Sirtuin 2 Regulates Protein LactoylLys Modifications.

Post-translational modifications (PTMs) play roles in both physiological and pathophysiological processes through the regulation of enzyme structure and function. We recently identified a novel PTM, lactoylLys, derived through a nonenzymatic mechanism from the glycolytic by-product, lactoylglutathione. Under physiologic scenarios, glyoxalase 2 prevents the accumulation of lactoylglutathione and thus lactoylLys modifications. What dictates the site-specificity and abundance of lactoylLys PTMs, however, remains unknown. Here, we report sirtuin 2 as a lactoylLys eraser. Using chemical biology and CRISPR-Cas9, we show that SIRT2 controls the abundance of this PTM both globally and on chromatin. These results address a major gap in our understanding of how nonenzymatic PTMs are regulated and controlled.

Ethanol Exposure Attenuates Immune Response in Sepsis via Sirtuin 2 Expression.

BACKGROUND: Sepsis and septic shock kill over 270,000 patients per year in the United States. Sepsis transitions from a hyper-inflammatory to a hypo-inflammatory phase. Alcohol dependence is a risk factor for mortality from sepsis. Ethanol (EtOH) exposure impairs pathogen clearance through mechanisms that are not fully understood. Sirtuin 2 (SIRT2) interferes with pathogen clearance in immune cells but its role in the effects of EtOH on sepsis is unknown. We studied the effect of EtOH exposure on hyper- and hypo-inflammation and the role of SIRT2 in mice. METHODS: We exposed C57Bl/6 (WT) mice to EtOH via drinking water and used intraperitoneal cecal slurry (CS)-induced sepsis to study: (i) 7-day survival, (ii) leukocyte adhesion (LA) in the mesenteric microcirculation during hyper- and hypo-inflammation, (iii) peritoneal cavity bacterial clearance, and (iv) SIRT2 expression in peritoneal macrophages. Using EtOH-exposed and lipopolysaccharide (LPS)-stimulated RAW 264.7 (RAW) cell macrophages for 4 hours or 24 hours, we studied: (i) tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and SIRT2 expression, and (ii) the effect of the SIRT2 inhibitor AK-7 on inflammatory response at 24 hours. Lastly, we studied the effect of EtOH on sepsis in whole body Sirt2 knockout (SIRT2KO) mice during hyper- and hypo-inflammation, bacterial clearance, and 7-day survival. RESULTS: WT EtOH-sepsis mice showed: (i) Decreased survival, (ii) Muted LA in the microcirculation, (iii) Lower plasma TNF-α and IL-6 expression, (iv) Decreased bacterial clearance, and (v) Increased SIRT2 expression in peritoneal macrophages versus vehicle-sepsis. EtOH-exposed LPS-stimulated RAW cells showed: (i) Muted TNF-α, IL-6, and increased IL-10 expression at 4 hours, (ii) endotoxin tolerance at 24 hours, and (iii) reversal of endotoxin tolerance with the SIRT2 inhibitor AK-7. EtOH-exposed SIRT2KO-sepsis mice showed greater 7-day survival, LA, and bacterial clearance than WT EtOH-sepsis mice. CONCLUSION: EtOH exposure decreases survival and reduces the inflammatory response to sepsis via increased SIRT2 expression. SIRT2 is a potential therapeutic target in EtOH with sepsis.

Sirt2 Inhibition Enhances Metabolic Fitness and Effector Functions of Tumor-Reactive T Cells.

Dysregulated metabolism is a key driver of maladaptive tumor-reactive T lymphocytes within the tumor microenvironment. Actionable targets that rescue the effector activity of antitumor T cells remain elusive. Here, we report that the Sirtuin-2 (Sirt2) NAD+-dependent deacetylase inhibits T cell metabolism and impairs T cell effector functions. Remarkably, upregulation of Sirt2 in human tumor-infiltrating lymphocytes (TILs) negatively correlates with response to TIL therapy in advanced non-small-cell lung cancer. Mechanistically, Sirt2 suppresses T cell metabolism by targeting key enzymes involved in glycolysis, tricarboxylic acid-cycle, fatty acid oxidation, and glutaminolysis. Accordingly, Sirt2-deficient murine T cells exhibit increased glycolysis and oxidative phosphorylation, resulting in enhanced proliferation and effector functions and subsequently exhibiting superior antitumor activity. Importantly, pharmacologic inhibition of Sirt2 endows human TILs with these superior metabolic fitness and effector functions. Our findings unveil Sirt2 as an unexpected actionable target for reprogramming T cell metabolism to augment a broad spectrum of cancer immunotherapies.

SIRT2 Inhibition Improves Functional Motor Recovery After Peripheral Nerve Injury.

Sirtuin-2 (Sirt2) is a member of the NAD (+)-dependent protein deacetylase family involved in neuroprotection, cellular metabolism, homeostasis, and stress responses after injury of the nervous system. So far, no data have been published describing the role of SIRT2 in motor functional recovery after damage. We found that SIRT2 expression and deacetylase activity were increased within motoneurons after axotomy. To shed light onto the biological relevance of this change, we combined in vitro and in vivo models with pharmacological and genetic ablation approaches. We found that SIRT2 KO (knockout) mice exhibited improved functional recovery after sciatic nerve crush. SIRT2 activity blockage, using AK7, increased neurite outgrowth and length in organotypic spinal cord cultures and human cell line models. SIRT2 blockage enhanced the acetyltransferase activity of p300, which in turn increased the levels of an acetylated form of p53 (Ac-p53 k373), histone 3 (Ac-H3K9), and expression of GAP43, a downstream marker of regeneration. Lastly, we verified that p300 acetyltransferase activity is essential for these effects. Our results suggest that bolstering an epigenetic shift that promotes SIRT2 inhibition can be an effective therapy to increase functional recovery after peripheral nerve injury.

SIRT2 deficiency prevents age-related bone loss in rats by inhibiting osteoclastogenesis.

Sirtuin 2 (SIRT2) is a deacetylase that belongs to class III family of histone deacetylases (HDACs). Although it is the most abundantly expressed member of HDAC-III in human bone tissues, it is unclear whether SIRT2 plays a role in bone metabolism. In this study, the role of SIRT2 in bone metabolism, and the underlying mechanism were investigated. In in vivo experiments, micro-CT analysis revealed that there were no differences in bone microstructures between SIRT2-KO and WT rats at 12 weeks of age. However, in 36-week-old rats, increased Tb. BMD, bone volume fraction (BV/TV) and trabecular number (Tb. N) of distal femurs were observed in SIRT2-KO rats, when compared with those of WT rats. Moreover, reduced serum ß-CTX was identified in the 36-week old rats. In in vitro studies, inhibition of SIRT2 with its specific inhibitor, AGK2, suppressed the differentiation of bone marrow-derived mononuclear cells (BMMs) into osteoclasts via reduction of the expressions of c-Fos and NFATc1. These results suggest that SIRT2 plays a role in age-related bone loss, probably by regulating osteoclastogenesis.

PGC-1α acts as an mediator of Sirtuin2 to protect annulus fibrosus from apoptosis induced by oxidative stress through restraining mitophagy.

Apoptosis of annulus fibrosus (AF) is observed widely in intervertebral disc degeneration (IVDD) which causes weaken of tension in the annulus of intervertebral disc. Previous studies reported that apoptosis of AF is induced mainly by oxidative stress. SIRT2 is a major regulator of mitochondria to mediate ROS production. However, the mechanism of SIRT2 in IVDD remains unclear. Here, the expression of SIRT2 was detected in AF cells exposed to tert-Butyl hydroperoxide (TBHP) by western blotting. Autophagic flux and apoptosis were assessed by western blotting, flow cytometry and immunofluorescence respectively. Safranin O staining, HE, and immunohistochemical were used to assess the IVDD after 3, 6 and 9 months of surgical procedure in vivo. The expression of SIRT2 was decreased in AF cells treated with TBHP. Repression of mitophagy alleviated the apoptosis of AF cells caused by TBHP. Overexpression of PGC-1α prevented AF cells from apoptosis and mitophagy after applying Lenti-PGC-1α to transfect AF cells. These protections of PGC-1α were reduced by FCCP. Furthermore, the expression of PGC-1α was reduced and the level of mitophagy was increased in IVDD models. In conclusion, this study indicates that the regulation of PGC-1α expression provide a new theoretical basis for the mechanism of IVDD.

SIRT2 knockout exacerbates insulin resistance in high fat-fed mice.

The NAD+-dependent deacetylase SIRT2 is unique amongst sirtuins as it is effective in the cytosol, as well as the mitochondria. Defining the role of cytosolic acetylation state in specific tissues is difficult since even physiological effects at the whole body level are unknown. We hypothesized that genetic SIRT2 knockout (KO) would lead to impaired insulin action, and that this impairment would be worsened in HF fed mice. Insulin sensitivity was tested using the hyperinsulinemic-euglycemic clamp in SIRT2 KO mice and WT littermates. SIRT2 KO mice exhibited reduced skeletal muscle insulin-induced glucose uptake compared to lean WT mice, and this impairment was exacerbated in HF SIRT2 KO mice. Liver insulin sensitivity was unaffected in lean SIRT2 KO mice. However, the insulin resistance that accompanies HF-feeding was worsened in SIRT2 KO mice. It was notable that the effects of SIRT2 KO were largely disassociated from cytosolic acetylation state, but were closely linked to acetylation state in the mitochondria. SIRT2 KO led to an increase in body weight that was due to increased food intake in HF fed mice. In summary, SIRT2 deletion in vivo reduces muscle insulin sensitivity and contributes to liver insulin resistance by a mechanism that is unrelated to cytosolic acetylation state. Mitochondrial acetylation state and changes in feeding behavior that result in increased body weight correspond to the deleterious effects of SIRT2 KO on insulin action.

The combination of NAD+-dependent deacetylase gene deletion and the interruption of gluconeogenesis causes increased glucose metabolism in budding yeast.

Metabolic engineering focuses on rewriting the metabolism of cells to enhance native products or endow cells with the ability to produce new products. This engineering has the potential for wide-range application, including the production of fuels, chemicals, foods and pharmaceuticals. Glycolysis manages the levels of various secondary metabolites by controlling the supply of glycolytic metabolites. Metabolic reprogramming of glycolysis is expected to cause an increase in the secondary metabolites of interest. In this study, we constructed a budding yeast strain harboring the combination of triple sirtuin gene deletion (hst3∆ hst4∆ sir2∆) and interruption of gluconeogenesis by the deletion of the FBP1 gene encoding fructose-1,6-bisphosphatase (fbp1∆). hst3∆ hst4∆ sir2∆ fbp1∆ cells harbored active glycolysis with high glucose consumption and active ethanol productivity. Using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) analysis, hst3∆ hst4∆ sir2∆ fbp1∆ cells accumulated not only glycolytic metabolites but also secondary metabolites, including nucleotides that were synthesized throughout the pentose phosphate (PP) pathway, although various amino acids remained at low levels. Using the stable isotope labeling assay for metabolites, we confirmed that hst3∆ hst4∆ sir2∆ fbp1∆ cells directed the metabolic fluxes of glycolytic metabolites into the PP pathway. Thus, the deletion of three sirtuin genes (HST3, HST4 and SIR2) and the FBP1 gene can allow metabolic reprogramming to increase glycolytic metabolites and several secondary metabolites except for several amino acids.

SIRT2 regulates oxidative stress-induced cell death through deacetylation of c-Jun NH2-terminal kinase.

c-Jun NH2-terminal kinases (JNKs) are responsive to stress stimuli and their activation regulate key cellular functions, including cell survival, growth, differentiation and aging. Previous studies demonstrate that activation of JNK requires dual phosphorylation by the mitogen-activated protein kinase kinases. However, other post-translational mechanisms involved in regulating the activity of JNK have been poorly understood. In this work, we studied the functional significance of reversible lysine acetylation in regulating the kinase activity of JNK. We found that the acetyl transferase p300 binds to, acetylates and inhibits kinase activity of JNK. Using tandem mass spectrometry, molecular modelling and molecular dynamics simulations, we found that acetylation of JNK at Lys153 would hinder the stable interactions of the negatively charged phosphates and prevent the adenosine binding to JNK. Our screening for the deacetylases found SIRT2 as a deacetylase for JNK. Mechanistically, SIRT2-dependent deacetylation enhances ATP binding and enzymatic activity of JNK towards c-Jun. Furthermore, SIRT2-mediated deacetylation favours the phosphorylation of JNK by MKK4, an upstream kinase. Our results indicate that deacetylation of JNK by SIRT2 promotes oxidative stress-induced cell death. Conversely, SIRT2 inhibition attenuates H2O2-mediated cell death in HeLa cells. SIRT2-deficient (SIRT2-KO) mice exhibit increased acetylation of JNK, which is associated with markedly reduced catalytic activity of JNK in the liver. Interestingly, SIRT2-KO mice were resistant to acetaminophen-induced liver toxicity. SIRT2-KO mice show lower cell death, minimal degenerative changes, improved liver function and survival following acetaminophen treatment. Overall, our work identifies SIRT2-mediated deacetylation of JNK as a critical regulator of cell survival during oxidative stress.

RNA interference-mediated knockdown of SIRT1 and/or SIRT2 in melanoma: Identification of downstream targets by large-scale proteomics analysis.

Melanoma is the most notorious and fatal of all skin cancers and the existing treatment options have not been proven to effectively manage this neoplasm, especially the metastatic disease. Sirtuin (SIRT) proteins have been shown to be differentially expressed in melanoma. We have shown that SIRTs 1 and 2 were overexpressed in melanoma and inhibition of SIRT1 imparts anti-proliferative responses in human melanoma cells. To elucidate the impact of SIRT 1 and/or 2 in melanoma, we created stable knockdowns of SIRTs 1, 2, and their combination using shRNA mediated RNA interference in A375 human melanoma cells. We found that SIRT1 and SIRT1&2 combination knockdown caused a decreased cellular proliferation in melanoma cells. Further, the knockdown of SIRT 1 and/or 2 resulted in a decreased colony formation in melanoma cells. To explore the downstream targets of SIRTs 1 and/or 2, we employed a label-free quantitative nano-LC-MS/MS proteomics analysis using the stable lines. We found aberrant levels of proteins involved in many vital cellular processes, including cytoskeletal organization, ribosomal activity, oxidative stress response, and angiogenesis. These findings provide clear evidence of cellular systems undergoing alterations in response to sirtuin inhibition, and have unveiled several excellent candidates for future study. SIGNIFICANCE: Melanoma is the deadliest form of skin cancer, due to its aggressive nature, metastatic potential, and a lack of sufficient treatment options for advanced disease. Therefore, detailed investigations into the molecular mechanisms of melanoma growth and progression are needed. In the search for candidate genes to serve as therapeutic targets, the sirtuins show promise as they have been found to be upregulated in melanoma and they regulate a large number of proteins involved in cellular processes known to affect tumor growth, such as DNA damage repair, cell cycle arrest, and apoptosis. In this study, we used a large-scale label-free comparative proteomics system to identify novel protein targets that are affected following knockdown of SIRT1 and/or 2 in A375 metastatic melanoma cell line. Our study offers important insight into the potential downstream targets of SIRTs 1 and/or 2. This may unravel new potential areas of exploration in melanoma research.

Loss of SIRT2 leads to axonal degeneration and locomotor disability associated with redox and energy imbalance.

Sirtuin 2 (SIRT2) is a member of a family of NAD+ -dependent histone deacetylases (HDAC) that play diverse roles in cellular metabolism and especially for aging process. SIRT2 is located in the nucleus, cytoplasm, and mitochondria, is highly expressed in the central nervous system (CNS), and has been reported to regulate a variety of processes including oxidative stress, genome integrity, and myelination. However, little is known about the role of SIRT2 in the nervous system specifically during aging. Here, we show that middle-aged, 13-month-old mice lacking SIRT2 exhibit locomotor dysfunction due to axonal degeneration, which was not present in young SIRT2 mice. In addition, these Sirt2-/- mice exhibit mitochondrial depletion resulting in energy failure, and redox dyshomeostasis. Our results provide a novel link between SIRT2 and physiological aging impacting the axonal compartment of the central nervous system, while supporting a major role for SIRT2 in orchestrating its metabolic regulation. This underscores the value of SIRT2 as a therapeutic target in the most prevalent neurodegenerative diseases that undergo with axonal degeneration associated with redox and energetic dyshomeostasis.

SIRT2 Acts as a Cardioprotective Deacetylase in Pathological Cardiac Hypertrophy.

BACKGROUND: Pathological cardiac hypertrophy induced by stresses such as aging and neurohumoral activation is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the roles of SIRT2 in aging-related and angiotensin II (Ang II)-induced pathological cardiac hypertrophy. METHODS: Male C57BL/6J wild-type and Sirt2 knockout mice were subjected to the investigation of aging-related cardiac hypertrophy. Cardiac hypertrophy was also induced by Ang II (1.3 mg/kg/d for 4 weeks) in male C57BL/6J Sirt2 knockout mice, cardiac-specific SIRT2 transgenic (SIRT2-Tg) mice, and their respective littermates (8 to ≈12 weeks old). Metformin (200 mg/kg/d) was used to treat wild-type and Sirt2 knockout mice infused with Ang II. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. RESULTS: SIRT2 protein expression levels were downregulated in hypertrophic hearts from mice. Sirt2 knockout markedly exaggerated cardiac hypertrophy and fibrosis and decreased cardiac ejection fraction and fractional shortening in aged (24-month-old) mice and Ang II-infused mice. Conversely, cardiac-specific SIRT2 overexpression protected the hearts against Ang II-induced cardiac hypertrophy and fibrosis and rescued cardiac function. Mechanistically, SIRT2 maintained the activity of AMP-activated protein kinase (AMPK) in aged and Ang II-induced hypertrophic hearts in vivo as well as in cardiomyocytes in vitro. We identified the liver kinase B1 (LKB1), the major upstream kinase of AMPK, as the direct target of SIRT2. SIRT2 bound to LKB1 and deacetylated it at lysine 48, which promoted the phosphorylation of LKB1 and the subsequent activation of LKB1-AMPK signaling. Remarkably, the loss of SIRT2 blunted the response of AMPK to metformin treatment in mice infused with Ang II and repressed the metformin-mediated reduction of cardiac hypertrophy and protection of cardiac function. CONCLUSIONS: SIRT2 promotes AMPK activation by deacetylating the kinase LKB1. Loss of SIRT2 reduces AMPK activation, promotes aging-related and Ang II-induced cardiac hypertrophy, and blunts metformin-mediated cardioprotective effects. These findings indicate that SIRT2 will be a potential target for therapeutic interventions in aging- and stress-induced cardiac hypertrophy.

Sirtuin 2 Regulates Microvascular Inflammation during Sepsis.

Objective. Sepsis and septic shock, the leading causes of death in noncoronary intensive care units, kill more than 200,000/year in the US alone. Circulating cell-endothelial cell interactions are the rate determining factor in sepsis inflammation. Sirtuin, a seven-member family of proteins (SIRT1-7), epigenetically controls inflammation. We have studied the roles of SIRTs 1, 3, and 6 in sepsis previously. In this project, we studied the role of SIRT2 on sepsis-related inflammation. Methods. Sepsis was induced in C57Bl/6 (WT), SIRT2 knockout (SIRT2KO), and SIRT2 overexpressing (SIRT2KI) mice by cecal ligation and puncture (CLP). We studied leukocyte/platelet adhesion using intravital microscopy and E-selectin/ICAM-1 adhesion molecule expression in the small intestine with immunohistochemistry (IHC) six hours post-CLP/sham surgery. We also studied 7-day survival rates in WT, SIRT2KO, and SIRT2KI sepsis mice. Results. Compared to WT mice, SIRT2KO mice show exaggeration while SIRT2KI mice show attenuation of cellular adhesion with sepsis in the small intestine. We also show that the small intestinal E-selectin and ICAM-1 expressions increased in SIRT2KO and decreased in SIRT2KI mice versus those in WT sepsis mice. We show that the 7-day survival rate is decreased in SIRT2KO and increased in SIRT2KI sepsis mice. Conclusion. SIRT2 modulates microvascular inflammation in sepsis and affects survival.

SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences.

Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed before mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age- and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age- and disease-associated genome instability.

Loss of NAD-Dependent Protein Deacetylase Sirtuin-2 Alters Mitochondrial Protein Acetylation and Dysregulates Mitophagy.

AIMS: Sirtuins connect energy generation and metabolic stress to the cellular acetylome. Currently, only the mitochondrial sirtuins (SIRT3-5) and SIRT1 have been shown to direct mitochondrial function; however, Aims: NAD-dependent protein deacetylase sirtuin-2 (SIRT2), the primary cytoplasmic sirtuin, is not yet reported to associate with mitochondria. RESULTS: This study revealed a novel physiological function of SIRT2: the regulation of mitochondrial function. First, the acetylation of several metabolic mitochondrial proteins was found to be altered in Sirt2-deficient mice, which was, subsequently, validated by immunoprecipitation experiments in which the acetylated mitochondrial proteins directly interacted with SIRT2. Moreover, immuno-gold electron microscopic images of mouse brains showed that SIRT2 associates with the inner mitochondrial membrane in central nervous system cells. The loss of Sirt2 increased oxidative stress, decreased adenosine triphosphate levels, and altered mitochondrial morphology at the cellular and tissue (i.e., brain) level. Furthermore, the autophagic/mitophagic processes were dysregulated in Sirt2-deficient neurons and mouse embryonic fibroblasts. INNOVATION: For the first time it is shown that SIRT2 directs mitochondrial metabolism. CONCLUSION: Together, these findings support that SIRT2 functions as a mitochondrial sirtuin, as well as a regulator of autophagy/mitophagy to maintain mitochondrial biology, thus facilitating cell survival. Antioxid. Redox Signal. 26, 849-863.

Downregulation of NAD-Dependent Deacetylase SIRT2 Protects Mouse Brain Against Ischemic Stroke.

Sirtuin 2 (SIRT2) is a member of NAD+-dependent protein deacetylases involved in a wide range of pathophysiological processes including myocardial injury, Parkinson's disease, and Huntington's disease. However, the direct implication of SIRT2 in ischemic stroke is still unclear. In the present study, we observed that SIRT2 protein was mainly expressed in the cytoplasm of neurons, but not in astrocyte and microglia. SIRT2 was upregulated in ischemic neurons in the oxygen-glucose deprivation cell model and in the transient middle cerebral artery occlusion (tMCAo) mouse model. Moreover, expression of SIRT2 was evaluated by immunohistochemistry in human brains consisting of ischemic penumbra of cerebral stroke, and their age-matched normal controls without diagnosed neurological disorders. The results revealed that SIRT2 was mainly expressed in the cytoplasm and neurites of neurons in the brains of normal subjects, while an elevated expression and nuclear translocation of SIRT2 were detected in the ischemic penumbra of cerebral stroke. Downregulation of SIRT2 using the SIRT2-specific inhibitor AGK2 or SIRT2 knockout had neuroprotective effects in tMCAo model, which could decrease the infract volume and neurological impairment scores. In summary, our findings revealed that SIRT2 was upregulated during neuronal ischemia and translocated into neuronal nuclei, while downregulation of SIRT2 could significantly protect neurons against cerebral ischemia.

SIRT2 deletion enhances KRAS-induced tumorigenesis in vivo by regulating K147 acetylation status.

The observation that cellular transformation depends on breaching a crucial KRAS activity threshold, along with the finding that only a small percentage of cellsharboring KRAS mutations are transformed, support the idea that additional, not fully uncovered, regulatory mechanisms may contribute to KRAS activation. Here we report that KrasG12D mice lacking Sirt2 show an aggressive tumorigenic phenotype as compared to KrasG12D mice. This phenotype includes increased proliferation, KRAS acetylation, and activation of RAS downstream signaling markers. Mechanistically, KRAS K147 is identified as a novel SIRT2-specific deacetylation target by mass spectrometry, whereas its acetylation status directly regulates KRAS activity, ultimately exerting an impact on cellular behavior as revealed by cell proliferation, colony formation, and tumor growth. Given the significance of KRAS activity as a driver in tumorigenesis, identification of K147 acetylation as a novel post-translational modification directed by SIRT2 in vivo may provide a better understanding of the mechanistic link regarding the crosstalk between non-genetic and genetic factors in KRAS driven tumors.

SIRT2- and NRF2-Targeting Thiazole-Containing Compound with Therapeutic Activity in Huntington's Disease Models.

There are currently no disease-modifying therapies for the neurodegenerative disorder Huntington's disease (HD). This study identified novel thiazole-containing inhibitors of the deacetylase sirtuin-2 (SIRT2) with neuroprotective activity in ex vivo brain slice and Drosophila models of HD. A systems biology approach revealed an additional SIRT2-independent property of the lead-compound, MIND4, as an inducer of cytoprotective NRF2 (nuclear factor-erythroid 2 p45-derived factor 2) activity. Structure-activity relationship studies further identified a potent NRF2 activator (MIND4-17) lacking SIRT2 inhibitory activity. MIND compounds induced NRF2 activation responses in neuronal and non-neuronal cells and reduced production of reactive oxygen species and nitrogen intermediates. These drug-like thiazole-containing compounds represent an exciting opportunity for development of multi-targeted agents with potentially synergistic therapeutic benefits in HD and related disorders.

Radiation-Induced Alteration of the Brain Proteome: Understanding the Role of the Sirtuin 2 Deacetylase in a Murine Model.

Whole brain radiotherapy (WBRT) produces unwanted sequelae, albeit via unknown mechanisms. A deacetylase expressed in the central nervous system, Sirtuin 2 (SIRT2), has been linked to neurodegeneration. Therefore, we sought to challenge the notion that a single disease pathway is responsible for radiation-induced brain injury in Sirt2 wild-type (WT) and knockout (KO) mice at the proteomic level. We utilized isobaric tag for relative and absolute quantitation to analyze brain homogenates from Sirt2 WT and KO mice with and without WBRT. Selected proteins were independently verified, followed by ingenuity pathway analysis. Canonical pathways for Huntington's, Parkinson's, and Alzheimer's were acutely affected by radiation within 72 h of treatment. Although loss of Sirt2 preferentially affected both Huntington's and Parkinson's pathways, WBRT most significantly affected Huntington's-related proteins in the absence of Sirt2. Identical protein expression patterns were identified in Mog following WBRT in both Sirt2 WT and KO mice, revealing a proteomic radiation signature; however, long-term radiation effects were found to be associated with altered levels of a small number of key neurodegeneration-related proteins, identified as Mapt, Mog, Snap25, and Dnm1. Together, these data demonstrate the principle that the presence of Sirt2 can have significant effects on the brain proteome and its response to ionizing radiation.

SIRT2 deficiency modulates macrophage polarization and susceptibility to experimental colitis.

BACKGROUND: SIRT2 belongs to a highly conserved family of NAD+-dependent deacylases, consisting of seven members (SIRT1-SIRT7), which vary in subcellular localizations and have substrates ranging from histones to transcription factors and enzymes. Recently SIRT2 was revealed to play an important role in inflammation, directly binding, deacetylating, and inhibiting the p65 subunit of NF-κB. METHODS: A Sirt2 deficient mouse line (Sirt2-/-) was generated by deleting exons 5-7, encoding part of the SIRT2 deacetylase domain, by homologous recombination. Age- and sex-matched Sirt2-/- and Sirt2+/+ littermate mice were subjected to dextran sulfate sodium (DSS)-induced colitis and analyzed for colitis susceptibility. RESULTS: Sirt2-/- mice displayed more severe clinical and histological manifestations after DSS colitis compared to wild type littermates. Notably, under basal condition, Sirt2 deficiency does not affect the basal phenotype and intestinal morphology Sirt2 deficiency, however, affects macrophage polarization, creating a pro-inflammatory milieu in the immune cells compartment. CONCLUSION: These data confirm a protective role for SIRT2 against the development of inflammatory processes, pointing out a potential role for this sirtuin as a suppressor of colitis. In fact, SIRT2 deletion promotes inflammatory responses by increasing NF-κB acetylation and by reducing the M2-associated anti-inflammatory pathway. Finally, we speculate that the activation of SIRT2 may be a potential approach for the treatment of inflammatory bowel disease.