Processing and trafficking of a prohormone convertase 2 active site mutant

Abstract

Processing of most PC zymogens is required for successful folding and/or passage through the secretory pathway; active site mutants are retained in the ER and degraded. We here report that the active site serine mutant of PC2 (PC2-S383A) was efficiently secreted as the intact zymogen in CHO-K1 cells, suggesting that its propeptide can productively insert into the mutated binding pocket without causing misfolding. In AtT-20 cells, PC2-S383A was cleaved at the secondary cleavage site within the propeptide; this cleavage event was pH-dependent and was inhibited by a proprotein convertase inhibitor. In vitro digestion of PC2-S383A with various convertases indicates that this site is accessible to in trans cleavage. Abundant immunoreactive S383A PC2 was found in secretory granules, supporting the idea that this protein is efficiently trafficked through the secretory pathway.

Knock-out mouse models of proprotein convertases: unique functions or redundancy?

“…The other neuroendocrine-specific member of the family is PC2. This enzyme is unique in its activation mechanism that requires the molecular chaperone 7B2 (11,56). The secretory protein 7B2 is a bifunctional molecule with an aminoterminal domain involved in proPC2 transport as well as activation and a carboxyterminal peptide that inhibits PC2 at nanomolar concentrations (57,58).…”

“…However, the propeptide remains associated to the enzyme until it reaches the trans-Golgi network (TGN), where the local pH and Ca 2+ -concentration facilitate a second autocatalytic internal cleavage and dissociation (10) ( Figure 3). The exception to the rule is PC2, which exits the ER as a zymogen and, furthermore, the internal cleavage of the propeptide can be performed in trans by other PCs but not PC2 (11). SKI-1 and NARC-1 are two functionally related members of subfamily S8A, which also cleave and activate proproteins, but at non-basic motifs.…”

Overexpressed proprotein convertase 1/3 induces an epithelial-mesenchymal transition in airway epithelium

“…We report here that PC1/3 expression is strongly increased in nasal polyps compared with normal nasal mucosa and induces the EMT-like process in airway epithelial cells. We also note that total PC activity was significantly higher in nasal polyps compared to normal nasal mucosa; the convertases furin, PC5/6 and PACE4 have a broad pH optimum with peak activity at neutral to weakly basic pHs (6.0-8.5 for furin, 7.0-8.0 for PC5/6 and 7.0-8.5 for PACE4), while PC1/3 is active within a much narrower pH range (5.0-6.5) [21]. Therefore, we speculate that a large portion of this enzyme activity under our PC enzyme assay conditions (pH 5.5) is originated from endogenous PC1/3.…”

Dynamic Modulation of Prohormone Convertase 2 (PC2)-mediated Precursor Processing by 7B2 Protein

“…1, left). Despite the fact that stable overexpression of 7B2 enhanced proPC2 processing to 68-kDa PC2 in AtT-20 cells (12), cleavage of proPC2 still occurred efficiently in primary cell cultures in the absence of 7B2, possibly by other convertases (30) (Fig. 1, right).…”