The Proprotein Convertase SKI-1/S1P

Abstract

Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketonepeptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations <100 M these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

The Proprotein Convertase SKI-1/S1P

“…Since the discovery of the proprotein convertase SKI-1/S1P (4,18), it was realized that this convertase can process a number of membranebound proteins in the cis/medial Golgi, including transcription factors (2,3,11,24,(37)(38)(39) and viral surface glycoproteins (6,12,14,40). Biosynthetic analysis of the zymogen-processing SKI-1 revealed an ordered autocatalytic activation process leading to the formation of intermediate B/BЈ forms in the ER and then an active C-form in the cis/medial Golgi (4,15,41,42).…”

“…In another experiment, we used triple transfections of 1 g each of rATF6 and SKI-1, along with either an empty 2 g of pIRES2 vector (control) or 2 g of cDNAs coding for either rSt-1 or rSt-2. 24 h later, cells were treated or not with 2 g/ml tunicamycin for 12 h. Thereafter, the cells were treated with ALLN (Sigma) at a final concentration of 25 g/ml for 1 h. The lysates were resolved on 6% SDS-PAGE and analyzed by Western blot using either a 1:5000 dilution of anti-FLAG M2 monoclonal antibody (Stratagene), as reported (6,24), or mAb/V5 at a 1:5000 dilution.…”

“…The eighth member is the pyrolysin-like subtilisin kexin isozyme-1 (SKI-1) (4), also known as site-1 protease (S1P) (5). It cleaves substrates at the consensus motif (R/K)X-(hydrophobic)-X2, where X is variable (6). The last member PCSK9 cleaves the sequence VFAQ 152 2 within its prosegment (7).…”

A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease

“…These systems have greatly contributed to our current understanding of SKI-1/S1P-mediated GPC processing. However, in some cases, these peptide-based systems failed to reproduce the SKI-1/S1P-mediated GPC processing observed in the context of viral infection, as illustrated by the lack of cleavage of peptides derived from LCMV or GTOV GPC (22,23). Since full-length GPCs of both LCMV and GTOV are readily cleaved in a range of mammalian cells (9,11,12), the lack of processing suggests that these otherwise powerful in vitro assays do not accurately recapitulate the authentic cellular environment.…”

“…Conventional approaches to the study of the processing of putative viral SKI-1/S1P substrates in a quantitative manner made use of homogeneous biochemical assays including synthetic peptides and soluble enzyme (22,23). These systems have greatly contributed to our current understanding of SKI-1/S1P-mediated GPC processing.…”