Browsing by Author "Hegde, Vijay"

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    Activation of APE/Ref-1 redox activity is mediated by reactive oxygen species and PKC phosphorylation
    (2001-05) Hsieh, Marlene M.; Hegde, Vijay; Kelley, Mark R.; Deutsch, Walter A.
    Reactive oxygen species (ROS) arise through normal cellular aerobic respiration, and, in combination with external sources such as ionizing radiation, cigarette tar and smoke, and particulate matter generated by combustion, can have a profound negative effect on cellular macromolecules such as DNA that may lead to a number of human pathological disorders including accelerated aging and cancer. A major end product of ROS damage to DNA is the formation of apurinic/apyrimidinic (AP) sites, which without removal are known to halt mRNA and DNA synthesis, or act as non-coding lesions resulting in the increased generation of DNA mutations. In human cells, the major enzyme in correcting the deleterious effects of AP sites in DNA is through the participation of AP endonuclease (APE), which initiates the removal of baseless sites in DNA through the catalytic scission of the phosphodiester bond 5′ and adjacent to an AP site. Interestingly, APE also possesses an activity (Ref-1) that controls the redox status of a number of transcription factors including Fos and Jun. The means by which APE/Ref-1 is directed to carry out such disparate roles are unknown. The presence of a number of phosphorylation sites scattered throughout both functional domains of APE/Ref-1 however offered one possible mechanism that we reasoned could play a role in dictating how this protein responds to different stimuli. Here we show that the in vitro redox activity of APE/Ref-1 is stimulated by PKC phosphorylation. Furthermore, when human cells were exposed to the PKC activator phorbol 12-myristate 13-acetate, an increase in redox activity was observed that corresponded to an increase in the phosphorylation status of APE/Ref-1. Importantly, human cells exposed to the oxidizing agent hypochlorite, followed by methyl methanesulfanate, responded with an increase in redox activity by APE/Ref-1 that also involved an increase in PKC activity and a corresponding increase in the phosphorylation of APE/Ref-1. These results suggest that the ability of APE/Ref-1 to perform its in vivo redox function is correlated to its susceptibility to PKC phosphorylation that notably occurs in response to DNA damaging agents.
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    Conversion of the Bifunctional 8-oxoguanine/β-δ AP DNA Repair Activities of Drosophila Ribosomal Protein S3 into the Human S3 Monofunctional β- elimination Catalyst Through a Single Amino Acid Change
    (2001-07) Hegde, Vijay; Kelley, Mark R.; Xu, Yi; Mian, Saira; Deutsch, Walter A.
    The Drosophila S3 ribosomal protein has important roles in both protein translation and DNA repair. In regards to the latter activity, it has been shown that S3 contains vigorous N-glycosylase activity for the removal of 8-oxoguanine residues in DNA that leaves baseless sites in their places. Drosophila S3 also possesses an apurinic/apyrimidinic (AP) lyase activity in which the enzyme catalyzes a β-elimination reaction that cleaves phosphodiester bonds 3′ and adjacent to an AP lesion in DNA. In certain situations, this is followed by a δ-elimination reaction that ultimately leads to the formation of a single nucleotide gap in DNA bordered by 5′- and 3′-phosphate groups. The human S3 protein, although 80% identical to its Drosophila homolog and shorter by only two amino acids, has only marginal N-glycosylase activity. Its lyase activity only cleaves AP DNA by a β-elimination reaction, thus further distinguishing itself from the Drosophila S3 protein in lacking a δ-elimination activity. Using a hidden Markov model analysis based on the crystal structures of several DNA repair proteins, the enzymatic differences between Drosophila and human S3 were suggested by the absence of a conserved glutamine residue in human S3 that usually resides at the cleft of the deduced active site pocket of DNA glycosylases. Here we show that the replacement of the Drosophila glutamine by an alanine residue leads to the complete loss of glycosylase activity. Unexpectedly, the δ-elimination reaction at AP sites was also abrogated by a change in the Drosophila glutamine residue. Thus, a single amino acid change converted the Drosophila activity into one that is similar to that possessed by the human S3 protein. In support of this were experiments executed in vivo that showed that human S3 and the Drosophila site-directed glutamine-changed S3 performed poorly when compared with Drosophilawild-type S3 and its ability to protect a bacterial mutant from the harmful effects of DNA-damaging agents.
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    E4orf1 Improves Lipid and Glucose Metabolism in Hepatocytes: A Template to Improve Steatosis & Hyperglycemia
    (Public Library of Science, 2012) Dhurandhar, Emily J.; Krishnapuram, Rashmi; Hegde, Vijay; Dubuisson, Olga; Tao, Rongya; Dong, X. Charlie; Ye, Jianping; Dhurandhar, Nikhil V.; Biochemistry and Molecular Biology, School of Medicine
    Hepatic steatosis often accompanies obesity and insulin resistance. The cornerstones of steatosis treatment include reducing body weight and dietary fat intake, which are marginally successful over the long term. Ad36, a human adenovirus, may offer a template to overcome these limitations. In vitro and in vivo studies collectively indicate that via its E4orf1 protein, Ad36 improves hyperglycemia, and attenuates hepatic steatosis, despite a high fat diet and without weight loss. Considering that hepatic insulin sensitivity, or the synthesis, oxidation, or export of fatty acid by hepatocytes are the key determinant of hepatic lipid storage, we determined the role of E4orf1 protein in modulating these physiological pathways. For this study, HepG2 cells, or mouse primary hepatocytes were transfected with E4orf1 or the null vector. Glucose output by hepatocytes was determined under gluconeogenic conditions (cAMP and dexamethasone, or glucagon exposure). Also, de-novo lipogenesis, palmitate oxidation, and lipid export as determined by apoB secretion were measured 48 h post transfection. Results show that compared to null vector transfected cells, E4orf1 significantly reduced glucose output in basal and gluconeogenic conditions. E4orf1 reduced de-novo lipogenesis by about 35%, increased complete fatty acid oxidation 2-fold (p<0.0001), and apoB secretion 1.5 fold(p<0.003). Response of key signaling molecules to E4orf1 transfection was in agreement with these findings. Thus, E4orf1 offers a valuable template to exogenously modulate hepatic glucose and lipid metabolism. Elucidating the underlying molecular mechanism may help develop therapeutic approaches for treating diabetes or non-alcoholic fatty liver disease(NAFLD).