Characterization of the S-adenosyl-L-methionine (SAM) binding domain of the yeast isoprenylcysteine carboxyl methyltransferase, Ste14p

Amanda R Logue, Purdue University

Abstract

Many eukaryotic proteins terminate with a –CaaX tetrapeptide motif, including most Ras, Rho, Ral and Rab proteins. Additionally, nuclear lamins, Rheb proteins, the γ subunits of heterotrimeric G proteins, and the yeast mating pheromone a-factor are all CaaX proteins. These proteins play essential roles in cellular events such as trafficking, transcription, cell division, cell proliferation and nuclear-envelope assembly and dissociation. K-Ras, a well-known CaaX protein, is the most frequently mutated oncogene found in ~30% of all cancers and is proposed to be mutated in ~90% of all pancreatic adenocarcinomas. Proteins terminating with a terminal CaaX motif contain a signal for a series of post-translational modifications, known as CaaX processing. In these proteins, "C" represents a cysteine, "a" represents any aliphatic amino acid and "X" represents one of several amino acids. CaaX processing encompasses three sequential post-translational modifications, including isoprenylation, proteolysis and methylesterification. These modifications are critical for the proper membrane localization and biological function of CaaX proteins. The research presented in this dissertation focuses on the integral membrane enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Icmt is responsible for the last step of CaaX processing and has been identified as a promising target for the development of anti-K-Ras chemotherapeutics. However, limited information is available on Icmt's biochemistry and mechanism of action. In order to efficiently target Icmt, we must first understand important enzyme characteristics. The work presented here describes the biochemical characterization of Icmt's cofactor binding site using S. cerevisiae as a model (Ste14p) for the construction of a library of 55 His10- myc3N-Ste14p mutants. From this subset, 31 conserved residues were identified as being essential for Ste14p activity. Of these residues, 14 are hypothesized to play a role in SAM binding, as described by Yang et al. (2011) and homology modeling. With the development of a novel SAM filter binding assay, L161, Y167, R171, P173, E213, E214, L217 and Y225 are predicted to be important for SAM recognition in Ste14p. Additional work discussed in this dissertation includes the testing of multiple inhibitor libraries in collaboration with the laboratory of Richard Gibbs (Purdue University) and the identification/characterization of the first submicromolar inhibitor of hIcmt (JM 4-68B) which lacks the carboxylate functionality of other substrate based analogs.

Degree

Ph.D.

Advisors

Hrycyna, Purdue University.

Subject Area

Pharmacology|Chemistry|Biochemistry

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