Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues.
Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with Rattus norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Some of the latter occur in the genomes of bacteria and viruses and may be important for pathogenesis, suggesting new targets for therapeutic intervention. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results, whereas the analogue synthesized from a C5 unit gave a different pattern of reactivity. Lastly, the substrate specificities of PFTases from three organisms (R. norvegicus, Saccharomyces cerevisiae, and Candida albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme.
Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Yen-Chih
YC
Wang
Jonathan K
JK
Dozier
Lorena S
LS
Beese
Mark D
MD
Distefano
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000818:Animals; D019151:Peptide Library; D011106:Polyisoprenyl Phosphates / Q000737:chemistry*; D051381:Rats; D013379:Substrate Specificity
D019151:Peptide Library; D011106:Polyisoprenyl Phosphates; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase