proPC2 Maturation Research Articles

1768-P: Sel1L-Hrd1 Endoplasmic Reticulum-Associated Degradation Regulates PC2 Function and Proglucagon Processing in Islet α-Cells

Pancreatic islets rely on cellular protein quality control to maintain glucoregulatory hormone production. Proglucagon, precursor of glucagon and glucagon-like peptide 1 (GLP1), is expressed in islet α cells, intestinal L-cells, and some neurons. Islet α cells predominantly convert proglucagon to glucagon through prohormone convertase 2 (PC2). Islet-derived GLP1 is increasingly recognized as an important contributor to islet function, but the factors controlling its production remain unclear. To understand how protein quality control affects proglucagon processing in α cells, we inactivated the highly conserved Sel1L-Hrd1 endoplasmic reticulum-associated degradation (ERAD) system using Cre-loxP recombination in mice. Compared to littermate controls, Sel1LΔGcg mice showed an age-related decline in glucagon secretion in vivo, associated with a 43% decrease in pancreatic glucagon content (76.1 +/- 14.1 vs. 134.4 +/- 14.9 ng/g). Pancreatic GLP1 content was unchanged in Sel1LΔGcg mice (779.4 +/- 60.7 vs. 773.4 +/- 128.6 pg/g), despite a 37% reduction in α cell mass (0.19 +/- 0.04 vs. 0.12 +/- 0.04 mg/pancreas). Expression of the precursor form of PC2 (proPC2) was increased in Sel1LΔGcg α cells, suggesting accumulation of misfolded protein and/or impaired maturation. To investigate the mechanism underlying these changes, we used CRISPR to delete Sel1L in αTC cells. ERAD-deficient αTC-ΔSel1L cells showed 1.75-fold higher expression of proPC2, in addition to appearance of a novel truncated (~56 kD) form of proPC2. Though expression of the mature (64 kD) PC2 form was unchanged in αTC-ΔSel1L cells, PC2 activity was reduced by half in a specific enzyme assay. This was accompanied by decreased expression of 7B2, a chaperone required for proPC2 transit and activation in the secretory pathway. These data show that Sel1L-Hrd1 ERAD is required to ensure proper proPC2 maturation and glucagon production in α cells, thus maintaining classic α cell identity. Disclosure R.B.Reinert: None. W.Zhu: None. L.Pan: None. A.Russo: None. R.Ray: None. N.Shrestha: None. I.Lindberg: None. L.Qi: None. Funding National Institutes of Health (K08DK129719, T32DK007245)

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH-dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.

Pro-protein convertase-2/carboxypeptidase-E mediated neuropeptide processing of RGC-5 cell after in vitro ischemia.

[This retracts the article DOI: 10.1007/s12264-009-1027-8.].

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).

A 36-Residue Peptide Contains All of the Information Required for 7B2-mediated Activation of Prohormone Convertase 2

The prohormone convertases (PCs) are serine proteinases responsible for the processing of secretory protein precursors. PC2 is the only member of this family whose activation requires intracellular interaction with a helper protein, the neuroendocrine protein 7B2. In order to gain a better understanding of the mechanism of proPC2 activation, we have characterized the structural determinants of 7B2 required for proPC2 activation. We had already identified a proline-rich binding determinant in the 21-kDa domain, the portion of 7B2 responsible for proPC2 activation. We have now investigated the function of the weakly conserved amino-terminal portion of 21-kDa 7B2 by sequential deletions. Mutant proteins were analyzed in four assays: binding to proPC2, facilitation of proPC2 maturation, and activation of proPC2 in vivo and in vitro. We found that the amino-terminal half of 7B2 is not involved in proPC2 activation, and we identified an active 36-residue peptide that contains the previously characterized proline-rich sequence as well as an alpha-helix and the only disulfide bond of 7B2. Mutation of the alpha-helix and of the cysteines demonstrated that these determinants are absolutely required for PC2 activation. Thus, the 186-residue full-length 7B2 rat protein can be functionally reduced to an internal segment of only 36 residues.

The Proteolytic Maturation of Prohormone Convertase 2 (PC2) is a pH-Driven Process

Recombinant proPC2 purified from the medium of CHO cells overexpressing both the prohormone convertase (PC) precursor proPC2 and the 21-kDa amino terminal portion of the neuroendocrine protein 7B2 can spontaneously convert to an active species. In the present report, we have characterized the proPC2 zymogen conversion process. Sequencing of the mature 66 kDa enzyme revealed a single site of cleavage at the paired basic site amino terminal to the GYRDI sequence. In contrast to mature PC2 activity, proPC2 conversion was inhibited neither by the eukaryotic subtilisin inhibitor pCMS nor by the specific PC2 inhibitor, 7B2 CT peptide, suggesting significant differences between the proPC2 conversion reaction and the hydrolysis of synthetic substrates by mature PC2. In support of this idea, proPC2 conversion was not calcium dependent and was unaffected by 5 mM EDTA. The rate of conversion of proPC2 remained similar with a 10-fold difference in zymogen concentration, implicating an intramolecular rather than intermolecular mechanism of activation.Interestingly, the rate of proPC2 conversion was extremely pH dependent, occurring most extensively between pHs 4.0 and 4.9. Taken together, our results suggest that cellular proPC2 maturation occurs via an autocatalytic, intramolecular process controlled not by 7B2 inhibition nor by calcium levels, but by the decreasing pH gradient along the secretory pathway.

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

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

Mapping of the domain in the neuroendocrine protein 7B2 important for its helper function towards prohormone convertase PC2

In the secretory pathway, the neuroendocrine polypeptide 7B2 transiently associates with its physiological target, the prohormone convertase PC2. The 7B2 protein carries two functional domains involved in the control of proPC2 maturation and activation, an inhibitory C-terminal region and an N-terminal domain displaying facilitative activity. Here, we show that a short segment in 7B2 (His 120–Pro 131) is important for its facilitative activity. Site-directed mutagenesis indicates that within this segment, Tyr 130 is particularly important for the ability of 7B2 to act as a helper protein for PC2.

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.

Identification of a Molluscan Homologue of the Neuroendocrine Polypeptide 7B2

In vertebrates, interaction of prohormone convertase 2 (PC2) with the highly conserved polypeptide 7B2 is essential for transport and maturation of proPC2 in the regulated secretory pathway. In vitro, 7B2 displays a strong inhibitory activity toward PC2. Here, we characterize a cDNA encoding the first invertebrate 7B2-related protein (L7B2) from the brain of the mollusc Lymnaea stagnalis. The overall amino acid sequence identity between L7B2 and its vertebrate counterparts is surprisingly low (29%) and is restricted to a few small stretches of amino acid residues. Of particular interest are a conserved proline-rich region in the middle portion of the L7B2 sequence and a repeated conserved region in the carboxyl-terminal domain. Synthetic peptides corresponding to the carboxyl-terminal regions inhibit Lymnaea PC2 enzyme activity in extracts of insulin-producing neurons, in which both L7B2 and Lymnaea PC2 are abundantly expressed. Moreover, the peptides inhibit mouse PC2 enzyme activity. Our cloning of invertebrate 7B2 helps to delineate residues that are important for 7B2-PC2 interaction.

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.

Dissociation of the Complex between the Neuroendocrine Chaperone 7B2 and Prohormone Convertase PC2 is not Associated with proPC2 Maturation

7B2 is a highly conserved neuroendocrine protein that is associated with the proform of the prohormone convertase PC2 in the early stages of the secretory pathway in intermediate pituitary cells of Xenopus laevis. Subsequent processing of 7B2 and dissociation of the 7B2/proPC2 complex is thought to be associated with the conversion of proPC2 to the mature enzyme. Here, we report that, in both Xenopus and mouse intermediate cells, proPC2 maturation does not take place when the proenzyme is associated with the 7B2 precursor and that, in contrast to the previous notion, dissociation of the complex between proPC2 and the N-terminal 7B2 fragment precedes, and is thus not directly linked to, proPC2 maturation. In vitro, conversion of newly synthesized proPC2 was efficiently blocked by recombinant 7B2 and studies with truncation mutants indicated that a short segment in the C-terminal region of 7B2 is necessary and sufficient for this inhibitory effect. Our results indicate that, after 7B2 precursor processing and dissociation of the N-terminal fragment, the C-terminal fragment of 7B2 may remain associated with proPC2, thereby preventing autocatalytic conversion of the proenzyme until the appropriate site for activation in the secretory pathway is reached.

7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity.

The prohormone convertase PC2, which is thought to mediate the proteolytic conversion of many peptide hormones, has recently been shown to interact with the neuroendocrine-specific polypeptide 7B2 in Xenopus intermediate lobe (Braks, J. A. M., and G. J. M. Martens. Cell. 78:263. 1994). In the present work we have stably transfected neuroendocrine cell lines with rat 7B2 constructs and found that overexpression of 27 kD 7B2 greatly facilitates the kinetics of maturation of proPC2, both in AtT-20/PC2 cells and in Rin5f cells. The half-life of conversion of proPC2 was reduced from 2.7 to 1.7 h in AtT-20/PC2 cells stably transfected with 27 kD 7B2 cDNA. The previously proposed "chaperone" domain was not sufficient for this facilitation event; however, a construct corresponding to the 21-kD 7B2 protein (which represents the naturally occurring maturation product) functioned well. A 7B2 construct in which maturation of 27 kD 7B2 to its 21-kD form was blocked was unable to facilitate maturation of proPC2. To correlate effects on PC2 maturation with the actual generation of PC2 enzymatic activity, a similar transfection of 21 kD 7B2 was performed using CHO cells previously amplified for the expression of proPC2. Enzymatic activity cleaving the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC was highly correlated with the expression of immunoreactive 21 kD 7B2 in the conditioned medium; medium obtained from the parent cell line was completely inactive. Enzymatic activity was identified as PC2 on the basis of inhibition by the carboxy-terminal peptide of 7B2, which has previously been shown to represent a potent and specific PC2 inhibitor. Taken together, our in vivo results indicate that the interesting secretory protein 7B2 is a bifunctional molecule with an amino-terminal domain involved in proPC2 transport as well as activation.

Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

PC2 and PC3, which is also known as PC1, are subtilisin-like proteases that are involved in the intracellular processing of prohormones and proneuropeptides. Both enzymes are synthesized as propolypeptides that undergo proteolytic maturation within the secretory pathway. An in vitro translation/translocation system from Xenopus egg extracts was used to investigate mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent rapid (t1/2 < 10 min) processing of the 88-kDa propolypeptide at the sequence RSKR83 to generate the 80-kDa active form of the enzyme. This processing was blocked when the active site aspartate was changed to asparagine, suggesting that an autocatalytic mechanism was involved. In this system, processing of pro-PC3 was optimal between pH 7.0 and 8.0 and was not dependent on additional calcium. These results are consistent with pro-PC3 maturation occurring at an early stage in the secretory pathway, possibly within the endoplasmic reticulum, where the pH would be close to neutral and the calcium concentration less than that observed in later compartments. Processing of pro-PC2 in the Xenopus egg extract was much slower than that of pro-PC3 (t1/2 = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K0.5 = 2-4 mM). The enzymatic properties of pro-PC2 processing were similar to that of the mature enzyme. Further studies using mutant pro-PC2 constructs suggested that cleavage of pro-PC2 was catalyzed by the mature 68-kDa PC2 molecule. The results were consistent with pro-PC2 maturation occurring within a late compartment of the secretory pathway that contains a high calcium concentration and low pH.

Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.