement of a polyproline helix-like (PPII) structure, with similarities to those present within SH3 domain ligands, in the interaction of 7B2 with proPC2. Site-directed point mutagenesis of this proline-rich region confirmed the involvement of this area. Replacement of prolines in positions critical to helix formation (Pro90, Pro91, Pro93, and Pro95) either severely impaired or totally abolished 7B2 bioactivity, as gauged by the four assays described. In addition, constructs longer than residues 1-121 were still functional, whereas those shorter than residues 1-109 were not. Computer-assisted analysis predicts the presence of an alpha-helix structure between residues 107 and 123. We conclude that both the proline-rich region and the alpha-helix contribute to 7B2 activity. Polyproline-containing peptides have been shown to be involved in cytoplasmic protein-protein interactions; our results suggest that the polyproline helix motif may also be used to mediate protein-protein interactions within the secretory pathway.

Animals↗

PubMed2000

Interaction of Drosophila melanogaster prohormone convertase 2 and 7B2. Insect cell-specific processing and secretion.

The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors. In Drosophila melanogaster, the homolog of prohormone convertase 2, dPC2 (amontillado), is required for normal hatching behavior, and immunoblotting data indicate that flies express 80- and 75-kDa forms of this protein. Because mouse PC2 (mPC2) requires 7B2, a helper protein for productive maturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open reading frame for this protein. dPC2 and d7B2 cDNAs were subcloned into expression vectors for transfection into HEK-293 cells; mPC2 and rat 7B2 were used as controls. Although active mPC2 was detected in medium in the presence of either d7B2 or r7B2, dPC2 showed no proteolytic activity upon coexpression of either d7B2 or r7B2. Labeling experiments showed that dPC2 was synthesized but not secreted from HEK-293 cells. However, when dPC2 and either d7B2 or r7B2 were coexpressed in Drosophila S2 cells, abundant immunoreactive dPC2 was secreted into the medium, coincident with the appearance of PC2 activity. Expression and secretion of dPC2 enzyme activity thus appears to require insect cell-specific posttranslational processing events. The significant differences in the cell biology of the insect and mammalian enzymes, with 7B2 absolutely required for secretion of dPC2 and zymogen conversion occurring intracellularly in the case of dPC2 but not mPC2, support the idea that the Drosophila enzyme has specific requirements for maturation and secretion that can be met only in insect cells.

Amino Acid Sequence↗

PubMed2004

The C-terminus of prohormone convertase 2 is sufficient and necessary for Raft association and sorting to the regulated secretory pathway.

Prohormone convertase 2 (PC2) is a member of the subtilisin family of proteases involved in prohormone maturation in the granules of the regulated secretory pathway (RSP). It has been suggested that targeting of this enzyme to the RSP is dependent on its association with lipid rafts in membranes at the trans-Golgi network. Here, we investigate the orientation of PC2 in granule membranes and the role of the C-terminus in sorting of the enzyme to the RSP. Molecular modeling and circular dichroism showed that this domain of PC2 forms an alpha-helix and inserts into artificial membranes. Furthermore, we show that the C-terminus of PC2 can be biotinylated at the C-terminus in intact chromaffin granules, indicating that it is a transmembrane protein. To determine if the PC2 C-terminus is necessary for raft association and sorting, we transfected a chimera of CPEDelta15 (carboxypeptidase E without the last 15 residues) and the last 25 residues of PC2 (CPEDelta15-PC2), and a truncated PC2 mutant with the last 6 residues deleted (PC2Delta6) into Neuro2a cells. Whereas CPEDelta15 was not raft-associated or sorted to the RSP, addition of the 25 residues of PC2 C-terminus to CPEDelta15 restored raft association and localization to the RSP granules, as determined by immunocytochemistry. Deletion of the last 6 residues of PC2 eliminated lipid raft association and sorting of PC2Delta6 to the RSP. These results showed that the PC2 C-terminus confers raft association and is sufficient and necessary for sorting PC2 to the RSP.

Amino Acid Sequence↗

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↗

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↗

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↗

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↗

PubMed2004

Perinatal food deprivation induces marked alterations of the hypothalamo-pituitary-adrenal axis in 8-month-old male rats both under basal conditions and after a dehydration period.

Dehydration is a classic homeostatic stressor in rats that leads to a series of endocrine responses including stimulation of the hypothalamo-pituitary-adrenal (HPA) axis. During the last decade, it has been well established that perinatal food restriction is associated with the onset of diseases in adults. Our previous demonstration of long-term alterations in HPA axis activity in both basal conditions and after a 72-hour dehydration period in 4-month-old rats exposed to a 50% maternal food restriction (FR50) in late gestation and lactation prompted us to investigate whether such perinatal undernutrition further affects HPA axis activity in mature animals. As previously described in 4-month-old rats under basal conditions, 8-month-old FR50 rats showed reduced body weight and an enhanced ratio between mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA levels in the hippocampus, as well as increased pro-opiomelanocortin (POMC) mRNA levels in the adenohypophysis. In addition, numerous additional alterations appeared in mature rats. In the hypothalamus, levels of vasopressin (VP) mRNAs were increased both in the paraventricular nucleus (PVN) and in the supraoptic nucleus (SON). In the adenohypophysis, GR and prohormone-convertase 2 (PC2) mRNA levels were significantly increased, whereas prohormone-convertase 1 (PC1) mRNA was not affected by maternal undernutrition. Interestingly, undernourished animals exhibited high plasma levels of total and free corticosterone in spite of normal corticotropin (ACTH) levels, an indication that HPA basal activity is enhanced by maternal undernutrition in 8-month-old animals. Dehydration for 72 h induced a rise in ACTH plasma levels, but did not modify total and free corticosterone plasma levels in 8-month-old FR50 animals. In the adenopituitary, POMC mRNA levels were decreased after dehydration but PC1 mRNA levels were unaffected. The present study indicates that maternal food restriction during the perinatal period dramatically affects the activity of the HPA axis until the age of 8 months. We speculate that higher basal HPA activity and an inadequate HPA response after dehydration in mature animals may contribute to diseases such as hypertension, known to develop with aging in perinatally growth-restricted rats.

Adrenocorticotropic Hormone↗

PubMed2004

The melanocortin receptor subtypes in chicken have high preference to ACTH-derived peptides.

1 Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. 2 To clarify the pharmacological impact of ligands acting in the MC system, we expressed the chicken MC1, MC2, MC3, MC4 and MC5 (cMC1-5) receptors in eukaryotic cells and performed comprehensive pharmacological characterization of the potency of endogenous and synthetic melanocortin peptides. 3 Remarkably, the cMC receptors displayed high affinity for ACTH-derived peptides and in general low affinity for alpha-MSH. It is evident that not only the cMC2 receptor but also the other cMC receptors interact with ACTH-derived peptide through an epitope beyond the sequence of alpha-MSH. 4 The synthetic ligand MTII was found to be a potent agonist whereas HS024 was a potent antagonist at the cMC4 receptor, indicating that these ligands are suitable for physiological studies in chicken. 5 We also show the presence of prohormone convertase 1 (PC1) and PC2 genes in chicken, and that these peptides are coexpressed with proopiomelanocortin (POMC) in various tissues.

Adrenocorticotropic Hormone↗