Proprotein convertases (PCs) are serine proteases containing a subtilisin-like catalytic domain that are involved in the conversion of hormone precursors into their active form. This study aims at designing small cyclic peptides that would specifically inhibit two members of this family of enzymes, namely, the neuroendocrine PC1/3 and the ubiquitously expressed furin. We studied peptide sequences related to the 18-residue loop identified as the active site of the 83 amino acid barley serine protease inhibitor 2 (BSPI-2). Peptides incorporating mutations at various positions in the sequence were synthesized on solid phase and purified by HPLC. Cyclization was achieved by the introduction of a disulfide bridge between the two Cys residues located at both the N- and C-terminal extremities. Peptides VIIA and VIIB incorporating P4Arg, P2Lys, P1Arg, and P2'Lys were the most potent inhibitors with K(i) around 4 microM for furin and around 0.5 microM for PC1/3. Whereas peptide VIIB behaved as a competitive inhibitor of furin, peptide VIIA acted as a noncompetitive one. However, all peptides were eventually cleaved after variable incubation times by PC1/3 or furin. To avoid this problem, we incorporated at the identified cleavage site a nonscissile aminomethylene bond (psi[CH(2)-NH]). Those pseudopeptides, in particular peptide VIID, were shown not to be cleaved and to inhibit potently furin. Conversely, they were not able to inhibit PC1/3 at all. Those results show the validity of this approach in designing new effective PC inhibitors showing a certain level of discrimination between PC1/3 and furin.

The human acid sphingomyelinase (ASM, EC 3.1.4.12), a lysosomal and secretory protein coded by the sphingomyelin phosphodiesterase 1 (SMPD-1) gene, catalyzes the degradation of sphingomyelin (SM) to ceramide and phosphorylcholine. We examined the structural-functional properties of its carboxyl-terminus (amino acids 462-629), which harbors approximately 1/3 of all mutations discovered in the SMPD-1 gene. We created four naturally occurring mutants (DeltaR608, R496L, G577A, and Y537H) and five serial carboxyl-terminal deletion mutants (N620, N590, N570, N510, and N490). Transient transfection of the His/V5-tagged wild-type and mutant recombinant ASM in Chinese hamster ovary cells showed that all the mutants were normally expressed. Nonetheless, none of them, except the smallest deletion mutant N620 that preserved all post-translational modifications, were found capable of secretion to the medium. Furthermore, only the N620 conserved functional integrity (100% activity of the wild type); all other mutants completely lost the ability to catalyze SM hydrolysis. Importantly, cell surface biotinylation revealed that mutant DeltaR608 transfected CHO cells and fibroblasts from a compound heterozygous Niemann-Pick disease type B (NPD-B) patient (DeltaR608 and R441X) have defective translocation to the plasma membrane. Furthermore, we demonstrated that the DeltaR608 and N590 were trapped in the endoplasmic reticulum (ER) quality control checkpoint in contrast to the wild-type lysosomal localization. Interestingly, while the steady-state levels of ubiquitination were minimal for the wild-type ASM, a significant amount of Lys63-linked polyubiquitinated DeltaR608 and N590 could be purified by S5a-affinity chromatography, indicating an important misfolding in the carboxyl-terminal mutants. Altogether, we provide evidence that the carboxyl-terminus of the ASM is crucial for its protein structure, which in turns dictates the enzymatic function and secretion.

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 PC1/3 preferentially cleaves its substrates in the dense core secretory granules of endocrine and neuroendocrine cells. Similar to most proteinases synthesized first as zymogens, PC1/3 is synthesized as a larger precursor that undergoes proteolytic processing of its signal peptide and propeptide. The N-terminally located propeptide has been shown to be essential for folding and self-inhibition. Furthermore, PC1/3 also possesses a C-terminal region (CT-peptide) which, for maximal enzymatic activity, must also be cleaved. To date, its role has been documented through transfection studies in terms of sorting and targeting of PC1/3 and chimeric proteins into secretory granules. In this study, we examined the properties of a 135-residue purified bacterially produced CT-peptide on the in vitro enzymatic activity of PC1/3. Depending on the amount of CT-peptide used, it is shown that the CT-peptide increases PC1/3 activity at low concentrations (nm) and decreases it at high concentrations (microm), a feature typical of an activator. Furthermore, we show that, contrary to the propeptide, the CT-peptide is not further cleaved by PC1/3 although it is sensitive to human furin activity. Based on these results, it is proposed that PC1/3, through its various domains, is capable of controlling its enzymatic activity in all regions of the cell that it encounters. This mode of self-control is unique among members of all proteinases families.

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