Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic β cell via two distinct site-specific endopeptidases

Abstract

Insulin is produced from an inactive precursor, proinsulin, through initial endoproteolytic cleavage at sites marked by pairs of basic amino-acid residues. We report here that lysates of insulin secretory granules contain two distinct Ca-dependent acidic endoproteases; one (type I) cleaving exclusively on the C-terminal side of Arg 31.Arg 32 (B-chain/C-peptide junction), the other (type II) preferentially on the C-terminal side of Lys 64.Arg 65 of proinsulin (C-peptide/A-chain junction). The Ca and pH requirements of these proteinases suggested that the type-II proteinase would be active in the Golgi apparatus and the secretory granule, whereas type-I activity would be compatible only with the intragranular environment. Kinetic analyses of (pro)insulin conversion intermediates in [35S]methionine-pulsed rat islets support this supposition. Our results suggest a simple mechanism whereby different dibasic sites can be cleaved in different cellular compartments. In conjunction with the regulation of the ionic composition of such compartments and the operation of post-Golgi segregation, our results also suggest how proteolytic conversion of diverse proproteins destined for different cellular sites can occur differentially and in a regulated manner.

Low serum 25-hydroxyvitamin D level predicts progression to type 2 diabetes in individuals with prediabetes but not with normal glucose tolerance

“…The direct effect of vitamin D might be mediated by binding of the circulating active form, 1.25(OH)D 3, to the pancreatic beta cell VDR, and activation of vitamin D within the beta cell by the 1-α-hydroxylase enzyme; alternatively, it may be brought about by an effect on insulin action through stimulating the expression of insulin receptors and enhancing insulin responsiveness for glucose transport [2]. The indirect effects of vitamin D can be mediated via regulating extracellular calcium and insulin secretion through calcium fluxes in the beta cells [26]. Vitamin D may also be involved in systemic inflammation processes, and improve insulin sensitivity and promote beta cell survival by directly modulating the effect of cytokines [2].…”

Proinsulin Targeting to the Regulated Pathway Is Not Impaired in Carboxypeptidase E-deficientCpe /Cpe Mice

“…NIT-2 and NIT-3 cells) confirmed that this CPE mutation leads to inhibition of proinsulin conversion while suggesting that at least some proinsulin was directed to granules. 2 The mere presence of proinsulin in a putative storage compartment is not proof in itself of sorting to the regulated pathway. This can only be addressed by following the kinetics of secretion of newly synthesized (labeled) proinsulin/insulin.…”

“…Acknowledgments-We thank Dr. Edward Leiter (The Jackson Laboratories, Bar Harbor, ME) for sending a copy of the study by Varlamov et al 2 prior to publication.…”

“…At least three enzymes are needed for generating native insulin, namely the conversion endoproteases PC1 (also known as PC3) and PC2 (2,3) and carboxypeptidase E (also called carboxypeptidase H or CPE 1 ) for trimming residual basic C-terminal residues (4 -6). Correct targeting to secretory granules is essential for the cell to produce and store bioactive insulin, which can then be released upon stimulation of the ␤-cell by glucose or other secretagogues.…”

Intracellular trafficking and metabolic turnover of yeast prepro-α-factor-SRIF precursors in GH3 cells

“…Prohormone convertases are specific to the regulated secretory pathway and cleave prohormones in secretory granules. The prohormone convertase family member PC2 may be involved in hybrid precursor processing in GH 3 cells since these cells express this enzyme (Seidah et al, 1990), which, although displaying optimum activity at acidic pH, is also active in neutral environments (Davidson et al, 1988). The other major candidate endoprotease likely to be involved in processing pro-α-factor in GH 3 cells is furin.…”