The PIs goals with this R15 proposal are studies on the thiamin diphosphate (ThDP)-dependent human 2-oxoglutarate dehydrogenase complex (OGDHc, also known as ?-ketoglutarate dehydrogenase; it comprises three component enzymes denoted with E1o-h, E2o-h and E3-h), the rate-limiting enzyme in the citric acid cycle (also known as the tricarboxylic or Krebs cycle), an enzyme critical for glucose metabolism in normal brain. OGDHc activity is diminished in the brain of Alzheimer's disease patients, which is characterized by reduced glucose metabolism and increased level of oxidative stress markers. A diminished OGDHc activity had also been correlated with other neurodegenerative diseases, including Parkinson's disease, Huntington disease, Wernike-Korsakoff disease and progressive subnuclear palsy. It is known that OGDHc is a sensitive target for a variety of oxidants generated during oxidative stress, including inhibition of OGDHc by peroxynitrite and nitric oxide, and by the reactive oxygen species (ROS), superoxide and H2O2, among other agents. OGDHc is not only a sensitive target of ROS, it can also produce superoxide and H2O2 in brain mitochondria. While earlier research attributed ROS generation by OGDHc to the E3-h component, the PI and collaborators have now shown definitively that (a) The ThDP-dependent E1o-h component in fact also produces superoxide anion and hydrogen peroxide, (b) Concomitantly, there is produced a ThDP-bound radical characterized by electron paramagnetic resonance spectroscopy (EPR). These products result from reaction of a ThDP-bound intermediate with dioxygen O2. Our contributions, and this proposal were made possible by the PI's group's recent success in producing full length, active recombinant versions of the human E1o-h and E2o-h components of this important complex in pure form and in significant amounts for the proposed research, while other groups address this issue mostly on intact brain mitochondrion, rather than with individual protein components of the human OGDHc. The OGDHc-h is located in the matrix of the mitochondrion where it is associated with the inner mitochondrial membrane, catalyzing the conversion of 2-oxoglutarate to succinyl-CoA according to: 2-oxoglutarate+NAD++CoA ? succinyl-CoA+NADH+H++CO2 (1) The overarching goal of our studies is an atomic-level explanation of: Which component (E1o-h and E3-h) is the major source of ROS production, consistent with the medical finding of reduced OGDHc activity in the brain with aging. Our long term goal is to provide an atomic explanation for the ROS findings in brain by identifying specific residues responsible for ROS formation, and those modified by ROS on all three components of the human OGDHc. Our immediate goals within the boundaries of the R15 program, are to initiate a logical sequence of projects, which will lead to achieving of our long-term goals, include: (a) Detection and kinetic characterization of thiamin-related intermediates on E1o-h and lipoamide-bound intermediates on E2o-h using a battery of kinetic and spectroscopic state-of-the-art methods developed in our group, as well as new methods EPR and ROS detection. We will determine the effect of OGDHc-h assembly, and of the contribution of the three individual components of OGDHc-h to ROS generation. No such studies are yet done on the human OGDHc. The mechanistic significance of these studies is to provide an understanding of how and where the ThDP-enamine radical formed by reaction of dioxygen with the enamine, receives special stabilization on the E1o-h; and which residues of E1o-h are the major contributors to ROS generation by OGDHc-h. (b) Development of an advanced laboratory course titled Introduction to Modern Biochemical Research to train both undergraduate and graduate students in state-of-the-art methodologies, with experiments strictly paralleling those proposed for the research program, and with the enzymes studied in our program; the tools to be taught will include, kinetics, spectroscopy (circular dichroism, MS, NMR, EPR), and basic tools of molecular biology and biochemistry. No such course exists at any Rutgers campus in Newark, hence it will serve the needs of both arts and sciences and the medical school in Newark. This course will also enable the Chemistry Department to at last offer a Biochemistry track; such a lab course is also highly recommended by the Am. Chem. Society. Significance and Innovation. An understanding of the mechanism by which OGDHc produces ROS, and how OGDHc activity could be altered by ROS is an important goal of the PI since it could provide insight to neurodegenerative diseases. This is the gap in our knowledge that our proposed research will help to fill. Methods for all experiments in the Specific Aim are in place i the PI's laboratories and are in published material, hence the proposed experiments carry little risk. Gary Gerfen at Albert Einstein College of Medicine has agreed to collaborate on the EPR studies. The close relationship between the course being proposed and the research being proposed is surely innovative and is in the spirit of the R-15 program.

Public Health Relevance

Title: Mechanistic Issues Related to Aging on Human 2-Oxoglutarate Dehydrogenase Complex. Narrative. The thiamin diphosphate (ThDP)-dependent 2-oxoglutarate dehydrogenase complex (OGDHc, also known as ?-ketoglutarate dehydrogenase) is critical for glucose metabolism in normal brain. OGDHc activity is diminished in the brain of Alzheimer's Disease patients, which is characterized by reduced glucose metabolism and increased level of oxidative stress markers. A diminished OGDHc activity had been correlated with other neurodegenerative diseases, including Parkinson's disease, Huntington disease, Wernike-Korsakoff disease and progressive subnuclear palsy. It is known that OGDHc is a sensitive target for a variety of oxidants generated during oxidative stress, including inhibition of OGDHc by peroxynitrite and nitric oxide, and by the reactive oxygen species (ROS), superoxide and H2O2, among other agents. The OGDHc is not only a sensitive target of ROS, it can also produce superoxide and H2O2 in brain mitochondria. While earlier research attributed ROS generation by OGDHc to its E3 component (dihydrolipoyl dehydrogenase), the PI and collaborators have now shown definitively that (a) The ThDP-dependent E1 component in fact is also a producer of superoxide anion and hydrogen peroxide, (b) Concomitantly, there is produced a ThDP-bound radical characterized by electron paramagnetic resonance spectroscopy (EPR). These products result from reaction of a ThDP-bound intermediate with dioxygen O2. Our recent contributions, and this proposal were made possible by the PI's group's recent success in producing full length, active recombinant versions of the human E1 and E2 components of this important enzyme in pure form and in significant amounts for the proposed research, while other groups work mostly on intact brain mitochondrion, rather individual protein components of this enzyme. Our long term goal is to provide an atomic explanation for the ROS findings in brain by identifying specific residues responsible for ROS formation, and those modified by ROS on all three components of the human OGDHc. Our immediate goals within the boundaries of the R15 program, are to initiate a logical sequence of projects, which will lead to achieving of our long-term goals, include: (a) Detection and kinetic characterization of thiamin-related intermediates on E1o-h and lipoamide-bound intermediates on E2o-h using a battery of kinetic and spectroscopic state-of-the-art methods developed in our group, as well as new methods EPR and ROS detection. We will determine the effect of OGDHc-h assembly, and of the contribution of the three individual components of OGDHc-h to ROS generation. No such studies are yet done on the human OGDHc. The mechanistic significance of these studies is to provide an understanding of how and where the ThDP- enamine radical formed by reaction of dioxygen with the enamine, receives special stabilization on the E1o-h; and which residues of E1o-h are the major contributors to ROS generation by OGDHc-h. (b) Development of an advanced laboratory course titled Introduction to Modern Biochemical Research to train both undergraduate and graduate students in state-of-the-art methodologies, with experiments strictly paralleling those proposed for the research program, and with the enzymes studied in our program; the tools to be taught will include, kinetics, spectroscopy (circular dichroism, MS, NMR, EPR), and basic tools of molecular biology and biochemistry. No such course exists at any Rutgers campus in Newark, hence it will serve the needs of both arts and sciences and the medical school in Newark. This course will also enable the Chemistry Department to at last offer a Biochemistry track; such a lab course is also highly recommended by the Am. Chem. Society. Significance and Innovation. An understanding of the mechanism by which OGDHc produces ROS, and how OGDHc activity could be altered by ROS is an important goal of the PI since it could provide insight to neurodegenerative diseases. This is the gap in our knowledge that our proposed research will help to fill.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
9R15GM116077-01A1
Application #
8957815
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
2015-08-01
Project End
2018-07-31
Budget Start
2015-08-01
Budget End
2018-07-31
Support Year
1
Fiscal Year
2015
Total Cost
$369,360
Indirect Cost
$129,360
Name
Rutgers University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
130029205
City
Newark
State
NJ
Country
United States
Zip Code
07102
Nemeria, Natalia S; Gerfen, Gary; Nareddy, Pradeep Reddy et al. (2018) The mitochondrial 2-oxoadipate and 2-oxoglutarate dehydrogenase complexes share their E2 and E3 components for their function and both generate reactive oxygen species. Free Radic Biol Med 115:136-145
Whitley, Matthew J; Arjunan, Palaniappa; Nemeria, Natalia S et al. (2018) Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the ?V138M variant of human pyruvate dehydrogenase. J Biol Chem 293:13204-13213
Chakraborty, Joydeep; Nemeria, Natalia S; Farinas, Edgardo et al. (2018) Catalysis of transthiolacylation in the active centers of dihydrolipoamide acyltransacetylase components of 2-oxo acid dehydrogenase complexes. FEBS Open Bio 8:880-896
DeColli, Alicia A; Nemeria, Natalia S; Majumdar, Ananya et al. (2018) Oxidative decarboxylation of pyruvate by 1-deoxy-d-xyulose 5-phosphate synthase, a central metabolic enzyme in bacteria. J Biol Chem 293:10857-10869
Nemeria, Natalia S; Gerfen, Gary; Yang, Luying et al. (2018) Evidence for functional and regulatory cross-talk between the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex and 2-oxoadipate dehydrogenase on the l-lysine, l-hydroxylysine and l-tryptophan degradation pathways from studies in vitro. Biochim Biophys Acta Bioenerg 1859:932-939
Nemeria, Natalia S; Gerfen, Gary; Guevara, Elena et al. (2017) The human Krebs cycle 2-oxoglutarate dehydrogenase complex creates an additional source of superoxide/hydrogen peroxide from 2-oxoadipate as alternative substrate. Free Radic Biol Med 108:644-654
Zhou, Jieyu; Yang, Luying; DeColli, Alicia et al. (2017) Conformational dynamics of 1-deoxy-d-xylulose 5-phosphate synthase on ligand binding revealed by H/D exchange MS. Proc Natl Acad Sci U S A 114:9355-9360
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
Ambrus, Attila; Wang, Junjie; Mizsei, Reka et al. (2016) Structural alterations induced by ten disease-causing mutations of human dihydrolipoamide dehydrogenase analyzed by hydrogen/deuterium-exchange mass spectrometry: Implications for the structural basis of E3 deficiency. Biochim Biophys Acta 1862:2098-2109
Nemeria, Natalia S; Shome, Brateen; DeColli, Alicia A et al. (2016) Competence of Thiamin Diphosphate-Dependent Enzymes with 2'-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin. Biochemistry 55:1135-48

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