Single Amino Acid Substitution in the PC1/3 Propeptide Can Induce Significant Modifications of Its Inhibitory Profile toward Its Cognate Enzyme

Abstract

The proprotein convertase PC1/3 is synthesized as a large precursor that undergoes proteolytic processing of the signal peptide, the propeptide and ultimately the COOH-terminal tail, to generate the mature form. The propeptide is essential for protease folding, and, although cleaved by an autocatalytic process, it remains associated with the mature form acting as an auto-inhibitor of PC1/3. To further assess the role of certain residues in its interaction with its cognate enzyme, we performed an alanine scan on two PC1/3 propeptide potential cleavable sites ( 50 RRSRR 54 and 61 KR 62 ) and an acidic region 65 DDD 67 conserved among species. Upon incubation with PC1/3, the ensuing peptides exhibit equal inhibitory potency, lower potency, or higher potency than the wild-type propeptide. The K i values calculated varied between 0.15 and 16.5 nM. All but one mutant exhibited a tight binding behavior. To examine the specificity of mutants, we studied their reactivity toward furin, a closely related convertase. The mutation of certain residues also affects the inhibition behavior toward furin yielding propeptides exhibiting K i ranging from 0.2 to 24 nM. Mutant propeptides exhibited against each enzyme either different mode of inhibition, enhanced selectivity in the order of 40-fold for one enzyme, or high potency with no discrimination. Hence, we demonstrate through single amino acid substitution that it is feasible to modify the inhibitory behavior of propeptides toward convertases in such a way as to increase or decrease their potency, modify their inhibitory mechanisms, as well as increase their selectivity.One of the most common methods used by cells to diversify the pool of their biologically active molecules is protein processing. Indeed, numerous secreted proteins are synthesized first as an inactive precursor, which is rendered biologically active upon cleavage at clusters of basic residues. Members of a family of proteins named proprotein convertases (PCs) 3 primarily perform this cleavage. To date, seven members were described: furin, PC1/3, PC2, PACE4, PC4, PC5/6, and PC7/ PC8/lymphoma proprotein convertase. Some of them are ubiquitously expressed such as furin, PACE4, and PC7, whereas others exhibit a more restricted expression pattern such as PC1/3 and PC2, which are solely present in endocrine and neuroendocrine tissues, and PC4, which is expressed only in germ cells. However, all of them belong to the larger family of serine proteases and are structurally related to bacterial subtilisin and yeast kexin (reviewed in Ref. 1). In terms of biological activities, numerous transfection experiments using recombinant enzymes and substrates, generation of knock-out animals as well as human cases of convertase deficiency pointed out the importance of convertases in crucial biological processes such as patterning during embryogenesis, angiogenesis, prohormone processing, tissue remodeling, and complement activation (2). Furthermore, some members are also implicated in many disease states, because they are able to...

The Proprotein Convertase SKI-1/S1P

“…We estimate that at least 5-10% of the fluorogenic peptide was digested within the analysis period. A similar phenomenon was also observed for the convertase PC1, where the lag phase could last up to 6 h (35).…”

Functional Consequences of a Novel Variant of PCSK1

“…The inhibitory role of the prodomain is of particular interest to this study when we consider the location of the R80Q (rs1799904) substitution within the secondary cleavage site of the prodomain ( Figure 1 ). Independent studies have shown that alteration of mouse proPC1/3 prodomain residues either within or surrounding cleavage motifs can affect propeptide processing; the in vitro proteolytic conversion of an R80A mutant propeptide (the same residue as the R80Q variant studied here) by wild-type enzyme was impaired compared to wild-type propeptide [44]. Given this finding, our lack of identification of propeptide-bearing R80Q PC1/3 is puzzling.…”

“…The proPC1/3 maturation process begins with the autocatalytic intramolecular cleavage of the pro-domain in the ER at the primary cleavage site, RSKR 107–110 [44], [45]. This cleavage yields an 87 kDa form of PC1/3 that, by analogy with the related enzyme furin [46] likely remains associated with its own prodomain through non-covalent interactions until its arrival at the trans-Golgi network.…”

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

“…Endogenous regulation of PC1/3 involves the propeptide of PC1/3 and inhibition by proSAAS-derived peptide. The propeptide segment inhibits PC1/3, as well as PC2 and furin (76,77). Furthermore, PC1/3 is inhibited by proSAAS-related peptides (78).…”