The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors.
Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides 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 < or = 100 microM these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.
Pasquato
Antonella
A
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada.
Pullikotil
Philomena
P
Asselin
Marie-Claude
MC
Vacatello
Manuela
M
Paolillo
Livio
L
Ghezzo
Francesca
F
Basso
Federica
F
Di Bello
Carlo
C
Dettin
Monica
M
Seidah
Nabil G
NG
D000595:Amino Acid Sequence; D002460:Cell Line; D002863:Chromogenic Compounds / Q000138:chemical synthesis / Q000378:metabolism; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007836:Lassa virus / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism; D043484:Proprotein Convertases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002863:Chromogenic Compounds; D003374:Coumarins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009842:Oligopeptides; D014759:Viral Envelope Proteins; C066508:glycoprotein gp1, lassa virus; C077198:glycoprotein gp2, lassa virus; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C115575:membrane-bound transcription factor peptidase, site 1