Mechanism and conformational dynamics of the yeast isoprenylcysteine carboxyl methyltransferase, Ste14p
Over 100 mammalian proteins fall into the diverse CaaX proteins family. These proteins are involved in many key processes, such as cellular proliferation, differentiation, trafficking, and survival. One CaaX protein that has received much attention is K-Ras, which is mutated in over 20% of human cancers. Interestingly, the oncogenic transformation of K-Ras is halted if the protein is not localized to the plasma membrane. Due to its C-terminal CaaX motif, Ras undergoes a series of post-translational modifications which aids in its proper localization. Through CaaX processing, KRas is farnesylated, the three terminal residues endoproteolytically cleaved, and the exposed cysteine methyl esterified. This final step is performed by isoprenylcysteine carboxyl methyltransferase (Icmt). Icmt deficiency causes KRas mislocalization in knockout mice, and inhibition of Icmt has been linked to a decrease in the downstream signaling of Ras. In this way, Icmt is an attractive target for Ras inhibition. Icmt requires the cofactor SAM for its activity, but lacks the conserved binding motif. Studies have focused on the founding member of the Icmt family found in Saccharomyces cerevisiae, Ste14p, as it shares high homology with the human enzyme, which has yet to be functionally purified. Herein, we have developed a library of 84 Ste14 variants and used a variety of biochemical techniques, such as in vitro activity and ligand binding assays, to elucidate what residues are essential for catalysis and binding of the cosubstrate. These studies will facilitate the generation of Icmt inhibitors for Ras-driven cancers.
Hrycyna, Purdue University.
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