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

ABSTRACT: 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.

SUBMITTER: Wang YC

PROVIDER: S-EPMC4136699 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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

Wang Yen-Chih YC Dozier Jonathan K JK Beese Lorena S LS Distefano Mark D MD

ACS chemical biology 20140605 8

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 v ...[more]

PMID: 24841702

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Project description:Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10-20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought.

Dataset Information

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

Publications

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

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