The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2.

Abstract

The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells.Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2.Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors Synthetic compounds (e.g., peptidyl chloromethanes containing basic amino acid residues) have been shown to inhibit the Kex2 enzyme and furin-mediated cleavage of the human immunodeficiency viral coat protein gpl60 (5, 6). In addition, the furin enzyme is inhibited by a1-antitrypsin genetically engineered to contain the consensus cleavage site of furin at its reactive site (7) and furin is moderately inhibited by the similarly mutated turkey ovomucoid third domain (8). However, as yet, potent naturally occurring inhibitors of the proprotein cleavage enzymes have not been identified.The neuroendocrine-specific polypeptide 7B2 was initially isolated from porcine anterior pituitary glands as a protein of ;21 kDa (9). Biosynthesis ofthe %21-kDa 7B2 protein occurs through carboxyl-terminal processing of a --27-kDa precursor protein (10,11) and only the cleaved form of 7B2 is released (10). Secretion of the 7B2 cleavage product could be regulated (10,12,13), establishing that, like PC1/PC3 and PC2, the polypeptide 7B2 is in the regulated secretory pathway. The 7B2 protein is highly conserved and widely distributed in the central nervous system and endocrine tissues (14)(15)(16) and was generated by PCR using specific primers; the 5' primer corresponded to nucleotides 107-129 with a BamHI site introduced at the 5' end and the 3' primer consisted of nucleotides 538-562 with an introduced 5' stop codon and 5' HindIII site. The recombinant 21-kDa 7B2 protein represents amino acids 1-151 of the human 7B2 protein and corresponds to the 7B2 cleavage product isolated from anterior pituitaries (9,11). The expression plasmid for the intact 27-kDa 7B2 precursor protein was constructed by replacing the =0.2-kb Kpn I-HindIII fiagment of the 21-kDa 7B2 construct by the -0.8-kb Kpn 1-HindIII fragment (encoding the carboxylterminal half of the precursor protein) of a full-length human 7B2 cDNA clone (18). Recombinant 7B2 was purified by Ni2+-NTA agarose affinity chromatography according to the instructions of the manufacturer (Qiagen, Chatsworth, CA).Preparation of PC1/PC3 and PC2 Enzymes. Active 87-kDa PC1/PC3 was purified from medium of overexpressing CHO cells as described (19). PC2 was obtained from the conditioned medium of ,BTC3 cells through immunopurification (20). One hundred milliliters of 16-h conditioned fffC3 cell culture medium (containing aprotinin at 100 jug/ml) was collected, centrifuged, and conce...

Mutations in the Catalytic Domain of Prohormone Convertase 2 Result in Decreased Binding to 7B2 and Loss of Inhibition with 7B2 C-terminal Peptide

“…Inhibition of PC2 by the 7B2 CT peptide strongly depends on the presence of a K-K site (40,42); other residues within the peptide are also critical to inhibitory potency (44). No information is available thus far on which PC2 residues confer the ability to be inhibited by the CT peptide; presumably, these residues are not shared by PC1, which is not inhibited by this peptide (45).…”

The neuroendocrine chaperone 7B2 can enhance in vitro POMC cleavage by prohormone convertase PC2

“…1A, lane 6). Intact 7B2 has been shown to act as a specific inhibitor of prohormone convertase PC2 in an in vitro enzyme assay [24]. Incubation of the lysate in the presence of both processed and intact 7B2 (both at-1 pM) also led to a complete block of POMC conversion, indicating that the intact form of 7B2 can abolish the stimulating effect of the processed form ( Fig.…”

“…Production and purification of the recombinant proteins was performed as described previously [24]. Recombinant intact 7B2 (amino acid residues 1-185; numbering refers to [26]), processed 7B2 (residues 1-151), the C-terminally truncated 7B2 mutants pNK171 (1-171), pNF177 (1-177), pNG162 (1-162), pNP131 (1 131), and pNG86 (1 86), and the N-terminally truncated 7B2 mutants pCP93 (93-185) and pCD125 (125-185) were used in the present study.…”

“…Based on its functional characteristics and the finding that the N-terminal half of 7B2 is distantly related to members of the Hsp60 family of chaperones, 7B2 has been referred to as a neuroendocrine chaperone [17]. In vitro, the recombinant intact form of 7B2 (and not its processed form) inhibits PC2 convertase activity and blocks autocatalytic conversion of proPC2 into its mature form [24,25]. These observations indicate that intact 7B2 regulates PC2 activity in neuroendocrine cells by preventing premature proPC2 conversion.…”

Functional analysis of human PACE4‐A and PACE4‐C isoforms: identification of a new PACE4‐CS isoform

“…3) which revealed that only PACE4-A could be detected in the medium. In order to further probe the intracellular enzymatic activity of either PACE4-A or PACE4-CS, we co-expressed these isoforms with pro7B2, a PC2-specific binding protein [20,28,29] known to be processed in the TGN by furin-like enzymes [20,30]. As shown in Fig.…”