Papers by Sailesh Surapureddi
Biochemical archives
ABSTRACT
Free Radical Biology and Medicine, 2009
8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most ... more 8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the beta-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human alpha-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial alpha-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.
American Journal of Respiratory Cell and Molecular Biology, 2006
Asbestos causes pulmonary toxicity in part by generating reactive oxygen species that cause DNA d... more Asbestos causes pulmonary toxicity in part by generating reactive oxygen species that cause DNA damage. We previously showed that the mitochondria-regulated (intrinsic) death pathway mediates alveolar epithelial cell (AEC) DNA damage and apoptosis. Because p53 regulates the DNA damage response in part by inducing intrinsic cell death, we determined whether p53-dependent transcriptional activity mediates asbestos-induced AEC mitochondrial dysfunction and apoptosis. We show that inhibitors of p53-dependent transcriptional activation (pifithrin and type 16-E6 protein) block asbestos-induced AEC mitochondrial membrane potential change (⌬⌿m), caspase 9 activation, and apoptosis. We demonstrate that asbestos activates p53 promoter activity, mRNA levels, protein expression, and Bax and p53 mitochondrial translocation. Further, pifithrin, E6, phytic acid, or 0 -A549 cells (cells incapable of mitochondrial reactive oxygen species production) block asbestosinduced p53 activation. Finally, we show that asbestos augments p53 expression in cells at the bronchoalveolar duct junctions of rat lungs and that phytic acid prevents this. These data suggest that p53-dependent transcription pathways mediate asbestos-induced AEC mitochondria-regulated apoptosis. This suggests an important interactive effect between p53 and the mitochondria in the pathogenesis of asbestos-induced pulmonary toxicity that may have broader implications for our understanding of pulmonary fibrosis and lung cancer.
Journal of Investigative Medicine, 2007
ABSTRACT
Hepatocyte nuclear factor 4α (HNF4α) is a liver-enriched receptor which regulates drug metabolism... more Hepatocyte nuclear factor 4α (HNF4α) is a liver-enriched receptor which regulates drug metabolism of drugs, glucose and lipids. HNF4α sites are important for crosstalk between HNF4α and the xenobiotic sensing receptors CAR (constitutive active receptor) and PXR (pregnane X receptor). To investigate the mechanism of this cross-talk, we have used adenovirally expressed short hairpin RNA (shRNA) constructs to silence expression of HNF4α and its coactivators. Activation of CYP2C9 promoter constructs by rifampicin in primary human metabolites is specifically repressed by mutation of HNF4 α sites or by transfection with shRNA constructs for HNF4α. Yeast two hybrid screens revealed a new HNF4α interacting protein, Med25, a protein sometimes associated with the Mediator complex which is required for transcription of most genes. GST-pull downs and mammalian two hybrid assays show that Med25 interacts with HNF4α through an LXXLL motif. CAR and HNF4α synergistically activate CYP2C9 and CYP3A4 ...
Febs Letters, 2000
We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsoma... more We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein-protein interaction may contribute to the regulation of LTC4 production.
Proceedings of the National Academy of Sciences, 2002
Peroxisome proliferator-activated receptor ␣ (PPAR␣) plays a central role in the cell-specific pl... more Peroxisome proliferator-activated receptor ␣ (PPAR␣) plays a central role in the cell-specific pleiotropic responses induced by structurally diverse synthetic chemicals designated as peroxisome proliferators. Transcriptional regulation by liganded nuclear receptors involves the participation of cofactors that form multiprotein complexes to achieve cell-and gene-specific transcription. Here we report the identification of such a transcriptionally active PPAR␣interacting cofactor (PRIC) complex from rat liver nuclear extracts that interacts with full-length PPAR␣ in the presence of ciprofibrate, a synthetic ligand, and leukotriene B 4, a natural ligand. The liganded PPAR␣-PRIC complex enhanced transcription from a peroxisomal enoyl-CoA hydratase͞L-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme gene promoter template that contains peroxisome proliferator response elements. Rat liver PRIC complex comprises some 25 polypeptides, and their identities were established by mass spectrometry and limited sequence analysis. Eighteen of these peptides contain one or more LXXLL motifs necessary for interacting with nuclear receptors. PRIC complex includes known coactivators or coactivator-binding proteins (CBP, SRC-1, PBP, PRIP, PIMT, TRAP100, SUR-2, and PGC-1), other proteins that have not previously been described in association with transcription complexes (CHD5, TOG, and MORF), and a few novel polypeptides designated PRIC300, -285, -215, -177, and -145. We describe the cDNA for PRIC285, which contains five LXXLL motifs. It interacts with PPAR␣ and acts as a coactivator by moderately stimulating PPAR␣-mediated transcription in transfected cells. We conclude that liganded PPAR␣ recruits a distinctive multiprotein complex from rat liver nuclear extracts. The composition of this complex may provide insight into the basis of tissue and species sensitivity to peroxisome proliferators.
The Mediator complex is vital for the transcriptional regulation of eukaryotic genes. Mediator b... more The Mediator complex is vital for the transcriptional regulation of eukaryotic genes. Mediator binds to nuclear receptors at target response elements and recruits chromatin modifying enzymes and RNA pol II. Here, we examine the involvement of Mediator subunit MED25 in the epigenetic regulation of human cytochrome P450 2C9 (CYP2C9)2. MED25 is recruited to the CYP2C9 promoter through association with liver-enriched HNF4α, and we show that MED25 influences the H3K27 status of the HNF4α binding region. This region was enriched for the activating marker H3K27ac and histone acetyltransferase CREBBP after MED25 overexpression, but was trimethylated when MED25 expression was silenced. The epigenetic regulator Polycomb repressive complex (PRC2), which represses expression by methylating H3K27, plays an important role in target gene regulation. Silencing MED25 correlated with increased association of PRC2 with not only the promoter region chromatin but HNF4α itself. We confirmed the involvement of MED25 for fully functional preinitiation complex recruitment and transcriptional output in vitro. Formaldehyde-assisted isolation of regulatory elements (FAIRE) revealed chromatin conformation changes that are reliant on MED25, indicating MED25 induced a permissive chromatin state that reflected increases in CYP2C9 mRNA. For the first time, we show evidence that a functionally relevant human gene is transcriptionally regulated by HNF4α via MED25 and PRC2. CYP2C9 is important for the metabolism of many exogenous chemicals including pharmaceutical drugs as well as endogenous substrates. Thus, MED25 is important for regulating the epigenetic landscape resulting in transcriptional activation of a highly inducible gene, CYP2C9.
Molecular and cellular biology, 2011
Hepatocyte nuclear factor 4␣ (HNF4␣) controls the expression of many critical metabolic pathways,... more Hepatocyte nuclear factor 4␣ (HNF4␣) controls the expression of many critical metabolic pathways, and the Mediator complex occupies a central role in recruiting RNA polymerase II (Pol II) to these gene promoters. An impaired transcriptional HNF4␣ network in human liver is responsible for many pathological conditions, such as altered drug metabolism, fatty liver, and diabetes. Here, we report that Med25, an associated member of the Mediator complex, is required for the association of HNF4␣ with Mediator, its several cofactors, and RNA Pol II. Further, increases and decreases in endogenous Med25 levels are reflected in the composition of the transcriptional complex, Pol II recruitment, and the expression of HNF4␣-bound target genes. A novel feature of Med25 is that it imparts "selectivity." Med25 affects only a significant subset of HNF4␣ target genes that selectively regulate drug and lipid metabolism. These results define a role for Med25 and the Mediator complex in the regulation of xenobiotic metabolism and lipid homeostasis.
Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drug... more Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tert-butylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions -2201 and -1930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQ-induced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes.
Molecular pharmacology, 2012
The CYP2C genes are extensively regulated at the transcriptional stage. The present study shows f... more The CYP2C genes are extensively regulated at the transcriptional stage. The present study shows for the first time that CYP2Cs are also regulated post-transcriptionally by microRNAs (miRNAs). By using online search engines, we found potential miRNA response elements (MREs) in the 3Ј-untranslated region (3Ј-UTR) of the CYP2C mRNAs. Among these were a MRE for the miRNAs miR-103 and miR-107 in the 3Ј-UTR of human CYP2C8. CYP2C8 protein levels (measured through immunoblot analyses) did not correlate with CYP2C8 mRNA levels (measured through quantitative polymerase chain reaction analyses) in human liver samples. The translation efficiency (protein/mRNA ratio) for CYP2C8 was inversely correlated with the expression of miR-103 and miR-107. When three copies of the putative MRE from CYP2C8 were inserted downstream from a luciferase expression reporter, transfection with precursors for miR-103 or miR-107 decreased lu-ciferase activity in primary hepatocytes, whereas transfection with antisense oligonucleotides (AsOs) for miR-103/miR-107 increased luciferase activity. As expected, there was no effect of the precursors or AsOs when three copies of the putative MRE were inserted in the reverse orientation. When precursors for miR-103/miR-107 were transfected into primary human hepatocytes, CYP2C8 protein levels were decreased, whereas AsOs increased CYP2C8 protein levels. Neither precursors nor AsOs affected CYP2C8 mRNA levels, which indicated that the effect was posttranscriptional. Putative MRE motifs were also found in the 3Ј-UTRs of CYP2C9 and CYP2C19, which suggested that the same miRNAs could regulate translation of other members of the CYP2C family, although to a lesser degree than CYP2C8. These results clearly show that CYP2Cs are regulated post-transcriptionally by miR-103 and miR-107.
Cell metabolism, Jan 1, 2009
Hepatic metabolic derangements are key components in the development of fatty liver, insulin resi... more Hepatic metabolic derangements are key components in the development of fatty liver, insulin resistance, and atherosclerosis. SIRT1, a NAD + -dependent protein deacetylase, is an important regulator of energy homeostasis in response to nutrient availability. Here we demonstrate that hepatic SIRT1 regulates lipid homeostasis by positively regulating PPARα, a nuclear receptor that mediates the adaptive response to fasting and starvation. Hepatocyte-specific deletion of SIRT1 impairs PPARα signaling and decreases fatty acid β-oxidation, whereas overexpression of SIRT1 induces the expression of PPARα targets. SIRT1 interacts with PPARα and is required to activate PPARα co-activator PGC-1α. When challenged with a high-fat diet, liver-specific SIRT1 knockout mice develop hepatic steatosis, hepatic inflammation, and endoplasmic reticulum stress. Taken together, our data indicate that SIRT1 plays a vital role in the regulation of hepatic lipid homeostasis, and that pharmacological activation of SIRT1 may be important for the prevention of obesity-associated metabolic diseases.
Proceedings of the …, Jan 1, 2002
Peroxisome proliferator-activated receptor ␣ (PPAR␣) plays a central role in the cell-specific pl... more Peroxisome proliferator-activated receptor ␣ (PPAR␣) plays a central role in the cell-specific pleiotropic responses induced by structurally diverse synthetic chemicals designated as peroxisome proliferators. Transcriptional regulation by liganded nuclear receptors involves the participation of cofactors that form multiprotein complexes to achieve cell-and gene-specific transcription. Here we report the identification of such a transcriptionally active PPAR␣interacting cofactor (PRIC) complex from rat liver nuclear extracts that interacts with full-length PPAR␣ in the presence of ciprofibrate, a synthetic ligand, and leukotriene B 4, a natural ligand. The liganded PPAR␣-PRIC complex enhanced transcription from a peroxisomal enoyl-CoA hydratase͞L-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme gene promoter template that contains peroxisome proliferator response elements. Rat liver PRIC complex comprises some 25 polypeptides, and their identities were established by mass spectrometry and limited sequence analysis. Eighteen of these peptides contain one or more LXXLL motifs necessary for interacting with nuclear receptors. PRIC complex includes known coactivators or coactivator-binding proteins (CBP, SRC-1, PBP, PRIP, PIMT, TRAP100, SUR-2, and PGC-1), other proteins that have not previously been described in association with transcription complexes (CHD5, TOG, and MORF), and a few novel polypeptides designated PRIC300, -285, -215, -177, and -145. We describe the cDNA for PRIC285, which contains five LXXLL motifs. It interacts with PPAR␣ and acts as a coactivator by moderately stimulating PPAR␣-mediated transcription in transfected cells. We conclude that liganded PPAR␣ recruits a distinctive multiprotein complex from rat liver nuclear extracts. The composition of this complex may provide insight into the basis of tissue and species sensitivity to peroxisome proliferators.
American journal of physiology. Gastrointestinal and liver physiology, 2011
Drug metabolism and disposition: the biological fate of chemicals, 2010
CYP2C enzymes are expressed constitutively and comprise ϳ20% of the total cytochrome P450 in huma... more CYP2C enzymes are expressed constitutively and comprise ϳ20% of the total cytochrome P450 in human liver. However, the factors influencing the transcriptional regulation of the CYP2C subfamily have only been addressed recently. In the present study, we used primary cultures of human hepatocytes to investigate the role of HNF4␣ in the pregnane X receptor (PXR)/rifampicin-mediated upregulation of CYP2C8, CYP2C9, and CYP2C19 gene expression. We first identified new proximal cis-acting HNF4␣ sites in the proximal CYP2C8 promoter [at ؊181 base pairs (bp) from the translation start site] and the CYP2C9 promoter (at ؊211 bp). Both sites bound HNF4␣ in gel shift assays. Thus, these and recent studies identified a total of three HNF4␣ sites in the CYP2C9 promoter and two in the CYP2C8 promoter. Mutational studies showed that the HNF4␣ sites are needed for up-regulation of the CYP2C8 and CYP2C9 promoters by rifampicin. Furthermore, silencing of HNF4␣ abolished transactivation of the CYP2C8 and CYP2C9 promoters by rifampicin. Constitutive promoter activity was also decreased. Quantitative polymerase chain reaction analysis demonstrated that silencing HNF4␣ reduced the constitutive expression of CYP2C8 (53%), CYP2C9 (55%), and CYP2C19 (43%) mRNAs and significantly decreased the magnitude of the rifampicin-mediated induction of CYP2C8 (6.6-versus 2.7-fold), CYP2C9 (3-versus 1.5-fold), and CYP2C19 . These results provide clear evidence that HNF4␣ contributes to the constitutive expression of the human CYP2C genes and is also important for upregulation by the PXR agonist rifampicin.
Molecular pharmacology, 2008
Pharmacological research : the official journal of the Italian Pharmacological Society, 2011
CYP2Cs and CYP3A4 sub family of enzymes of the Cytochrome P-450 super family metabolize clinicall... more CYP2Cs and CYP3A4 sub family of enzymes of the Cytochrome P-450 super family metabolize clinically prescribed therapeutics. Constitutive and induced expressions of these enzymes are under the control of HNF4α and rifampicin activated PXR. In the present study, we show a mechanism for ligand dependent synergistic cross talk between PXR and HNF4α. Two-hybrid screening identified NCOA6 as a HNF4α interacting protein. NCOA6 was also found to interact with PXR through the first LXXLL motif in GST pull down and mammalian two hybrid assays. NCOA6 enhances the synergistic activation of CYP2C9 and CYP3A4 promoter activity by PXR and HNF4α in the presence of rifampicin. However silencing NCOA6 abrogated the synergistic activation and induction of CYP2C9 by PXR-HNF4α but not of CYP3A4. ChIP analysis revealed that NCOA6 could bridge HNF4α and PXR binding sites of the CYP2C9 promoter. Our results indicate that NCOA6 is responsible for the synergistic activation of CYP2C9 by HNF4α and PXR and NCOA6 differentially regulates CYP2C9 and CYP3A4 gene expression though both the genes are regulated by the same nuclear receptors.
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Papers by Sailesh Surapureddi