The proprotein convertases are synthesized as zymogens that acquire activity upon autocatalytic removal of their NH 2 -terminal prosegment. Based on the convertase furin, to fold properly and gain activity, the convertases PC5A, PACE4, and PC7 are presumed to undergo two sequential prosegment cleavages in the endoplasmic reticulum and then in the trans-Golgi network. However, biochemical and immunocytochemical experiments revealed that mouse PC5A is complexed to its prosegment at the plasma membrane. This labeling is lost upon treatment with heparin and is increased by overexpressing members of the syndecan family and CD44, suggesting attachment of secreted PC5A-prosegment complex to heparan sulfate proteoglycans. Following stimulation of Y1 cells with adrenocorticotropic hormone or 8-bromo-cyclic AMP, the cell surface labeling of the prosegment of PC5A is greatly diminished, whereas the signal for mature PC5A is increased. Moreover, after stimulation, the protease activity of PC5A is enhanced, as evidenced by the cleavage of the PC5A substrates Lefty, ADAMTS-4, endothelial lipase, and PCSK9. Our data suggest a novel mechanism for PC5A activation and substrate cleavage at the cell surface, through a regulated removal of its prosegment. A similar mechanism may also apply to the convertase PACE4, thereby extending our knowledge of the molecular details of the zymogen activation and functions of these heparan sulfate proteoglycan-bound convertases.
eThe proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1 [IL-1]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg 41 2 (where the down arrow indicates the cleavage location), and potentially by PCs at Arg 41 XXXXArg 46 2. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg 46 2. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIVinduced neuroinflammation, viral infection, and potentially the development of HAND.
Although the processing profile of the membrane-bound epidermal growth factor precursor (pro-EGF) is tissue-specific, it has not been investigated at the cellular level nor have the cognate proteinases been defined. Among the proprotein convertases (PCs), only the membrane-bound PC7, the most ancient and conserved basic amino acid-specific PC family member, induces the processing of pro-EGF into an ϳ115-kDa transmembrane form (EGF-115) at an unusual VHPR 290 2A motif. Because sitedirected mutagenesis revealed that Arg 290 is not critical, the generation of EGF-115 by PC7 is likely indirect. This was confirmed by testing a wide range of protease inhibitors, which revealed that the production of EGF-115 is most probably achieved via the activation by PC7 of a latent serine and/or cysteine protease(s). EGF-115 is more abundant at the cell surface than pro-EGF and is associated with a stronger EGF receptor (EGFR) activation, as evidenced by higher levels of phosphorylated ERK1/2. This suggests that the generation of EGF-115 represents a regulatory mechanism of juxtacrine EGFR activation. Thus, PC7 is distinct from the other PCs in its ability to enhance the activation of the cell surface EGFR.
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