Modulation of PC1/3 Activity by Self-Interaction and Substrate Binding

Abstract

Prohormone convertase (PC)1/3 is a eukaryotic serine protease in the subtilase family that participates in the proteolytic maturation of prohormone and neuropeptide precursors such as proinsulin and proopiomelanocortin. Despite the important role of this enzyme in peptide synthesis, how PC1/3 activity is regulated is still poorly understood. Using ion exchange chromatography and two-dimensional gel electrophoresis we found that natural PC1/3 present in AtT-20 cells and bovine chromaffin granules, as well as recombinant PC1/3 secreted from overexpressing Chinese hamster ovary cells, exists as multiple ionic forms. Gel filtration and cross-linking studies revealed that protein oligomerization and aggregation contribute greatly to variability in surface charge. The most acidic forms of PC1/3 contained both inactive aggregates as well as oligomerized 87-kDa PC1/3 that exhibited stable activity which was partially latent and could be revealed by dilution. No such latency was observed for the more basic, 66/74-kDa forms of PC1/3. Fractions containing these species were stabilized by preincubation with micromolar concentrations of either fluorogenic substrate or peptides containing pairs of basic residues. In addition, the most active form of 87-kDa PC1/3, a probable homodimer, was activated by preincubation with these same peptides. Cleavage by PC1/3 is often the initiating step in the biosynthetic pathway for peptide hormones, implying that this is a natural step for regulation. Our data suggest that enzyme oligomerization and peptide stabilization represent important contributing factors for the control of PC1/3 activity within secretory granules.

Revisiting PC1/3 Mutants: Dominant-Negative Effect of Endoplasmic Reticulum-Retained Mutants

“…PC1/3 forms are known to strongly oligomerize; indeed, previous work from our laboratory has shown that 87-kDa mPC1/3 naturally forms dimers and other oligomers (39). In cotransfection experiments using MG132, the 94-kDa zymogen form of wildtype PC1/3 was specifically increased by the presence of ER-retained mutants, and co-IP data demonstrated interaction of mutant and wild-type zymogen forms.…”

“…Previous experiments have shown that PC1/3 species can interact (29,39). To examine this possibility, we performed co-IP experiments using Flag-tag affinity resin to pull down interacting proteins and HA immunoblotting to visualize only wild-type protein.…”

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

“…Mouse 87-kDa PC1/3 and mouse pro-PC2 were purified from the conditioned medium of stably transfected, methotrexate-amplified Chinese hamster ovary cells as described previously (Hoshino et al, 2011). Pro-PC2 was activated before use by dilution in reaction buffer.…”

PCSK1 Mutations and Human Endocrinopathies: From Obesity to Gastrointestinal Disorders

“…In the acidic environment of the secretory granules, 87-kDa PC1/3 is further intermolecularly cleaved at the C-terminal region, which results in the formation two truncated forms: 74-kDa and 66-kDa PC1/3 ( Figure 1C). In contrast to the 87-kDa species, which possesses a relative low enzymatic activity compared to other convertases (12), and tends to form dimers, oligomers and aggregates (75), these truncated forms (especially 66-kDa PC1/3) are more active, exhibit higher calcium dependence and narrower pH optimum (5.0 -5.5), and are mostly present as monomers (75)(76)(77)(78). The 74/66-kDa forms, however, are much less stable than the nontruncated 87-kDa species (76 -78).…”

“…Both endogenous and recombinant PC1/3 have been shown to be present in multimeric forms in CHO and AtT20 cells, and bovine chromaffin granules (75). PC1/3 is present as monomers, dimers, and oligomers/aggregates, the latter having no activity.…”