Modulating farnesyltransferase substrate specificity: Tools for altering protein function in vivo
Abstract
Farnesylation is the post-translational lipid modification that results in a C15 farnesyl isoprenoid attachment to the C-terminus of a substrate protein. Farnesyltransferase inhibitors are currently in Phase III chemotherapeutic clinical trials. However, after a decade of research, it is still unclear what are the crucial proteins in cancer cells whose farnesylation is inhibited by FTIs. The determination of the total set of farnesylated cellular proteins has been an active ongoing area of research. Numerous mechanistic and structural studies have revealed that FTase catalyzes farnesylation through a complex and unique mechanism. Protein substrates are recognized by a C-terminal CaaX sequence where the C is the cysteine to which the farnesyl, provided by farnesyl diphosphate (FPP) is attached, a is usually an aliphatic amino acid, and X is typically serine, methionine, or glutamine. We have utilized the synthesis of CaaX peptide libraries and medium-throughput screening to expand the knowledge of FTase CaaX specificity and generate a list of potentially farnesylated proteins. Due to the complex reaction mechanism of FTase, FPP analogs may serve as modulators of FTase activity. These prenyl tools would be very useful for the development of cellular probes to investigate the role of lipidated proteins in the cell. In order to gain insight into the FPP analog-induced CaaX specificity, we have utilized a combinatorial screen of FPP analogs and CaaX peptides. Through the combinatorial screening of a library of FPP analogs with the CaaX peptide libraries, we have shown that modified FPP analogs can act as either substrates for, or inhibitors of, FTase for different CaaX peptides. We term these FPP analogs "selective modulators". Furthermore, we have shown that these analogs can select for one CaaX peptide in the presence of another. In developing these biochemical tools into biological probes, a tagging-via-substrate method for the identification of proteins farnesylated by alkynyl-based FPP analogs has been developed. We are now able to begin applying these tools to modulate the lipidation of a protein and study the protein,s function in a more selective manner.
Degree
Ph.D.
Advisors
Gibbs, Purdue University.
Subject Area
Cellular biology
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