Yeast Ste23p shares functional similarities with mammalian insulin‐degrading enzymes

Abstract

The S. cerevisiae genome encodes two M16A enzymes: Axl1p and Ste23p. Of the two, Ste23p shares significantly higher sequence identity with M16A enzymes from other species, including mammalian insulin-degrading enzymes (IDEs). In this study, recombinant Ste23p and R. norvegicus IDE (RnIDE) were isolated from E. coli, and their enzymatic properties compared. Ste23p was found to cleave established RnIDE substrates, including the amyloid-β peptide (Aβ1–40) and insulin B-chain. A novel internally quenched fluorogenic substrate (Abz–SEKKDNYIIKGV–nitroY-OH) based on the polypeptide sequence of the yeast P2 a-factor mating propheromone was determined to be a suitable substrate for both Ste23p and RnIDE, and was used to conduct comparative enzymological studies. Both enzymes were most active at 37 °C, in alkaline buffers and in high salt environments. In addition, the proteolytic activities of both enzymes towards the fluorogenic substrate were inhibited by metal chelators, thiol modifiers, inhibitors of cysteine protease activity and insulin. Characteristics of STE23 expression were also evaluated. Our analysis indicates that the 5′ terminus of the STE23 gene has been mischaracterized, with the physiologically relevant initiator corresponding to residue M53 of the publicly annotated protein sequence. Finally, we demonstrate that, unlike haploid-specific Axl1p, Ste23p is expressed in both haploid and diploid cell types. Our study presents the first comprehensive biochemical analysis of a yeast M16A enzyme, and provides evidence that S. cerevisiae Ste23p has enzymatic properties that are highly consistent with mammalian IDEs and other M16A enzymes.

Ste24p Mediates Proteolysis of Both Isoprenylated and Non-prenylated Oligopeptides

“…Previously described plasmids used were pRS316 (CEN URA3) (36), pSM1282 (2 URA3 P PGK -HIS-HA-ScSTE24) (2), pWS804 (pT7-HIS-RnIDE M42-L1019 ) (27), pWS335 (2 URA3 P PGK -HIS-HA-HsRce1⌬22), and pWS479 (2 URA3 P PGK -ScRCE1-HA) (4). pWS1275 (2 URA3 P PGK -HA-HsSTE24) was created by PCR-directed, plasmid-based recombination (37).…”

“…To obtain reaction rates and other kinetic parameters, rate values (relative fluorescence units/min) were converted to product amounts per minute (mol/min) by referencing two standard curves (27,35,42). First, the amount of fluorescence observed was corrected for intermolecular quenching using standard curves for fluorophore alone and a 1:1 mixture of fluorophore and quencher pair (Fig.…”

“…1) (16). Moreover, yeast Ste24p and M16A proteases (Ste23p and Axl1p) cleave the a-factor precursor at different sites that are each distal from the CAAX motif, indicating that they can cleave non-prenylated regions of the same peptide, albeit at different locations (27,28,32). M16A proteases cleave a variety of small peptides, potentially up to ϳ80 amino acids in length (20,21,43).…”

“…The human M16A protease IDE cleaves numerous small peptides and has been implicated in the clearance of neurotoxic A␤ peptides and other amyloidogenic peptides (22)(23)(24)(25)(26). The yeast M16A protease Ste23p also cleaves amyloidogenic peptides (27). Coincidentally, the yeast M16A proteases Axl1p and Ste23p are both involved in a-factor production (28).…”

“…These findings provide potential insight into the biological function of Ste24p and suggest that its major biological role may not be as a CAAX protease. (27,35). Trans,trans-farnesyl bromide was prepared as 100 mM stock in DMSO.…”