Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues

Yen Chih Wang, Jonathan K. Dozier, Lorena S. Beese, Mark D Distefano

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a 2X 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.

Original languageEnglish (US)
Pages (from-to)1726-1735
Number of pages10
JournalACS Chemical Biology
Volume9
Issue number8
DOIs
StatePublished - Aug 15 2014

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Peptide Library
Diphosphates
Terpenes
Substrate Specificity
Libraries
Substrates
Peptides
Alkynes
Proteins
Candida albicans
Screening
Protein Prenylation
Click Chemistry
Saccharomyces cerevisiae Proteins
Solid-Phase Synthesis Techniques
Streptavidin
Azides
Candida
Enzymes
Biotin

Cite this

Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues. / Wang, Yen Chih; Dozier, Jonathan K.; Beese, Lorena S.; Distefano, Mark D.

In: ACS Chemical Biology, Vol. 9, No. 8, 15.08.2014, p. 1726-1735.

Research output: Contribution to journalArticle

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