Purification, characterization, and inhibition of the Saccharomyces cerevisiae isoprenylcysteine carboxylmethyltransferase, Ste14p

Jessica Lynn Anderson, Purdue University

Abstract

Many eukaryotic proteins terminate with a C-terminal CaaX sequence where C is cysteine, a is an aliphatic amino acid, and X can be one of many amino acids. These proteins are modified by a trio of post-translational modifications. The final methylesterification step is carried out by isoprenylcysteine carboxylmethyltransferase (Icmt). Icmt is thought to be a good target for anti-cancer chemotherapeutic drugs because it methylates Ras proteins. Ras proteins are mutated in 30% of human tumors and recent studies have shown that oncogenic forms of Ras are unable to transform cells that lack Icmt activity. The Icmt family is the only known family of integral membrane methyltransferases and they lacks all of the sequence motifs commonly found in S-adenosylmethionine (SAM)-dependent methyltransferases. Due to their membrane localization, the enzymes have been difficult to purify, precluding further in-depth characterization of the enzyme. Here, we report the first purification of a histidine-tagged version of the Icmt from S. cerevisiae, Ste14p (His-Ste14p) to homogeneity. We determined that His-Ste14p recognizes farnesylated and geranylgeranylated substrates equally. Partially purified, unmethylated His-Ras2p was found to be a substrate for purified and reconstituted His-Ste14p, showing definitively that this in vivo protein is a substrate in vitro. Purified His-Ste14p was also found to be a metalloenzyme. We developed a novel hydrophobic chelator (Cholesterol-Lys-NTA) and showed that it inhibited both human and yeast Icmt. Our studies also revealed that His-Ste14p arginine residues are important for enzymatic activity and that cysteine residues are not. We also report the ability of a series of prenylcysteine analogs to act as Ste14p substrates and inhibitors. The lead compounds in these studies will be further optimized in the future to obtain efficient small molecule inhibitors of Icmt. These data led to the conclusion that His-Ste14p was tolerant of bulk in 3-position of the isoprenyl chain of the substrate. We were also able to compare the differences in the ability of the lead compounds to act as substrates and inhibitors of His-hIcmt (human Icmt) expressed in S. cerevisiae.

Degree

Ph.D.

Advisors

Hrycyna, Purdue University.

Subject Area

Biochemistry

COinS