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J. Biol. Chem., Vol. 281, Issue 33, 23471-23481, August 18, 2006

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The Proprotein Convertase SKI-1/S1P

IN VITRO ANALYSIS OF LASSA VIRUS GLYCOPROTEIN-DERIVED SUBSTRATES AND EX VIVO VALIDATION OF IRREVERSIBLE PEPTIDE INHIBITORS^*

Antonella Pasquato, Philomena Pullikotil, Marie-Claude Asselin, Manuela Vacatello^¶, Livio Paolillo^¶, Francesca Ghezzo, Federica Basso, Carlo Di Bello, Monica Dettin, and Nabil G. Seidah¹

From the Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada, the Department of Chemical Process Engineering, University of Padova, 35131 Padova, Italy, and the ^¶Department of Chemistry, University of Naples "Federico II", Complesso Universitario di Monte S. Angelo, 80126 Naples, Italy

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 100 µM these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

Received for publication, December 22, 2005 , and in revised form, June 19, 2006.

^* This work was supported by Canadian Institutes of Health Research Grant MOP-36496 and a private donation from the Strauss Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 110 Pine Ave., West Montreal, QC H2W 1R7, Canada. Tel.: 514-987-5609; Fax: 514-987-5542; E-mail: seidahn{at}ircm.qc.ca.

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				P. Pullikotil, S. Benjannet, J. Mayne, and N. G. Seidah The Proprotein Convertase SKI-1/S1P: ALTERNATE TRANSLATION AND SUBCELLULAR LOCALIZATION J. Biol. Chem., September 14, 2007; 282(37): 27402 - 27413. [Abstract] [Full Text] [PDF]