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J Biol Chem

Title:		Yeast as a tractable genetic system for functional studies of the insulin-degrading enzyme
Author(s):		Kim S; Lapham AN; Freedman CG; Reed TL; Schmidt WK;
Address:		"Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA"
Journal Title:		J Biol Chem
Year:		2005
Volume:		20050608
Issue:		30
Page Number:		27481 - 27490
DOI:		10.1074/jbc.M414192200
ISSN/ISBN:		0021-9258 (Print) 0021-9258 (Linking)
Abstract:		"We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A beta peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity"
Keywords:		"Alleles Amino Acid Motifs Amino Acid Sequence Animals Conserved Sequence Electrophoresis, Polyacrylamide Gel Fungal Proteins/genetics *Genetic Techniques Genotype Green Fluorescent Proteins/metabolism Haploidy Insulin/metabolism Insulysin/*chemistry/*gene;"
Notes:		"MedlineKim, Seonil Lapham, Andrea N Freedman, Christopher G K Reed, Tiffany L Schmidt, Walter K eng Research Support, Non-U.S. Gov't 2005/06/10 J Biol Chem. 2005 Jul 29; 280(30):27481-90. doi: 10.1074/jbc.M414192200. Epub 2005 Jun 8"