PROTEIN PRENYLATION BY GERANYLGERANYLTRANSFERASE TYPE I (GGTASE-I): UNDERSTANDING THE SUBSTRATE RECOGNITION AND ACTIVITY REGULATION OF A MULTISPECIFIC ENZYME
Date of Award
Doctor of Philosophy (PhD)
James L. Hougland
cellular geranylgeranylation, enzyme reengineering, fluorescence microscopy, GGTase-I, Prenylation, substrate selectivity
Physical Sciences and Mathematics
Protein prenylation is a post-translational modification wherein a substrate protein is modified by the attachment of a 20- or 15- carbon isoprenoid group to a cysteine residue near its C-terminus, by protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) respectively. This modification provides a hydrophobic group that can aid protein association with cellular membranes and enable protein-protein interactions. These enzymes recognize their substrates using a C-terminal Ca1a2X sequence where C denotes the cysteine residue being modified and the a1, a2 and X residues dictate substrate specificity for GGTase-I or FTase. Crystallographic studies have provided a structural model of the a2 residue binding pocket within GGTase-I, but the functional roles of many of the proposed enzyme-substrate interactions in the active site remain to be defined. We have characterized amino acid residues in the active site of GGTase-I that engender substrate specificity at the a2 position using techniques such as site-directed and saturation mutagenesis, GGTase-I mutant library screening, and peptide substrate structure-activity studies. GGTase-I substrate specificity can be reengineered by mutating a small number of amino acids in the active site, similar to the malleable substrate selectivity observed with FTase. Our work supports the idea that tunable substrate specificity may be a general property of multispecific enzymes involved in post-translational modification. The mutant GGTase-I enzymes described in this work can be used as unique tools, to potentially probe the effects of prenylation pathway modifications on target protein localization and function without altering the larger prenylated proteome.
To assess geranylgeranylation in a biologically relevant context, we have developed tools for determining GGTase-I prenylation activity inside a cell. Based on predictive algorithms and in vitro peptide reactivities, many Ca1a2X sequences have been identified as potential substrates for GGTase-I although, the ability of these sequences to be geranylgeranylated within the cell is still unknown. To address this question, we have developed a fluorescence reporter protein series that spans several orders in reactivity with GGTase-I and determined the minimum reactivity required for this reporter protein to be geranylgeranylated inside an intact mammalian cell. Further, we have demonstrated application of this calibrated GGTase-I sensor to monitor changes in GGTase-I activity in response to inhibitors or genetic mutations. Determination of the GGTase-I activity within the cell will help in defining the prenylated proteome and identifying GGTase-I dependent pathways for therapeutic targeting.
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Gangopadhyay, Soumyashree Ashok, "PROTEIN PRENYLATION BY GERANYLGERANYLTRANSFERASE TYPE I (GGTASE-I): UNDERSTANDING THE SUBSTRATE RECOGNITION AND ACTIVITY REGULATION OF A MULTISPECIFIC ENZYME" (2016). Dissertations - ALL. 574.
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