The focus of this study is on the human pyruvate dehydrogenase complex (PDHc-h) consisting of six proteins including thiamin diphosphate (ThDP)-dependent pyruvate dehydrogenase (E1, ?22-heterotetramer), the dihydrolipoamide transacetylase (E2, with two lipoyl domains), dihydrolipoamide dehydrogenase (E3), a unique E3-binding protein (E3BP) and two regulatory enzymes pyruvate dehydrogenase kinases (PDK1-4) and phosphatases (PDP1-2). Specifically, the regulation of PDHc via phosphorylation of E1 by PDK1 will be pursued, as PDK1 has been strongly implicated as an overexpressed culprit in multiple cancer cells; hence inhibition of the interaction of PDK1 with the E1 or E2?E3BP, could provide novel targets for the design of anti- cancer agents. Since the PDKs reside on (have a stronger binding to) the E2?E3BP core of the entire complex, specific inhibition of the PDK1-E2?E3BP interaction provides a possible new approach to treatment of cancer. Recently, the Jordan group published the first paper defining the interaction loci between the PDK1 (and PDK2) and the E2?E3BP core by two complementary methods, HD-exchange mass spectrometry (HDX-MS) and multinuclear nuclear magnetic resonance (NMR) spectroscopy in a special issue of Biochemistry titled `Frontiers in Kinase Research' in January 2015. The first principal goal of the proposal is to determine where along the complex multistep pathway the effect of E1 phosphorylation by PDK1 is expressed; in this goal the detection of ThDP intermediates along the complex pathway will be performed by established methods, namely, CD spectroscopy, the Tittmann-Hubner (TH) NMR assignments and stopped-flow CD. The second principal goal of the proposal will focus on follow-up experiments to validate the sites on E2?E3BP identified to interact with PDK1 in the recent Jordan group publication, which will be accomplished by two consecutive steps: (i) site- directed mutagenesis studies on the amino acids identified in the interaction maps of E2?E3BP as `hot spots' in the recent paper, followed by (ii) protein-protein interaction studies on the substituted E2?E3BP with PDK1 and PDK2 using HDX-MS, isothermal titration calorimetry (ITC) and fluorescence titrations. These measurements will confirm whether the `hot spot' interrogated indeed is important for the interaction of E2?E3BP with the PDK1, and whether it is specific to PDK1 by comparing such measurements with PDK2. These experiments will identify `real' hot spots, against which rational drug design could be undertaken.
The proposed research concentrates on human pyruvate dehydrogenase multi-enzyme complex (PDHc), and will provide fundamental knowledge regarding issues directly related to human health. This enzyme complex is subject to regulation/inhibition by covalent phosphorylation (at the E1 component turning off the overall reaction), an inhibition that could be reversed by specific protein phosphatases. However, dysfunctions in this covalent regulation lead to genetic defects causing type-2 diabetes and some forms of cancers. The overarching goal of this proposal is to understand two issues regarding PDK1, the first of the four kinases: (a) where in the PDHc pathway does the PDK1 inhibit the enzyme; and (b) identify the loci of interaction of the PDK1 with the E2?E3BP components of the PDHc. Once we understand these issues, involving regulatory inhibition and the protein-protein interactions, there is a potential for rational drug design by inhibiting interaction of PDHc with PDK1, an interaction that is implicated in several forms of cancer.
|Guevara, Elena L; Yang, Luying; Birkaya, Barbara et al. (2017) Global view of cognate kinase activation by the human pyruvate dehydrogenase complex. Sci Rep 7:42760|