Investigation of peptide substrate specificity and design of novel bisubstrate inhibitors of protein geranylgeranyl transferase-I
Abstract
Protein prenylation is an important post-translational modification that governs membrane trafficking and function of more than a hundred human proteins. This modification is catalyzed by three prenyltransferases – farnesyltransferase (FTase), geranylgeranyltransferase-I (GGTase-I) and geranylgeranyltransferase-II (Rab-GGTase). FTase and GGTase-I transfer the C15 and C20 isoprenoids respectively to the C-terminal CaaX sequence of several cellular signaling proteins. Prenylated proteins are involved in variety of signaling pathways and are implicated in oncogenesis and several other diseases. Therefore, CaaX-prenyltransferases have emerged as promising targets to develop novel therapeutics for the treatment of cancer. This dissertation is aimed at gaining a better understanding of the substrate specificity trends of CaaX-prenyltransferases, and to use this information to design selective inhibitors of GGTase-I. Although the substrate selectivity patterns for both prenyltransferases can be predicted through either crystal structure or database analysis, there is a scarcity of experimental data that confirm these predictions. We have compiled an exhaustive list of Ca1a2X proteins that are predicted substrates of GGTase-I and have diverse residues at the a1 and a2 positions. Selected libraries of dansyl-GCaaX peptides were synthesized by Fmoc solid phase synthesis methodology. These peptide libraries (X = methionine, leucine and phenylalanine, isoleucine and valine) were then screened with both GGTase-I and FTase using a well-established fluorescence assay. In vitro screening analysis of dansyl-GCaaX peptides not only confirmed the substrate ability of various known CaaX sequences and the ones that were hypothesized as GGTase-I substrates. Additionally, we have also correlated the substrate activity of these CaaX sequences with log P of the capped a1+a2 dipeptide – a structural descriptor that is easily obtained and accounts for inherent hydrophobicity of CaaX peptides. The results of this screening have revealed interesting and unusual trends with implications for the development of GGTase-I inhibitors.
Degree
Ph.D.
Advisors
Gibbs, Purdue University.
Subject Area
Biochemistry|Organic chemistry|Pharmacy sciences
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