Deacetylation of the retinoblastoma tumour suppressor protein by SIRT1

The activity of Rb (retinoblastoma protein) is regulated by phosphorylation and acetylation events. Active Rb is hypophosphorylated and acetylated on multiple residues. Inactivation of Rb involves concerted hyper-phosphorylation by cyclin-CDK (cyclin-dependent kinase) complexes combined with deacetylation of appropriate lysine residues within Rb. In the present study, using in vivo co-immunoprecipitation experiments, we identified mammalian SIRT1 (sirtuin 1) as a binding partner for Rb and its family members p107 and p130. Formation of Rb-SIRT1 complexes required the pocket domain of Rb. p300 catalysed the acetylation of Rb, and SIRT1 was a potent deacetylase for Rb. The ability of SIRT1 to catalyse the deacetylation of Rb was dependent on NAD and was inhibited by the SIRT1 inhibitor nicotinamide. Deacetylated lysine residues within Rb formed a domain similar to the SIRT1-targeted domain of the p53 tumour suppressor protein. Cultures of arrested cells, via contact inhibition or DNA damage, exhibited decreased Rb phosphorylation and increased Rb acetylation. Overexpression of SIRT1 in either confluent or etoposide-treated cells resulted in a significant reduction in Rb acetylation, which was restored with nicotinamide. Gene knockdown of SIRT1 by siRNA (short interfering RNA) produced an accumulation of acetylated Rb. This increase was augmented further when siRNA against SIRT1 was used in conjunction with nicotinamide. In conclusion, our results demonstrate that SIRT1 is an in vitro and in vivo deacetylase for the Rb tumour suppressor protein.     

NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10

A member of the sirtuin family of NAD⁺-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD⁺-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3^−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD⁺-dependent deacetylase, SIRT3.     

Aromatase Inhibition Attenuates Desflurane-Induced Preconditioning against Acute Myocardial Infarction in Male Mouse Heart In Vivo

The volatile anesthetic desflurane (DES) effectively reduces cardiac infarct size following experimental ischemia/reperfusion injury in the mouse heart. We hypothesized that endogenous estrogens play a role as mediators of desflurane-induced preconditioning against myocardial infarction. In this study, we tested the hypothesis that desflurane effects local estrogen synthesis by modulating enzyme aromatase expression and activity in the mouse heart. Aromatase metabolizes testosterone to 17β- estradiol (E2) and thereby significantly contributes to local estrogen synthesis. We tested aromatase effects in acute myocardial infarction model in male mice. The animals were randomized and subjected to four groups which were pre-treated with the selective aromatase inhibitor anastrozole (A group) and DES alone (DES group) or in combination (A+DES group) for 15 minutes prior to surgical intervention whereas the control group received 0.9% NaCl (CON group). All animals were subjected to 45 minutes ischemia following 180 minutes reperfusion. Anastrozole blocked DES induced preconditioning and increased infarct size compared to DES alone (37.94±15.5% vs. 17.1±3.62%) without affecting area at risk and systemic hemodynamic parameters following ischemia/reperfusion. Protein localization studies revealed that aromatase was abundant in the murine cardiovascular system with the highest expression levels in endothelial and smooth muscle cells. Desflurane application at pharmacological concentrations efficiently upregulated aromatase expression in vivo and in vitro. We conclude that desflurane efficiently regulates aromatase expression and activity which might lead to increased local estrogen synthesis and thus preserve cellular integrity and reduce cardiac damage in an acute myocardial infarction model.     

Sirtuin-3 (SIRT3) Protein Attenuates Doxorubicin-induced Oxidative Stress and Improves Mitochondrial Respiration in H9c2 Cardiomyocytes

Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.     

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica

A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This Δgut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to β-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal β-oxidation. Additional deletion of the POX1 to POX6 genes in the Δgut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.     

Deficiency of a Lipid Droplet Protein,Perilipin 5, Suppresses Myocardial Lipid Accumulation,Thereby Preventing Type 1 Diabetes-Induced Heart Malfunction

Lipid droplet (LD) is a ubiquitous organelle that stores triacylglycerol and other neutral lipids. Perilipin 5 (Plin5), a member of the perilipin protein family that is abundantly expressed in the heart, is essential to protect LDs from attack by lipases, including adipose triglyceride lipase. Plin5 controls heart metabolism and performance by maintaining LDs under physiological conditions. Aberrant lipid accumulation in the heart leads to organ malfunction, or cardiomyopathy. To elucidate the role of Plin5 in a metabolically disordered state and the mechanism of lipid-induced cardiomyopathy, we studied the effects of streptozotocin-induced type 1 diabetes in Plin5-knockout (KO) mice. In contrast to diabetic wild-type mice, diabetic Plin5-KO mice lacked detectable LDs in the heart and did not exhibit aberrant lipid accumulation, excessive reactive oxygen species (ROS) generation, or heart malfunction. Moreover, diabetic Plin5-KO mice exhibited lower heart levels of lipotoxic molecules, such as diacylglycerol and ceramide, than wild-type mice. Membrane translocation of protein kinase C and the assembly of NADPH oxidase 2 complex on the membrane were also suppressed. The results suggest that diabetic Plin5-KO mice are resistant to type 1 diabetes-induced heart malfunction due to the suppression of the diacylglycerol/ceramide-protein kinase C pathway and of excessive ROS generation by NADPH oxidase.     

SIRT3 Enhances Glycolysis and Proliferation in SIRT3-Expressing Gastric Cancer Cells

SIRT3 is a key NAD⁺-dependent protein deacetylase in the mitochondria of mammalian cells, functioning to prevent cell aging and transformation via regulation of mitochondrial metabolic homeostasis. However, SIRT3 is also found to express in some human tumors; its role in these SIRT3-expressing tumor cells needs to be elucidated. This study demonstrated that the expression of SIRT3 was elevated in a group of gastric cancer cells compared to normal gastric epithelial cells. Although SIRT3 expression levels were increased in the gastric tumor tissues compared to the adjacent non-tumor tissues, SIRT3 positive cancer cells were more frequently detected in the intestinal type gastric cancers than the diffuse type gastric cancers, indicating that SIRT3 is linked with subtypes of gastric cancer. Overexpression of SIRT3 promoted cell proliferation and enhanced ATP generation, glucose uptake, glycogen formation, MnSOD activity and lactate production, which were inhibited by SIRT3 knockdown, indicating that SIRT3 plays a role in reprogramming the bioenergetics in gastric tumor cells. Further analysis revealed that SIRT3 interacted with and deacetylated the lactate dehydrogenase A (LDHA), a key protein in regulating anaerobic glycolysis, enhancing LDHA activity. In consistence, a cluster of glycolysis-associated genes was upregulated in the SIRT3-overexpressing gastric tumor cells. Thus, in addition to the well-documented SIRT3-mediated mitochondrial homeostasis in normal cells, SIRT3 may enhance glycolysis and cell proliferation in SIRT3-expressing cancer cells.     

Adipophilin在动脉粥样硬化病变和脂质负荷细胞中的作用研究(英)

Adipophilin是细胞内脂质聚集和与脂质聚集有关疾病的标志物,巨噬细胞源性泡沫细胞的形成是动脉粥样硬化性疾病发生的重要环节.为了探讨adipophilin在动脉粥样硬化性疾病的作用,通过高胆固醇饲料喂养新西兰白兔12周,复制动脉粥样硬化疾病模型,同时测定血脂的变化和动脉壁胆固醇,使用HE染色、苏丹Ⅳ染色观察动脉粥样硬化病变的形成,使用免疫组织化学的方法观察动脉粥样硬化病变处和动物肝脏中adipophilin的表达.结果发现,高胆固醇饲料喂养组血清总胆固醇、低密度脂蛋白胆固醇和动脉壁胆固醇明显增高,动脉粥样硬化病变面积增加到(40.06±7.29)%,动脉粥样硬化病变处adipophilin表达呈阳性;而adipophilin在肝脏中的表达无论是高胆固醇饲料喂养组或对照组均为阴性.使用80 mg/L OxLDL与小鼠腹膜巨噬细胞共孵育,复制脂质负荷细胞,然后把构建的1 mmol/L adipophilin反义寡核苷酸与该细胞共孵育.结果发现,使用油红O染色观察的细胞内脂滴明显减少,生化测定细胞内胆固醇酯显著降低,与对照组相比,差别有显著性.说明adipophilin与动脉粥样硬化病变有密切的关系,控制adipophilin的表达能够减少巨噬细胞细胞内胆固醇酯的聚集.     

Adipophilin在动脉粥样硬化病变和脂质负荷细胞中的作用研究(英文)

Adipophilin是细胞内脂质聚集和与脂质聚集有关疾病的标志物 ,巨噬细胞源性泡沫细胞的形成是动脉粥样硬化性疾病发生的重要环节 .为了探讨adipophilin在动脉粥样硬化性疾病的作用 ,通过高胆固醇饲料喂养新西兰白兔 12周 ,复制动脉粥样硬化疾病模型 ,同时测定血脂的变化和动脉壁胆固醇 ,使用HE染色、苏丹Ⅳ染色观察动脉粥样硬化病变的形成 ,使用免疫组织化学的方法观察动脉粥样硬化病变处和动物肝脏中adipophilin的表达 .结果发现 ,高胆固醇饲料喂养组血清总胆固醇、低密度脂蛋白胆固醇和动脉壁胆固醇明显增高 ,动脉粥样硬化病变面积增加到 (40 0 6± 7 2 9) % ,动脉粥样硬化病变处adipophilin表达呈阳性 ;而adipophilin在肝脏中的表达无论是高胆固醇饲料喂养组或对照组均为阴性 .使用80mg/L OxLDL与小鼠腹膜巨噬细胞共孵育 ,复制脂质负荷细胞 ,然后把构建的 1mmol/Ladipophilin反义寡核苷酸与该细胞共孵育 .结果发现 ,使用油红O染色观察的细胞内脂滴明显减少 ,生化测定细胞内胆固醇酯显著降低 ,与对照组相比 ,差别有显著性 .说明adipophilin与动脉粥样硬化病变有密切的关系 ,控制adipophilin的表达能够减少巨噬细胞细胞内胆固醇酯的聚集     

Icariin attenuates excessive alcohol consumption-induced susceptibility to atrial fibrillation through SIRT3 signaling

Excessive alcohol consumption has long been identified as a risk factor for adverse atrial remodeling and atrial fibrillation (AF). Icariin is a principal active component from traditional Chinese medicine Herba Epimedii and has been demonstrated to exert potential antiarrhythmic effect. The present study was designed to evaluate the effect of icariin against alcohol-induced atrial remodeling and disruption of mitochondrial dynamics and furthermore, to elucidate the underlying mechanisms. Excessive alcohol-treated C57BL/6 J mice were infected with serotype 9 adeno-associated virus (AAV9) carrying mouse SIRT3 gene or negative control virus. Meanwhile, icariin (50 mg/kg/d) was administered to the animals in the presence or absence of AAV9 carrying SIRT3 shRNA. We noted that 8 weeks of icariin treatment effectively attenuated alcohol consumption-induced atrial structural and electrical remodeling as evidenced by reduced AF inducibility and reversed atrial electrical conduction pattern as well as atrial enlargement. Furthermore, icariin-treated group exhibited significantly enhanced atrial SIRT3-AMPK signaling, decreased atrial mitoSOX fluorescence and mitochondrial fission markers, elevated mitochondrial fusion markers (MFN1, MFN2) as well as NRF-1-Tfam-mediated mitochondrial biogenesis. Importantly, these beneficial effects were mimicked by SIRT3 overexpression while abolished by SIRT3 knockdown. These data revealed that targeting atrial SIRT3-AMPK signaling and preserving mitochondrial dynamics might serve as the novel therapeutic strategy against alcohol-induced AF genesis. Additionally, icariin ameliorated atrial remodeling and mitochondrial dysfunction by activating SIRT3-AMPK signaling, highlighting the use of icariin as a promising antiarrhythmic agent in this circumstance.     

Neuritin Attenuates Cognitive Function Impairments in Tg2576 Mouse Model of Alzheimer's Disease

Neuritin, also known as CPG15, is a neurotrophic factor that was initially discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in the process of neural development and synaptic plasticity, although its binding receptor(s) and downstream signaling effectors remain unclear. In this study, we found that the cortical and hippocampal expression of neuritin is reduced in the brains of Alzheimer''s disease (AD) patients and demonstrated that viral-mediated expression of neuritin in the dentate gyrus of 13-month-old Tg2576 mice, an AD animal model, attenuated a deficit in learning and memory as assessed by a Morris water maze test. We also found that neuritin restored the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neuron cultures prepared from Tg2576 mice. It was also shown that viral-mediated expression of neuritin in the dentate gyrus of 7-week-old Sprague-Dawley rats increased neurogenesis in the hippocampus. Taken together, our results demonstrate that neuritin restores the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neurons from Tg2576 neurons, and also attenuates cognitive function deficits in Tg2576 mouse model of AD, suggesting that neuritin possesses a therapeutic potential for AD.