Development of mass spectrometric tools for the cellular characterization of protein prenylation

Jiao Song, Purdue University

Abstract

Protein prenylation is a critical post-translational process, occurring on ∼ 1-2% of all mammalian proteins, including Ras proteins. This process is typically executed by either farnesyltransferase or geranylgeranyltransferase I, which catalyze the transfer of a C15 or C20 isoprenoid to the cysteine sulfur of the C-terminal CaaX. Farnesylation has been studied extensively as an area of drug development due to its important role in diverse cellular activities, various pathologies and particularly carcinogenesis. Mutated Ras was found in approximately 30% of human cancers and 90% of the pancreatic cancers. However, the biological results seen with farnesyltransferase inhibitors are complex and unexpected, and thus it is important to emphasize that there are many crucial cellular proteins other than Ras that are farnesylated. Therefore, this has led to significant interest in both determining the entire set of mammalian farnesylated proteome, and quantitating the response of individual farnesylated proteins to drug treatment. A SWISSPROT database search indicated that there are over 300 different proteins bearing a CaaX motif in the human genome. It is hypothesized that a number of important farnesylated proteins remain to be identified and characterized, and that their discovery will lead to a much deeper understanding of the full biological ramifications of protein farnesylation. We view the development of FPP-based tools to selectively alter the prenylation of certain proteins in a cell as an important step to elucidate the function of the farnesyl groups. Our lab has discovered multiple isoprenoid analogs that may specifically farnesylate some cellular protein substrates. However, no quantitative method had been established to evaluate their efficacy at prenylating proteins in cells. My first research project entailed the development of a metabolomic tool to quantify protein farnesylation altered by native and synthetic isoprenoids. In my second thesis project, utilizing alkynyl FPP analogs coupled with ‘click chemistry’ aided proteomic tools, we were able to generate the most comprehensive profile for the farnesylated proteome yet reported. The accomplishment of both the metabolomic and proteomic projects has provided valuable tools that will allow for the more detailed and selective investigation of the biological consequences of protein farnesylation, a key protein post-translational modification with clear therapeutic implications.

Degree

Ph.D.

Advisors

Gibbs, Purdue University.

Subject Area

Biochemistry

COinS