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.
Highlights
The insulin-degrading enzyme (IDE1; EC 3.4.24.56) has broad substrate specificity, being able to cleave and inactivate a number of small molecules, including the A peptides and insulin [1,2,3]
We 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
IDE Can Be Heterologously Expressed in Yeast—Many of the yeast enzymes required for a-factor production can be functionally replaced with orthologs from other eukaryotic species (29 – 34)
Summary
Cysteine is exactly 67 amino acids distal to the extended motif (HXXEHX69EX6EX67C). This distal cysteine is not conserved in Escherichia coli Protease III, a bacterial M16A enzyme that is insensitive to thiol modifiers [17]. The sequence similarity between IDE, Ste23p, and Axl1p has prompted us to investigate the hypothesis that these proteins have conserved enzymatic properties and substrate specificity. To this end, we present evidence indicating that rat IDE can substitute for Axl1p and Ste23p in a-factor production but not for the other known functions of Axl1p.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have