Glyceraldehyde-3-phosphate dehydrogenase as a model to investigate protein-protein interactions, non-canonical protein-RNA interactions, and post-translational modifications
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Author/Creator ORCID
Date
2016-01-01
Type of Work
Department
Chemistry & Biochemistry
Program
Chemistry
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This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
Distribution Rights granted to UMBC by the author.
Distribution Rights granted to UMBC by the author.
Abstract
The topic of this dissertations is a highly collaborative and multifaceted look into clinically relevant aspects of the interactions of the homotetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work details the importance of basic and translational research centered on GAPDH structure and function as the enzyme has been linked to several cardiovascular, diabetic, neurologic, and tumorigenic disorders. The project consists of three components, outlined below. (1) Developing a model system based on GAPDH to study biomolecular interactions using coherent X-ray scattering (BICXS). BICXS is a technique based on Small Angle X-ray Scattering (SAXS) signatures of gold nanoparticle (GNP) reporter agents that are indicative of an interaction between target biomolecules. This is of importance as there is a need for innovative methodologies to identify and characterize biomolecular interactions in vivo to complement those currently in use. Detailed here is a proof-of-concept study describing the utility of GNPs as reporter agents, demonstrating the process by which the fraction of interacting species within a sample can be determined. Also discussed is an analysis of the development of antibody conjugated GNPs targeted against GAPDH to test the ability of the technique in the identification and characterization of protein-protein interactions. (2) Elucidating the binding site and regulatory mechanisms of TNF-? mRNA in GAPDH for therapeutic intervention. GAPDH binds many clinically relevant mRNAs and alters their stability and/or translation. Most of these interactions occur via GAPDH binding to adenine-uridine rich elements (AREs) within their 3? untranslated regions (UTRs). The exact site and mechanism of binding, though, remain elusive. Through a collaborative approach, we found that GAPDH binds to the core AREs of the tumor necrosis factor-? mRNA 3? UTR via a sequential two-step mechanism. A single point mutation at the GAPDH dimer interface resulted in reduced binding affinity and structural alteration of the bound RNA. These results lead to the novel hypothesis that the dimer interface may be a crucial region for RNA binding. (3) Dveloping a high-throughput assay for the identification of small molecules that modulate GAPDH oxidoreductase activity. Due to the multifunctional nature of GAPDH and its crucial role in metabolism, it is a promising target for cancer therapy. Dithiolethione compounds possess chemopreventive, cytoprotective, and antiproliferative properties. In collaboration with researchers at the National Cancer Institute (NIH-NCI) we have determined that the dithiolethione, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ACS1), disrupts glycolysis in cancer cells and modifies GAPDH. However, while ACS1 may modify GAPDH in cells, we definitively show that it does not affect GAPDH oxidoreductase activity in vitro. Accordingly, we have repurposed our high-throughput kinetic assay, and applied it to the identification of novel GAPDH modulators using the commercially available compound library LOPAC (library of pharmacologically active compounds) with the National Center for Advancing Translational Sciences (NIH-NCATS). From our initial screen we identified eight high efficacy inhibitors of GAPDH oxidoreductase activity that can be further validated and used in secondary assays.