Sharda P. Singh, Ph.D.
Ph.D. – Central Drug Research Institute, Lucknow / Agra University, Agra, India, 1992
Since early during my post-doctoral training, my major research interest has been the physiological and toxicological role(s) of products of oxidative stress, in particular lipid peroxidation products. Specifically, we studied the metabolism and signaling functions of the electrophilic product of lipid peroxidation, 4-hydroxynonenal (4-HNE). My initial studies on 4-HNE metabolism were aimed at understanding the structure-function relationships of the protein mGSTA4-4 (an alpha class glutathione S-transferase), which is known to have high catalytic efficiency for 4-HNE. Using a number of different techniques, including site-directed mutagenesis of mGsta4 cDNA and immunohistochemistry, we characterized the enzymatic properties of mGSTA4-4 (conjugating lipid hydroperoxides and/or 4-hydroxyalkenals) and determined its intracellular localization. Our studies then focused on the roles of 4-HNE, as a model lipid peroxidation product and signaling molecule and allowed us to determine how this lipid peroxidation product could affect both lifespan and the rate of aging. We modulated 4-HNE levels by genetically altering 4-HNE-conjugating enzyme activities in both mouse and C. elegans models, and characterized the resulting phenotypes. We generated a mouse model of impaired 4-HNE metabolism, the mGsta4-/- (or knockout mutant) mouse and studied the effects in a number of tissues, including most recently in the heart.
In the C. elegans model, we showed that experimental lowering of tissue 4-HNE levels extends lifespan, whereas excessive 4-HNE shortens it and results in obesity. Our studies with the mGsta4-/- mouse model demonstrated that 4-HNE also triggers obesity in mammals, indicating an evolutionarily conserved process. In our more recent studies, we have tried to understand how elevated 4-HNE levels could result in obesity. Our recent findings show that 4-HNE partially inhibits several key tricarboxylic acid (TCA) cycle enzymes, resulting in an increased level of citrate and related metabolites leading to the allosteric activation of acetyl-CoA carboxylase (ACC). We also examined the possible role of 4-HNE in inhibition of AMPK, either by inhibition of an upstream kinase, inhibition of adiponectin secretion, or by withdrawal of the cofactor lipoic acid, that may result in activation of ACC through attenuated phosphorylation. Overall, our results show that 4-HNE, which is cyto- and genotoxic at supraphysiological concentrations, is also a signaling molecule at physiological levels and modulates a great variety of cellular and organismal functions.
Recently, we have focused our attention on the role of electrophile signaling in the activation of the Nrf2 transcription factor, the key player in one of the most important cytoprotective pathways. Nrf2 is a critical regulator of genes encoding antioxidant and anti-electrophile enzymes in all animals. Doxorubicin (DOX) is a potent, broad-spectrum anti-cancer drug, widely used to treat breast cancer and a great variety of other cancers. But DOX frequently produces life-threatening cardiomyopathy due to its induction of oxidative stress and subsequent accumulation of the damaging lipid peroxidation product, 4-HNE. We demonstrated that absence of a functional Gsta4 gene product in mice results in upregulation of Nrf2 activity in cardiac tissue, mediating protection against DOX cardiotoxicity. In our pilot studies leading to this proposal, we observed a similar Nrf2-driven protection against DOX toxicity in cardiac H9c2 cells, preconditioned with the non-toxic and electrophilic phytochemical, sulforaphane (SFN). SFN is known to upregulate activity of Nrf2 and its target genes. However, our laboratory is the first to show that this SFN-mediated protective effect involves a novel mechanism of Nrf2 activation, mediated by a recently discovered protein, PGAM5. We also demonstrated not only that administration of SFN to DOX-treated mice induces a similar protection from DOX toxicity, but that a novel posttranslational modification of PGAM5 is necessary for the induction of protective Nrf2 activity. These findings are critical to the highly innovative studies that we propose: the unique characterization of differential SFN action in normal cardiomyocytes versus breast cancer cells both in vitro and in a tumor-bearing mouse model.
Recent Research Support
Ongoing Research Support
AHA 14GRNT18890084 (01/01/14 – 12/31/16)
Mitochondrial-nuclear signaling for protection against doxorubicin cardiotoxicity
Completed Research Support
Pilot proposal on P20 GM109005-01A1 (PI) (10/01/15 – 09/30/16)
Prevention of Doxorubicin Cardiomyopathy in Cancer Treatment
NIA/NIH R01 AG 032643 (09/01/09 – 09/30/16)
Causative role of lipid peroxidation products in obesity and aging
NIH/NIA 1P30AG028718-01A2 (12/01/11 – 12/31/12)
Modulation by 4‑hydroxynonenal (4‑HNE) of the phosphorylation status of acetyl-CoA carboxylase (ACC) as a potential determinant of ectopic fat levels in skeletal muscle
R01 AG 028088 (02/01/07 – 01/31/13)
Role of glutathione transferases in life span extension of C. elegans
NIH/NIA R01AG 18845-01 (CoI) (03/01/02 – 02/28/07)
Role of lipid peroxidation and 4hydroxynonenal in aging
Boerma M, Singh P, Sridharan V, Tripathi P, Sharma S, Singh SP. Effects of Local Heart Irradiation in a Gluthathione S-Transferase Alpha 4 Null Mouse Model. Radiat Res. 183(6):610-619, 2015 (PMID: 26010708).
Singh P, Sharma R, McElhanon K, Allen CD, Megyesi JK, Benes H, Singh SP. Sulforaphane Protects the Heart from Doxorubicin-induced Toxicity. Free Radical Biology and Medicine. 86 (2015): 90-101, 2015 (PMID: 26025579).
Benes H, Vuong MK, Boerma M, McElhanon KE, Siegel ER, Singh SP. Protection from Oxidative and Electrophilic Stress in the Gsta4-null Mouse Heart (2013. Cardiovasc Toxicol. 13(4):347-56, 2013 (PMID: 23690225).
McElhanon KE, Bose C, Sharma R, Wu L, Awasthi YC, Singh SP. Gsta4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity. Open J. Apoptosis. 2(1): 1-11, 2013 (PMID: 24353929).
Singh SP, Zimniak L, Zimniak P. The human hGSTA5 gene encodes an enzymatically active protein. Biochimica et Biophysica Acta. 1800: 16-22, 2010 (PMID: 19664689).