Keywords

protein structure, Ras protein, S-adenosyl-L-methionine, site-directed mutagenesis

Presentation Type

Poster

Research Abstract

Ninety percent of pancreatic cancers are attributed to mutations in the Ras protein, making it paramount to concentrate on Ras activity. This study focuses on Ras activity by targeting a post-translational modifying enzyme of Ras called Isoprenylcysteine carboxyl methyltransferase (Icmt). Elucidation of the binding site of Icmt will allow the development of therapeutics that effectively inhibit Icmt causing the mislocalization of Ras, and in turn, aid in the treatment of Ras driven cancers. Currently, the hydrophobic substrate binding site of Icmt is unknown. In order to characterize the substrate binding site of Icmt, site-directed mutagenesis was used to design mutations in the yeast homolog of Icmt, Ste14p, and these mutants were tested on substrate specificity. Residues L33, L34, L40, L176, L190, and L195 were mutated to alanine and residue F80 was mutated to tyrosine. When tested with a methyltransferase assay, all of the mutants lost activity as compared to wild type (WT). Noticeably, L190A had only 44% WT activity. This suggests that L190A is important for either substrate binding or the overall structural integrity of Ste14p. The results from trypsin digestion show that all mutants have cleavage patterns similar to WT. This indicates that the structural integrity of Ste14p remains intact regardless of these mutations. Substrate specificity and photolabeling experiments should be conducted in the future to elucidate if these residues are vital for the substrate binding site of Icmt. These results could be utilized to design more potent and effective drug therapies to minimize Ras signaling in cancer cells.

Session Track

Health

Included in

Biochemistry Commons

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Aug 4th, 12:00 AM

Mutational and Biochemical Analysis of Isoprenylcysteine Carboxyl Methyltransferase

Ninety percent of pancreatic cancers are attributed to mutations in the Ras protein, making it paramount to concentrate on Ras activity. This study focuses on Ras activity by targeting a post-translational modifying enzyme of Ras called Isoprenylcysteine carboxyl methyltransferase (Icmt). Elucidation of the binding site of Icmt will allow the development of therapeutics that effectively inhibit Icmt causing the mislocalization of Ras, and in turn, aid in the treatment of Ras driven cancers. Currently, the hydrophobic substrate binding site of Icmt is unknown. In order to characterize the substrate binding site of Icmt, site-directed mutagenesis was used to design mutations in the yeast homolog of Icmt, Ste14p, and these mutants were tested on substrate specificity. Residues L33, L34, L40, L176, L190, and L195 were mutated to alanine and residue F80 was mutated to tyrosine. When tested with a methyltransferase assay, all of the mutants lost activity as compared to wild type (WT). Noticeably, L190A had only 44% WT activity. This suggests that L190A is important for either substrate binding or the overall structural integrity of Ste14p. The results from trypsin digestion show that all mutants have cleavage patterns similar to WT. This indicates that the structural integrity of Ste14p remains intact regardless of these mutations. Substrate specificity and photolabeling experiments should be conducted in the future to elucidate if these residues are vital for the substrate binding site of Icmt. These results could be utilized to design more potent and effective drug therapies to minimize Ras signaling in cancer cells.