Browsing by Author "Surendran, Sneha"

Now showing 1 - 6 of 6
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    Acetylation of Replication Protein A (RPA) Improves its DNA Binding Property
    (Office of the Vice Chancellor for Research, 2016-04-08) Surendran, Sneha; Ononye, Onyeka; Balakrishnan, Lata
    Genome maintenance is critical for cellular survival and growth. Replication Protein A (RPA), a single-strand DNA (ssDNA) binding protein, is vital for various aspects of genome maintenance such as replication, recombination, repair and checkpoint activation. RPA binding to ssDNA protects it from degradation by cellular nucleases, prevents secondary structure formation and from illegitimate recombination. Within the cell, RPA is subject to many post-translational modifications including phosphorylation, SUMOylation and ribosylation. These modifications regulate the activity of RPA with DNA and other binding partners. RPA has been reported to be also modified by acetylation. We found that human RPA (hRPA) can be in vitro acetylated by p300, an acetyl transferase (AT). To study the effect of this modification on its ssDNA binding function, we made use of electro-mobility gel shift assay (EMSA) and bio-layer interferometry (BLI) technology. Using various length oligos, we tested the binding property of unmodified and acetylated RPA. Our results showed that acetylation of RPA increased its binding affinity compared to unmodified RPA. Interestingly, the acetylated form was also able to bind more stably to shorter length oligos compared to the unmodified form. This suggests that the acetylation of RPA improves its ssDNA binding function. This alteration in its enzymatic activity would have significant implications in maintenance of genome fidelity since improved DNA binding function of RPA will protect the genome from both endogenous and exogenous stresses. Additionally, using mass spectrometry analysis we have identified the lysine residues that get modified by the acetyl group both in vitro and in vivo. We are currently studying the factors that trigger this post-translational modification in the cell.
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    Gene targets of mouse miR-709: regulation of distinct pools
    (Nature, 2016-01) Surendran, Sneha; Jideonwo, Victoria N.; Merchun, Chris; Ahn, Miwon; Murray, John; Ryan, Jennifer; Dunn, Kenneth W.; Kota, Janaiah; Morral, Núria; Department of Medical & Molecular Genetics, IU School of Medicine
    MicroRNA (miRNA) are short non-coding RNA molecules that regulate multiple cellular processes, including development, cell differentiation, proliferation and death. Nevertheless, little is known on whether miRNA control the same gene networks in different tissues. miR-709 is an abundant miRNA expressed ubiquitously. Through transcriptome analysis, we have identified targets of miR-709 in hepatocytes. miR-709 represses genes implicated in cytoskeleton organization, extracellular matrix attachment, and fatty acid metabolism. Remarkably, none of the previously identified targets in non-hepatic tissues are silenced by miR-709 in hepatocytes, even though several of these genes are abundantly expressed in liver. In addition, miR-709 is upregulated in hepatocellular carcinoma, suggesting it participates in the genetic reprogramming that takes place during cell division, when cytoskeleton remodeling requires substantial changes in gene expression. In summary, the present study shows that miR-709 does not repress the same pool of genes in separate cell types. These results underscore the need for validating gene targets in every tissue a miRNA is expressed.
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    Impact of silencing hepatic SREBP-1 on insulin signaling
    (PLOS, 2018-05-03) Jideonwo, Victoria; Hou, Yongyong; Ahn, Miwon; Surendran, Sneha; Morral, Núria; Medical and Molecular Genetics, School of Medicine
    Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that plays a central role in regulating expression of genes of carbohydrate and fatty acid metabolism in the liver. SREBP-1 activity is essential for the control of insulin-induced anabolic processes during the fed state. In addition, SREBP-1 regulates expression of key molecules in the insulin signaling pathway, including insulin receptor substrate 2 (IRS2) and a subunit of the phosphatidylinositol 3-kinase (PI3K) complex, PIK3R3, suggesting that feedback mechanisms exist between SREBP-1 and this pathway. Nevertheless, the overall contribution of SREBP-1 activity to maintain insulin signal transduction is unknown. Furthermore, Akt is a known activator of mTORC1, a sensor of energy availability that plays a fundamental role in metabolism, cellular growth and survival. We have silenced SREBP-1 and explored the impact on insulin signaling and mTOR in mice under fed, fasted and refed conditions. No alterations in circulating levels of insulin were observed. The studies revealed that depletion of SREBP-1 had no impact on IRS1Y612, AktS473, and downstream effectors GSK3αS21 and FoxO1S256 during the fed state. Nevertheless, reduced levels of these molecules were observed under fasting conditions. These effects were not associated with changes in phosphorylation of mTOR. Overall, our data indicate that the contribution of SREBP-1 to maintain insulin signal transduction in liver is modest.
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    Lack of liver glycogen causes hepatic insulin resistance and steatosis in mice
    (American Society for Biochemistry and Molecular Biology, 2017-06-23) Irimia, Jose M.; Meyer, Catalina M.; Segvich, Dyann M.; Surendran, Sneha; DePaoli-Roach, Anna A.; Morral, Nuria; Roach, Peter J.; Biochemistry and Molecular Biology, School of Medicine
    Disruption of the Gys2 gene encoding the liver isoform of glycogen synthase generates a mouse strain (LGSKO) that almost completely lacks hepatic glycogen, has impaired glucose disposal, and is pre-disposed to entering the fasted state. This study investigated how the lack of liver glycogen increases fat accumulation and the development of liver insulin resistance. Insulin signaling in LGSKO mice was reduced in liver, but not muscle, suggesting an organ-specific defect. Phosphorylation of components of the hepatic insulin-signaling pathway, namely IRS1, Akt, and GSK3, was decreased in LGSKO mice. Moreover, insulin stimulation of their phosphorylation was significantly suppressed, both temporally and in an insulin dose response. Phosphorylation of the insulin-regulated transcription factor FoxO1 was somewhat reduced and insulin treatment did not elicit normal translocation of FoxO1 out of the nucleus. Fat overaccumulated in LGSKO livers, showing an aberrant distribution in the acinus, an increase not explained by a reduction in hepatic triglyceride export. Rather, when administered orally to fasted mice, glucose was directed toward hepatic lipogenesis as judged by the activity, protein levels, and expression of several fatty acid synthesis genes, namely, acetyl-CoA carboxylase, fatty acid synthase, SREBP1c, chREBP, glucokinase, and pyruvate kinase. Furthermore, using cultured primary hepatocytes, we found that lipogenesis was increased by 40% in LGSKO cells compared with controls. Of note, the hepatic insulin resistance was not associated with increased levels of pro-inflammatory markers. Our results suggest that loss of liver glycogen synthesis diverts glucose toward fat synthesis, correlating with impaired hepatic insulin signaling and glucose disposal.
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    Role of microRNA-709 in murine liver
    (2014) Surendran, Sneha; Morral, Nuria; Herbert, Brittney-Shea; Ivan, Mircea; Considine, Robert V.; Carlesso, Nadia
    MicroRNAs are small RNA molecules that regulate expression of genes involved in development, cell differentiation, proliferation and death. It has been estimated that in eukaryotes, approximately 0.5 to 1% of predicted genes encode a microRNA, which in humans, regulate at least 30% of genes at an average of 200 genes per miRNA. Some microRNAs are tissue-specific, while others are ubiquitously expressed. In liver, a few microRNAs have been identified that regulate specialized functions. The best known is miR-122, the most abundant liver-specific miRNA, which regulates cholesterol biosynthesis and other genes of fatty acid metabolism; it also regulates the cell cycle through inhibition of cyclin G1. To discover other miRNAs with relevant function in liver, we characterized miRNA profiles in normal tissue and identified miR-709. Our data indicates this is a highly abundant hepatic miRNA and is dysregulated in an animal model of type 2 diabetes. To understand its biological role, miR-709 gene targets were identified by analyzing the transcriptome of primary hepatocytes transfected with a miR-709 mimic. The genes identified fell within four main categories: cytoskeleton binding, extracellular matrix attachment, endosomal recycling and fatty acid metabolism. Thus, similar to miR-122, miR-709 downregulates genes from multiple pathways. This would be predicted, given the abundance of the miRNA and the fact that the estimated number of genes targeted by a miRNA is in the hundreds. In the case of miR-709, these suggested a coordinated response during cell proliferation, when cytoskeleton remodeling requires substantial changes in gene expression. Consistently, miR-709 was found significantly upregulated in an animal model of hepatocellular carcinoma. Likewise, in a mouse model of liver regeneration, mature miR-709 was increased. To study the consequences of depleting miR-709 in quiescent and proliferating cells, primary hepatocytes and hepatoma cells were cultured with antagomiRs (anti-miRs). The presence of anti-miR-709 caused cell death in proliferating cells. Quiescent primary hepatocytes responded by upregulating miR-709 and its host gene, Rfx1. These studies show that miR-709 targets genes relevant to cystokeleton structural genes. Thus, miR-709 and Rfx1 may be needed to facilitate cytoskeleton reorganization, a process that occurs after liver injury and repopulation, or during tumorigenesis.
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    Sterol regulatory element-binding protein-1 (SREBP-1) is required to regulate glycogen synthesis and gluconeogenic gene expression in mouse liver
    (ASBMB, 2014-01-07) Ruiz, Rafaela; Jideonwo, Victoria; Ahn, Miwon; Surendran, Sneha; Tagliabracci, Vincent S.; Hou, Yongyong; Gamble, Aisha; Kerner, Janos; Irimia-Dominguez, Jose M.; Puchowicz, Michelle A.; Hoppel, Charles; Roach, Peter; Morral, Nuria; Department of Medical & Molecular Genetics, IU School of Medicine
    Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor that regulates genes in the de novo lipogenesis and glycolysis pathways. The levels of SREBP-1 are significantly elevated in obese patients and in animal models of obesity and type 2 diabetes, and a vast number of studies have implicated this transcription factor as a contributor to hepatic lipid accumulation and insulin resistance. However, its role in regulating carbohydrate metabolism is poorly understood. Here we have addressed whether SREBP-1 is needed for regulating glucose homeostasis. Using RNAi and a new generation of adenoviral vector, we have silenced hepatic SREBP-1 in normal and obese mice. In normal animals, SREBP-1 deficiency increased Pck1 and reduced glycogen deposition during fed conditions, providing evidence that SREBP-1 is necessary to regulate carbohydrate metabolism during the fed state. Knocking SREBP-1 down in db/db mice resulted in a significant reduction in triglyceride accumulation, as anticipated. However, mice remained hyperglycemic, which was associated with up-regulation of gluconeogenesis gene expression as well as decreased glycolysis and glycogen synthesis gene expression. Furthermore, glycogen synthase activity and glycogen accumulation were significantly reduced. In conclusion, silencing both isoforms of SREBP-1 leads to significant changes in carbohydrate metabolism and does not improve insulin resistance despite reducing steatosis in an animal model of obesity and type 2 diabetes.