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J. Biol. Chem., Vol. 281, Issue 11, 7556-7567, March 17, 2006

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

Nadia Rabah¹, Dany Gauthier, Brian C. Wilkes, Daniel J. Gauthier¹, and Claude Lazure²

From the Neuropeptides Structure and Metabolism and the Chemical Biology and Peptide Research Units, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada

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 (⁵⁰RRSRR⁵⁴ and ⁶¹KR⁶²) and an acidic region ⁶⁵DDD⁶⁷ 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.

Received for publication, September 28, 2005 , and in revised form, December 1, 2005.

^* This work was supported in part by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. 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 Recipients of a Fonds de la Recherche en Santé du Québec studentship award and registered at the Division of Experimental Medicine of McGill University.

² To whom correspondence should be addressed: Neuropeptides Structure and Metabolism Research Laboratory, Institut de Recherches Cliniques de Montréal (Affiliated to the University of Montreal), 110 Pine Ave. W., Montréal, Québec H2W 1R7, Canada. Tel.: 514-987-5593; Fax: 514-987-5542; E-mail: lazurec{at}ircm.qc.ca.

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