| Title: | a-Factor Analogues Containing Alkyne- and Azide-Functionalized Isoprenoids Are Efficiently Enzymatically Processed and Retain Wild-Type Bioactivity |
| Author(s): | Diaz-Rodriguez V; Hsu ET; Ganusova E; Werst ER; Becker JM; Hrycyna CA; Distefano MD; |
| Address: | "Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States. Department of Chemistry, Purdue University , 560 Oval Drive, West Lafayette, Indiana 47907, United States. Department of Microbiology, University of Tennessee , Circle Park Drive, Knoxville, Tennessee 37996, United States" |
| DOI: | 10.1021/acs.bioconjchem.7b00648 |
| ISSN/ISBN: | 1520-4812 (Electronic) 1043-1802 (Print) 1043-1802 (Linking) |
| Abstract: | "Protein prenylation is a post-translational modification that involves the addition of one or two isoprenoid groups to the C-terminus of selected proteins using either farnesyl diphosphate or geranylgeranyl diphosphate. Three crucial enzymatic steps are involved in the processing of prenylated proteins to yield the final mature product. The farnesylated dodecapeptide, a-factor, is particularly useful for studies of protein prenylation because it requires the identical three-step process to generate the same C-terminal farnesylated cysteine methyl ester substructure present in larger farnesylated proteins. Recently, several groups have developed isoprenoid analogs bearing azide and alkyne groups that can be used in metabolic labeling experiments. Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. Herein, we describe the preparation and use of prenylated a-factor analogs, and precursor peptides, to investigate two key questions. First, a-factor analogues containing modified isoprenoids were prepared to evaluate whether the non-natural lipid group interferes with the biological activity of the a-factor. Second, a-factor-derived precursor peptides were synthesized to evaluate whether they can be efficiently processed by the yeast proteases Rce1 and Ste24 as well as the yeast methyltransferase Ste14 to yield mature a-factor analogues. Taken together, the results reported here indicate that metabolic labeling experiments with azide- and alkyne-functionalized isoprenoids can yield prenylated products that are fully processed and biologically functional. Overall, these observations suggest that the isoprenoids studied here that incorporate bio-orthogonal functionality can be used in metabolic labeling experiments without concern that they will induce undesired physiological changes that may complicate data interpretation" |
| Keywords: | Alkynes/chemical synthesis/*chemistry/metabolism Azides/chemical synthesis/*chemistry/metabolism Cell Line Mating Factor/chemical synthesis/*chemistry/metabolism Protein Prenylation Proteolysis Saccharomyces cerevisiae/*chemistry/metabolism Saccharomyces; |
| Notes: | "MedlineDiaz-Rodriguez, Veronica Hsu, Erh-Ting Ganusova, Elena Werst, Elena R Becker, Jeffrey M Hrycyna, Christine A Distefano, Mark D eng R01 GM106082/GM/NIGMS NIH HHS/ R01 GM022087/GM/NIGMS NIH HHS/ T32 GM008347/GM/NIGMS NIH HHS/ T32 GM008700/GM/NIGMS NIH HHS/ R01 GM084152/GM/NIGMS NIH HHS/ R56 GM022087/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S. 2017/12/01 Bioconjug Chem. 2018 Feb 21; 29(2):316-323. doi: 10.1021/acs.bioconjchem.7b00648. Epub 2017 Dec 20" |
