Academic literature on the topic 'Hyperacetylation'

Diabetes mellitus (DM) is considered to be associated with an increased risk of colorectal cancer. Recent studies have also revealed that tubulin hyperacetylation is caused by a diabetic status and we have reported previously that, under microtubule hyperacetylation, a microtubule severing protein, katanin-like (KL) 1, is upregulated and contributes to tumorigenesis. To further explore this phenomenon, we tested the effects of the ketone bodies, acetoacetate and β-hydroxybutyrate, in colon and fibroblast cells. Both induced microtubule hyperacetylation that responded differently to a histone deacetylase 3 knockdown. These two ketone bodies also generated intracellular reactive oxygen species (ROS) and hyperacetylation was commonly inhibited by ROS inhibitors. In a human fibroblast-based microtubule sensitivity test, only the KL1 human katanin family member showed activation by both ketone bodies. In primary cultured colon epithelial cells, these ketone bodies reduced the tau protein level and induced KL1- and α-tubulin acetyltransferase 1 (ATAT1)-dependent micronucleation. Resveratrol, known for its tumor preventive and tubulin deacetylation effects, inhibited this micronucleation. Our current data thus suggest that the microtubule hyperacetylation induced by ketone bodies may be a causal factor linking DM to colorectal carcinogenesis and may also represent an adverse effect of them that needs to be controlled if they are used as therapeutics.

Abstract Histone deacetylase 6 (HDAC6) is a heat shock protein 90 (hsp90) deacetylase. Treatment with pan-HDAC inhibitors or depletion of HDAC6 by siRNA induces hyperacetylation and inhibits ATP binding and chaperone function of hsp90. Treatment with 17-allylamino-demothoxy geldanamycin (17-AAG) also inhibits ATP binding and chaperone function of hsp90, resulting in polyubiquitylation and proteasomal degradation of hsp90 client proteins. In this study, we determined the effect of hsp90 hyperacetylation on the anti-hsp90 and antileukemia activity of 17-AAG. Hyperacetylation of hsp90 increased its binding to 17-AAG, as well as enhanced 17-AAG–mediated attenuation of ATP and the cochaperone p23 binding to hsp90. Notably, treatment with 17-AAG alone also reduced HDAC6 binding to hsp90 and induced hyperacetylation of hsp90. This promoted the proteasomal degradation of HDAC6. Cotreatment with 17-AAG and siRNA to HDAC6 induced more inhibition of hsp90 chaperone function and depletion of BCR-ABL and c-Raf than treatment with either agent alone. In addition, cotreatment with 17-AAG and tubacin augmented the loss of survival of K562 cells and viability of primary acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) samples. These findings demonstrate that HDAC6 is an hsp90 client protein and hyperacetylation of hsp90 augments the anti-hsp90 and antileukemia effects of 17-AAG.

Genomic characterization of various euchromatic regions in higher eukaryotes has revealed that domain-wide hyperacetylation (over several kb) occurs at a range of loci, including individual genes, gene family clusters, compound clusters, and more general clusters of unrelated genes. Patterns of long-range histone hyperacetylation are strictly conserved within each unique cellular system studied and they reflect biological variability in gene regulation. Domain-wide histone acetylation consists generally of nonuniform peaks of enriched hyperacetylation of specific core histones, histone isoforms, and (or) histone variants against a backdrop of nonspecific acetylation across the domain in question. Here we review the characteristics of long-range histone acetylation in some higher eukaryotes and draw special attention to recent literature on the multiple effects that histone hyperacetylation has on chromatin’s structural integrity and how they affect transcription. These include the thermal, ionic, cumulative, and isoform-specific (H4 K16) consequences of acetylation that result in a more dynamic core complex and chromatin fiber.

Recently a model for eukaryotic transcriptional activation has been proposed in which histone hyperacetylation causes release of nucleosomal supercoils, and this unconstrained tension in turn stimulates transcription (V. G. Norton, B. S. Imai, P. Yau, and E. M. Bradbury, Cell 57:449-457, 1989; V. G. Norton, K. W. Marvin, P. Yau, and E. M. Bradbury, J. Biol. Chem. 265:19848-19852, 1990). These studies analyzed the effect of histone hyperacetylation on the change in topological linking number which occurs during nucleosome assembly in vitro. We have tested this model by determining the effect of histone hyperacetylation on the linking number change which occurs during assembly in vivo. We find that butyrate treatment of cells infected with simian virus 40 results in hyperacetylation of the histones of the extracted viral minichromosome as expected. However, the change in constrained supercoils of the minichromosome DNA is minimal, a result which is inconsistent with the proposed model. These results indicate that the proposed mechanism of transcriptional activation is unlikely to take place in the cell.

Recently a model for eukaryotic transcriptional activation has been proposed in which histone hyperacetylation causes release of nucleosomal supercoils, and this unconstrained tension in turn stimulates transcription (V. G. Norton, B. S. Imai, P. Yau, and E. M. Bradbury, Cell 57:449-457, 1989; V. G. Norton, K. W. Marvin, P. Yau, and E. M. Bradbury, J. Biol. Chem. 265:19848-19852, 1990). These studies analyzed the effect of histone hyperacetylation on the change in topological linking number which occurs during nucleosome assembly in vitro. We have tested this model by determining the effect of histone hyperacetylation on the linking number change which occurs during assembly in vivo. We find that butyrate treatment of cells infected with simian virus 40 results in hyperacetylation of the histones of the extracted viral minichromosome as expected. However, the change in constrained supercoils of the minichromosome DNA is minimal, a result which is inconsistent with the proposed model. These results indicate that the proposed mechanism of transcriptional activation is unlikely to take place in the cell.

Sepsis-induced liver dysfunction (SILD) is a common event and is strongly associated with mortality. Establishing a causative link between protein post-translational modification and diseases is challenging. We studied the relationship among lysine acetylation (Kac), sirtuin (SIRTs), and the factors involved in SILD, which was induced in LPS-stimulated HepG2 cells. Protein hyperacetylation was observed according to SIRTs reduction after LPS treatment for 24 h. We identified 1449 Kac sites based on comparative acetylome analysis and quantified 1086 Kac sites on 410 proteins for acetylation. Interestingly, the upregulated Kac proteins are enriched in glycolysis/gluconeogenesis pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) category. Among the proteins in the glycolysis pathway, hyperacetylation, a key regulator of lactate level in sepsis, was observed at three pyruvate kinase M2 (PKM2) sites. Hyperacetylation of PKM2 induced an increase in its activity, consequently increasing the lactate concentration. In conclusion, this study is the first to conduct global profiling of Kac, suggesting that the Kac mechanism of PKM2 in glycolysis is associated with sepsis. Moreover, it helps to further understand the systematic information regarding hyperacetylation during the sepsis process.

Enhanced microtubule acetylation has been identified as a negative prognostic indicator in breast cancer. We reported previously that primary cultured human mammary epithelial cells manifest breast cancer-related aneuploidization via the activation of severing protein katanin-like (KL)1 when tau is deficient. To address in this current study whether microtubule hyperacetylation is involved in breast carcinogenesis through mitosis, the effects of tubacin on human mammary epithelial cells were tested using immunofluorescence techniques. Tau-knockdown cells showed enhancement of KL1-dependent events, chromosome-bridging and micronucleation in response to tubacin. These enhancements were suppressed by further expression of an acetylation-deficient tubulin mutant. Consistently, using a rat fibroblast-based microtubule sensitivity test, it was confirmed that KL1 also shows enhanced activity in response to microtubule hyperacetylation as well as katanin. It was further observed in rat fibroblasts that exogenously expressed KL1 results in more micronucleation under microtubule hyperacetylation conditions. These data suggest that microtubule acetylation upregulates KL1 and induces more aneuploidy if tau is deficient. It is thus plausible that microtubule hyperacetylation promotes tumor progression by enhancing microtubule sensitivity to KL1, thereby disrupting spindle microtubules and this process could be reversed by the microtubule-binding and microtubule protective octapeptide NAPVSIPQ (NAP) which recruits tau to the microtubules.

The role of histone hyperacetylation in regard to growth, differentiation, and apoptosis in colon cancer cells was assessed in an in vitro model system. HT-29 cells were grown in ±10% fetal bovine serum with either 5 mM sodium butyrate or 0.3 μM trichostatin A [single dose (T) or 3 doses 8 h apart (TR)] for 24 h. Serum-starved HT-29 cells were further treated with epidermal growth factor or insulin-like growth factor I for an additional 24 h. Apoptosis was quantified with propidium iodide and characterized by electron microscopy. Northern blot analyses were performed with cDNA probes specific for intestinal alkaline phosphatase, Na-K-2Cl cotransporter, the cell cycle inhibitor p21, and the actin control. Flow cytometric analysis revealed a time-dependent growth suppression along with early induction of p21 mRNA in the butyrate, T, and TR groups. Histone hyperacetylation, assessed by acid-urea-triton gel electrophoresis, was transient in the T group but persisted for up to 24 h in the butyrate and TR groups. Induction of apoptosis, growth factor unresponsiveness, and differentiation occurred in the butyrate- and TR-treated cells but not those treated with a single dose of trichostatin A. Thus transient hyperacetylation of histones is sufficient to induce p21 expression and produce cellular growth arrest, but prolonged histone hyperacetylation is required for induction of the programs of differentiation, apoptosis, and growth factor unresponsiveness.

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This “mito-aggresome” is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

Bnip3 is a BH3-only member of the Bcl-2 family of apoptotic proteins. Our laboratory has previously shown that Bnip3 induces a unique pathway of cardiac myocyte cell death, characterized by mitochondrial dysfunction, cytochrome c release and DNA fragmentation. Bnip3 is induced by hypoxia and the death pathway is activated by concurrent acidosis. We have shown that hypoxia-acidosis creates an environment that is permissive to calpain but not caspase activation and is characterized by enhanced DNase(s) activity as evidenced by genomic DNA fragmentation. This dissertation describes the nuclear consequences of Bnip3 activation by hypoxia-acidosis. Chapter 3 presents my evidence that hypoxia with progressive acidosis in cardiac myocytes results in a biphasic activation of DNases. In phase 1, [pH]o 6.9-6.7, apoptosis-inducing factor (AIF) is released from the mitochondria and translocates to the nucleus. AIF release coincided with the loss of mitochondrial membrane potential and with the release of cytochrome c from the mitochondria. In Phase II, [pH]o 6.3-6.0, DNase II translocates from the cytoplasm to the nuclear compartment. Nuclear localization of DNase II was associated with the collapse of endosomal pH gradients, indicated by diffuse Lysotracker Red staining and with single strand DNA nicks. Both phases of DNase release were independent of Bnip3, the mPTP and calpains. Neither phase involved activation of caspase-dependent DNases. Chapter 4 describes a unique role for Bnip3 in the modulation of histone acetylation. I found that hypoxia with acidosis in cardiac myocytes but not hypoxia alone stimulated a global increase in the acetylation of histones H3 and H4. Acetylation was initiated at [pH]o ~ 6.8 and increased as the pH declined. Histone hyperacetylation was associated with an increase in histone acetyltransferase (HAT) activity but no change in deacetylase (HDAC) activity. Knockdown of Bnip3 protein expression with siRNA dramatically reduced both histone H3 and H4 acetylation levels and HAT activity indicating an essential role for Bnip3 in this process. Components of the hypoxia-acidosis death pathway including the mPTP and calpains are not required for Bnip3-mediated histone hyperacetylation. These results reveal a novel role for Bnip3 in regulating HAT activity and histone acetylation which may lead to altered cardiac gene expression.

Orientador: Joaquim Mansano Garcia
Banca: Lígia Veiga Pereira
Banca: Irina Kerkis
Resumo: O estudo dos processos de diferenciação em células tronco embrionárias (CTE) representa uma importante ferramenta para o entendimento das vias moleculares que os regem, apresentando grande aplicação tanto na ciência básica quanto na engenharia de tecidos e medicina regenerativa. Pouco é conhecido sobre as marcas epigenéticas existentes na cromatina destas células, e de que forma a regulação da expressão gênica ocorre no momento da diferenciação. O presente trabalho teve como objetivo o estudo dos efeitos da hiperacetilação das histonas causada pela droga tricostatina A (TSA), uma inibidora das enzimas histona desacetilases, sobre a diferenciação destas células em estádios iniciais e avançados. Para tanto, a hiperacetilação induzida pela droga foi estimada por reações de imunocitoquímica para AcLys9H3. Os efeitos anti-proliferativos da TSA foram mensurados pelo teste de TUNEL e contagem de células. Ainda, foram conduzidos experimentos de diferenciação in vitro de CTE e análise da expressão de proteínas características de linhagens celulares diferenciadas por reações de imunocitoquímica (Oct3/4, nestina, âIII tubulina, desmina e troponina I), em cultivos tratados com TSA em diferentes concentrações e em diferentes momentos. Desta forma, foi estimada a população de tipos celulares oriundos dos folhetos embrionários ectodérmico e mesodérmico, como neurônios, e células musculares, quando foi promovida a hiperacetilação das histonas nas CTE, em diferentes momentos da diferenciação celular in vitro. A TSA induziu apoptose em níveis superiores aos do grupo controle, e retardou/inibiu a divisão celular. Promoveu hiperacetilação dose-dependente nos períodos estudados, e estimulou a diferenciação de precursores mesodérmicos (50nM d5) e ectodérmicos (15nMd0-5 e 50nMd5), cardiomiócitos (50nMd5 e 100nMd13) e neurônios (15nMd0-5, 50nMd5, 100nMd5, 100nMd13).
Abstract: Studies on embryonic stem cells (ESC) differentiation represents an important tool leading to understanding of its molecular pathways, with many applications both on basic research and tissue engineering / regenerative medicine. Little is known about epigenetic marks on ESC chromatin, and how gene expression occurs at differentiation time. The aim of this work was to study effects of histone hiperacetylation, induced by cell treatment with trichostatin A (TSA), an histone deacetylase inhibitor, on both initial and late differentiation. For that, drug-induced hyperacetylation was studied by AcLys9H3 immunocitochemistry. TSA anti-proliferative effects were analysed by TUNEL test and cell counts. Experiments on ESC in vitro differentiation and immunocitochemistry for specific cell types proteins (Oct3/4, nestin, âIII tubulin, desmin and troponin I) were performed, in treated and control groups, at different moments. This analysis showed specific cell types populations derived from embryonic ectodermal and mesodermal, such as neurons and cardiomyocytes, when histone hyperacetylation were induced, on both initial and late diferentiation. Our results showed that TSA induces apoptosis and inhibits cellular proliferation. Also, TSA promoted dose-dependent histone hyperacetylation at studied moments, and stimulated mesodermal (50nM d5) and ectodermal (15nMd0-5 e 50nMd5) precursors, cardiomyocytes (50nMd5 e 100nMd13) and neurons (15nMd0-5, 50nMd5, 100nMd5, 100nMd13) differentiation.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O estudo dos processos de diferenciação em células tronco embrionárias (CTE) representa uma importante ferramenta para o entendimento das vias moleculares que os regem, apresentando grande aplicação tanto na ciência básica quanto na engenharia de tecidos e medicina regenerativa. Pouco é conhecido sobre as marcas epigenéticas existentes na cromatina destas células, e de que forma a regulação da expressão gênica ocorre no momento da diferenciação. O presente trabalho teve como objetivo o estudo dos efeitos da hiperacetilação das histonas causada pela droga tricostatina A (TSA), uma inibidora das enzimas histona desacetilases, sobre a diferenciação destas células em estádios iniciais e avançados. Para tanto, a hiperacetilação induzida pela droga foi estimada por reações de imunocitoquímica para AcLys9H3. Os efeitos anti-proliferativos da TSA foram mensurados pelo teste de TUNEL e contagem de células. Ainda, foram conduzidos experimentos de diferenciação in vitro de CTE e análise da expressão de proteínas características de linhagens celulares diferenciadas por reações de imunocitoquímica (Oct3/4, nestina, âIII tubulina, desmina e troponina I), em cultivos tratados com TSA em diferentes concentrações e em diferentes momentos. Desta forma, foi estimada a população de tipos celulares oriundos dos folhetos embrionários ectodérmico e mesodérmico, como neurônios, e células musculares, quando foi promovida a hiperacetilação das histonas nas CTE, em diferentes momentos da diferenciação celular in vitro. A TSA induziu apoptose em níveis superiores aos do grupo controle, e retardou/inibiu a divisão celular. Promoveu hiperacetilação dose-dependente nos períodos estudados, e estimulou a diferenciação de precursores mesodérmicos (50nM d5) e ectodérmicos (15nMd0-5 e 50nMd5), cardiomiócitos (50nMd5 e 100nMd13) e neurônios (15nMd0-5, 50nMd5, 100nMd5, 100nMd13).
Studies on embryonic stem cells (ESC) differentiation represents an important tool leading to understanding of its molecular pathways, with many applications both on basic research and tissue engineering / regenerative medicine. Little is known about epigenetic marks on ESC chromatin, and how gene expression occurs at differentiation time. The aim of this work was to study effects of histone hiperacetylation, induced by cell treatment with trichostatin A (TSA), an histone deacetylase inhibitor, on both initial and late differentiation. For that, drug-induced hyperacetylation was studied by AcLys9H3 immunocitochemistry. TSA anti-proliferative effects were analysed by TUNEL test and cell counts. Experiments on ESC in vitro differentiation and immunocitochemistry for specific cell types proteins (Oct3/4, nestin, âIII tubulin, desmin and troponin I) were performed, in treated and control groups, at different moments. This analysis showed specific cell types populations derived from embryonic ectodermal and mesodermal, such as neurons and cardiomyocytes, when histone hyperacetylation were induced, on both initial and late diferentiation. Our results showed that TSA induces apoptosis and inhibits cellular proliferation. Also, TSA promoted dose-dependent histone hyperacetylation at studied moments, and stimulated mesodermal (50nM d5) and ectodermal (15nMd0-5 e 50nMd5) precursors, cardiomyocytes (50nMd5 e 100nMd13) and neurons (15nMd0-5, 50nMd5, 100nMd5, 100nMd13) differentiation.

博士
國防醫學院
生命科學研究所
93
Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, has been known to block cellular growth, induce apoptosis and inhibit cell migration in human cancer cell lines. In human hepatocellular carcinoma (HCC) cell lines, the effects of TSA treatment on cell migration are still unknown. In order to investigate the roles of epigenetic modulations, especially histone acetylation, on cancer genes participated in HCC metastasis, HCC cell lines were treated with different concentrations of TSA and in vitro migration activity was measured by using the transwell migration assay. The results indicated that the cell migration activity can be substantially enhanced by TSA treatment in a dose-dependent manner for HCC cell lines Huh7, PLC5 and Hep3B. By RT-PCR and Western blot analysis, the TSA induction on two migration-related gene families, matrix metalloproteinases (MMPs) and integrins, was first examined for revealing the molecular pathways in the enhancement of Hep3B cell migration. The results indicated that the enhancement of Hep3B cell migration was associated with up-regulation of several MMPs and integrin subunits including MMP-1, -2, -9, -10, -13 and integrin subunits of a4, b2, b6 in both mRNA and protein levels. Addition of specific inhibitors or neutralizing antibodies to those MMPs or integrins blocked the TSA-induced cell migration activity in the Hep3B cells. Up-regulated MMP-1, -2 and -9 were also clinicopathologically associated with HCC tumorigenesis (P<0.05) by quantitative RT-PCR analysis. In conclusion, the results suggest that TSA has a novel effect on enhancing migration activities and that histone acetylation plays an important role in HCC cell migration. With further evidence of epigenetic modulation in HCC tissues, TSA treatment or its revealed target pathways will add to the understanding of molecular mechanisms of HCC metastasis and treatment.

Over the last century the major causes of human deaths and ailments has shifted from acute infectious diseases to chronic aging related disorders like cancer and type-2 diabetes. The aging process is a universal property of most organisms, accompanied by a subtle, progressive, and often irreversible decline of physiological and reproductive functions resulting in an increased vulnerability to environmental challenge and a growing risk of disease and death. In molecular terms, it can be understood as a decline of the homeostatic mechanisms that ensure the function of cells, tissues, organs, and organ systems. However, caloric restriction (CR), i.e. reduced intake of food has been shown to ameliorate the effects of aging to some extent. One of the ways through CR slows down aging is by activating a group of enzymes known as Sirtuins, are a family of NAD+ dependant Class III histone deacetylases, which were first characterized in yeast. In S. cerevisiae, overexpression of Sir2, the founding member of the family, was shown to extend the replicative lifespan, while its depletion decreased lifespan. In mammals, there are seven orthologues of Sir2 (SIRT1-7) with a conserved catalytic domain. They differ in their enzymatic activity, subcellular localisation and therefore their binding partners and target molecules. SIRT1, 6 and 7 are localized in nucleus, SIRT3, 4 and 5 are mitochondrial sirtuins. SIRT2, which is mostly cytoplasmic but translocate to the nucleus during G2/M transition phase in cell cycle, has a NAD+-dependent deacetylase activity along with Mono-ADP-Ribosyl transferase activity. SIRT2 regulates microtubule dynamics of the cell by acetylation at K40 residue of tubulin. Although, generally deemed to be a tumour suppressor as it regulates cell cycle progression, it was observed to be upregulated in some cancers. One of the major targets of SIRT2 are FoxO family of transcription factors, which are involved in regulation of processes as varied as cell death, adipocyte differentiation and autophagy. By deacetylating them, SIRT2 can regulate their activity, stability and sub-cellular localization. However, the role of SIRT2 in cardiac diseases, oxidative stress response and regulation of insulin resistance is unknown. In this work, we studied the role of SIRT2 in cardiac hypertrophy, cell death and glucose homeostasis. PART 1: Role of SIRT2 in cardiac hypertrophy To find out if SIRT2 plays a role in cardiac hypertrophy, we treated mice with Isoproterenol, a well-accepted model of pathological hypertrophy. We found SIRT2 protein levels to be significantly downregulated in Isoproterenol-treated mice hearts as compared to the controls. In vitro experiments with primary cardiomyocytes yielded the same results. SIRT2 knockout mice spontaneously developed age dependent cardiac hypertrophy and fibrosis. We found that SIRT2 binds to and deacetylates the transcription factor NFATc2. NFATc2 activation is a well-known modulator of cardiac fetal-programme and cardiac hypertrophy. SIRT2 deficiency leads to hyperacetylation of NFATc2, while acetylation enhances nuclear localization of NFATc2. Further, we have demonstrated that GSK3 activity is markedly reduced in Sirt2 knockout mice, a hallmark of hypertrophic hearts. It has been shown that cardiac specific GSK3 overexpression ameliorates cardiac hypertrophy. We found GSK3 activity increased by SIRT2 mediated deacetylation of Lys246 and Lys183 of GSK3α and GSK3β respectively. Interestingly, reduced GSK activity in SIRT2-deficient mice was independent of inhibitory phosphorylation at Ser9. Moreover, GSK3 is required for the anti-hypertrophic function of SIRT2. PART 2: Role of SIRT2 in regulating oxidative stress-induced cell death Our body is continually exposed to variety of exogenous or endogenous stresses. Chronic stresses can disrupt nearly every system in our body and can eventually lead to serious life-threatening illnesses such as heart attacks, kidney disease and cancer. Sirtuins are known to regulate cell death. SIRT1 increases cell survival, by modulating activity of p53 and FOXO3 deacetylation mediated degradation. SIRT3 decreases cell death by improving mitochondrial function. However, role of SIRT2 in cell death is not well understood. In this study we found that SIRT2-depleted cells are resistant to oxidative stress and show enhanced survival. Similarly, SIRT2-KO mice showed enhanced resistance to acetaminophen-induced hepatocyte cell death. Mechanistically, acetylation of JNK at K153 by p300 acetyl transferase reduced JNK phosphorylation and activity, whereas deacetylation of JNK by SIRT2 promoted its phosphorylation as well its activity. Our molecular simulation and biological assays demonstrated that acetylation of JNK at K153 impairs ATP binding, hence reduces its activity. Our results indicate that deacetylation of JNK by SIRT2 promotes oxidative stress-induced cell death. PART 3: Understanding the role of SIRT2 in glucose homeostasis Glucose is one of the major sources of energy for human body, hence regulation of glucose is tightly regulated. Skeletal muscles play a key role in glucose homeostasis. Skeletal muscles comprise around 40-50% of the total body mass in humans and around 80% of glucose in the body is utilized by skeletal muscles. Impaired glucose uptake by muscle cells leads to insulin resistance. SIRT2 improves glucose uptake in hepatocytes through deacetylation of glucokinase regulatory protein (GKRP) or SIRT2 mediated phosphoenolpyruvate carboxy kinase (PEPCK) deacetylation, thus modulating gluconeogenesis in liver. However, role of SIRT2 in glucose homeostasis in muscle tissue is not well studied. Upon induction of high fat diet (HFD) mediated insulin resistance, SIRT2 levels were increased in skeletal muscle of mice. We found SIRT2-KO mice to be highly resistant to HFD induced insulin resistance as revealed by markedly higher glucose clearance rate. Mechanistically, SIRT2 mediated deacetylation of IRS1 was found to promote the inhibitory phosphorylation of IRS1 at Ser307 through activate JNK, which is associated with impaired insulin signalling. We found that SIRT2 overexpression reduced the insulin-stimulated membrane localization of GLUT4 transporter. Our results further suggest that inhibition of SIRT2 reverses the palmitate-induced reduction in cellular glucose uptake and promotes glucose uptake in insulin resistant myotubes and therefore ameliorates the effects of insulin resistance. In the present work, we have elucidated the role of SIRT2 cardiac myocytes, hepatocytes and skeletal myotubes. We believe that modulation of SIRT2 activity could be a potential therapeutic strategy to treat diseases related to aging