| Title: | Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes |
| Author(s): | Hahne K; Vervacke JS; Shrestha L; Donelson JL; Gibbs RA; Distefano MD; Hrycyna CA; |
| Address: | "Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA" |
| Journal Title: | Biochem Biophys Res Commun |
| DOI: | 10.1016/j.bbrc.2012.05.089 |
| ISSN/ISBN: | 1090-2104 (Electronic) 0006-291X (Print) 0006-291X (Linking) |
| Abstract: | "Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and Saccharomyces cerevisiae (Ste14p) catalyze the alpha-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast a-factor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the a-factor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition. Furthermore, these data suggest that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. Our data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites" |
| Keywords: | Acetylcysteine/*analogs & derivatives/chemistry Benzophenones/chemistry Binding Sites Biotinylation Enzyme Inhibitors/chemistry Humans Mating Factor Peptides/*chemistry Photoaffinity Labels/chemistry Protein Methyltransferases/*antagonists & inhibitors/*c; |
| Notes: | "MedlineHahne, Kalub Vervacke, Jeffrey S Shrestha, Liza Donelson, James L Gibbs, Richard A Distefano, Mark D Hrycyna, Christine A eng R01 GM058842/GM/NIGMS NIH HHS/ R01 GM084152/GM/NIGMS NIH HHS/ R01 GM106082/GM/NIGMS NIH HHS/ 2012/05/29 Biochem Biophys Res Commun. 2012 Jun 22; 423(1):98-103. doi: 10.1016/j.bbrc.2012.05.089. Epub 2012 May 23" |
