Regulation of the G Protein βγ Subunits through the Covalent Modification of Gβ: From Random Discovery to Rational Design

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2009.Cells possess the ability to respond to external stimuli. In order to relay information from membrane receptors to internal effectors, they have developed diverse mechanisms. One involves the heterotrimeric G proteins, comprised of Gα and Gβγ subunits, which dissociate from each other after activation by cell surface G protein-coupled receptors (GPCRs). Both Gα and Gβγ are able to transmit signals that regulate cell function. Overactivation of Gβγ has been implicated in the pathophysiology of several diseases, such as cancer, heart failure and inflammation, and for this reason, inhibitors of Gβγ might be of pharmacological value. Our group has used a combination of high-throughput and manual screening to find small molecules that non-covalently bind to a protein-protein interaction “hot spot” on Gβγ and inhibit interactions with effectors. We found that some of the compounds identified in these screens covalently modify Gβγ through a redox-dependent mechanism. The diselenide selenocystamine, for instance, attaches a selenocysteamine moiety to a cysteine, C204, in the “hot spot.” These covalent modifiers produce more effective, and in some cases, more potent effects than the non-covalent binders, but they lack specificity. In order to obtain covalent modifiers with greater selectivity for Gβγ, we conceived the idea of incorporating non-selective covalent modifiers into Gβγ-selective non-covalent binders that would act as carrier molecules. We took advantage of the co-crystal structure of Gβγ with the selective, non-covalent binding peptide SIGKAFKILGYPDYD. The side chain of the first lysine of this peptide is in close proximity to C204. With the purpose of incorporating a disulfide bridge into this side chain, we mutated the lysine to cysteine and coupled a cysteamine moiety to it. The resulting peptide, which we call for short SIGC(-cysteamine), modifies Gβγ more potently than other disulfides, and more efficiently and specifically than selenocystamine. Explicitly, SIGC(-cysteamine) delivers a cysteamine moiety to C204. These data support the concept that Gβγ can be regulated by redox-dependent mechanisms that may be physiologically relevant, and that this can be exploited in the design of targeted covalent modifiers