Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation.

Abstract

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and aTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When '251-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.The eukaryotic subtilisin family of serine proteases is involved in the processing of prohormone and other precursor proteins through cleavage at paired or multiple basic residues (1-3). Prohormone convertase 2 (PC2), a member of this proteinase family, is believed to participate in the later stages of prohormone processing (4-7). It has recently been shown that the protein known as 7B2, whose expression is restricted to the central nervous system and to endocrine tissues (8-10), is intimately involved in proPC2 maturation (11,12). This function appears to require the amino-terminal 21-kDa portion of the molecule (12). The portion of this protein (i.e., the last 31 amino acids of 7B2) that represents a potent inhibitor of PC2 and of the activation of immunopurified proPC2 (13, 14) is here termed the carboxyl-terminal (CT) peptide.

Impaired Prohormone Convertases in Cpe fat/Cpe fat Mice

“…7B2 contains two domains, a 21-kDa N-terminal domain required for PC2 maturation and a CT region that inhibits PC2 at nanomolar concentrations. There is evidence that the 7B2 CT peptide is cleaved at the internal paired basic site, most likely by PC2 itself (38). It is likely that the resultant peptide (CT peptide 1-18) remains associated with PC2, inhibiting the enzyme.…”

“…It has been previously shown that CT peptide 1-18 is a powerful inhibitor of PC2, whereas CT peptide 1-16 (lacking the Cterminal Lys-Lys) displays little inhibitory effect even at extremely high concentrations (38). Next, we examined the effect of altered PC1 and PC2 levels on the extent of processing of two peptide precursors, namely ProDyn and POMC.…”

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in Cpe fat mice associated with a carboxypeptidase E mutation

“…It was hypothesized that proinsulin was poorly processed to insulin in these mice due to inactive PC2, a prohormone convertase that processes proinsulin (22). CPE has been shown in vitro to be able to cleave the C-terminal basic residues of a fragment of 7B2, a PC2 inhibitor, causing it to release from binding to PC2 and thereby activating the enzyme (23). While this phenomenon may also be occurring, our present study indicates a defect in the intracellular routing of peptide hormones to the regulated secretory pathway in these mice, which can account for the lack of processing and secretion of large amounts of proinsulin into the circulation (16).…”

Deficiencies in Pro-thyrotropin-releasing Hormone Processing and Abnormalities in Thermoregulation in CpeMice

“…Both endoproteases are synthesized as pro-enzymes and must be processed to attain full activity (1, 2). It has been proposed that CPE may participate in the full activation of these convertases through removal of C-terminal basic amino acids or by inactivation of endogenous inhibitors (27)(28)(29). Recently, it has been reported that protein levels and enzymatic activities of both enzymes are altered in the brains of the Cpe fat/fat mice (30).…”