去乙酰化酶SIRT1的表达及活性调控机制

SIRT1是哺乳动物中重要的NAD+依赖性去乙酰化酶,参与许多重要的生理和病理过程,如衰老、细胞死亡和肿瘤发生。如何精确调节SIRT1的表达和活性对SIRT1执行其生物学功能至关重要。文章以基因表达的不同阶段为切入点,对调控SIRT1表达及活性的机制进行了论述。     

Upregulation of SIRT1 deacetylase in phenylephrine-treated cardiomyoblasts

The sirtuin SIRT1 is an ubiquitous NAD⁺ dependent deacetylase that plays a role in biological processes such as longevity and stress response. In cardiac models, SIRT1 is associated to protection against many stresses. However, the link between SIRT1 and heart hypertrophy is complex and not fully understood. This study focuses specifically on the response of SIRT1 to the α-adrenergic agonist phenylephrine in H9c2 cardiac myoblasts, a cell model of cardiac hypertrophy. After 24 and 48 h of phenylephrine treatment, SIRT1 expression and deacetylase activity were significantly increased. SIRT1 upregulation by phenylephrine was not associated to changes in NAD⁺ levels, but was blocked by inhibitors of AMP-activated Protein Kinase (AMPK) or by AMPK knockdown by siRNA. When SIRT1 was inhibited with sirtinol or downregulated by siRNA, H9c2 cell viability was significantly decreased following phenylephrine treatment, showing that SIRT1 improves cell survival under hypertrophic stress. We so then propose that the increase in SIRT1 activity and expression in H9c2 cells treated with phenylephrine is an adaptive response to the hypertrophic stress, suggesting that adrenergic stimulation of heart cells activates hypertrophic programming and at the same time also promotes a self-protecting and self-regulating mechanism.     

Emerging role of SIRT3 in mitochondrial dysfunction and cardiovascular diseases

As a nicotinamide adenine dinucleotide (NAD)⁺-dependent protein deacetylase, SIRT3 is highly expressed in tissues with high metabolic turnover and mitochondrial content. It has been demonstrated that SIRT3 plays a critical role in maintaining normal mitochondrial biological function through reversible protein lysine deacetylation. SIRT3 has a variety of substrates that are involved in mitochondrial biological processes such as energy metabolism, reactive oxygen species production and clearance, electron transport chain flux, mitochondrial membrane potential maintenance, and mitochondrial dynamics. In the suppression of SIRT3, functional deficiencies of mitochondria contribute to the development of various cardiovascular disorders. Activation of SIRT3 may represent a promising therapeutic strategy for the improvement of mitochondrial function and the treatment of relevant cardiovascular disorders. In the current review, we discuss the emerging roles of SIRT3 in mitochondrial derangements and subsequent cardiovascular malfunctions, including cardiac hypertrophy and heart failure, ischemia-reperfusion injury, and endothelial dysfunction in hypertension and atherosclerosis.     

Substrate specificity of SIRT1-catalyzed lysine N-deacetylation reaction probed with the side chain modified N-acetyl-lysine analogs

Peptides containing l-N^ε-acetyl-lysine (l-AcK) or its side chain modified analogs were prepared and assayed using SIRT1, the prototypical human silent information regulator 2 (Sir2) enzyme. While previous studies showed that the side chain acetyl group of l-AcK can be extended to bulkier acyl groups for Sir2 (including SIRT1)-catalyzed lysine N^ε-deacylation reaction, our current study suggested that SIRT1-catalyzed deacetylation reaction had a very stringent requirement for the distance between the α-carbon and the side chain acetamido group, with that found in l-AcK being optimal. Moreover, our current study showed that SIRT1 catalyzed the stereospecific deacetylation of l-AcK versus its d-isomer. The results from our current study shall constitute another piece of important information to be considered when designing inhibitors for SIRT1 and Sir2 enzymes in general.     

Dual role for Zn2+ in maintaining structural integrity and inducing DNA sequence specificity in a promiscuous endonuclease

We describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.     

Macromolecular crowding effect is critical for maintaining SIRT1's nuclear localization in cancer cells

SIRT1 is a principle class III histone deacetylase which exhibits versatile functions in stress response, development, and pathological processes including cancer. Although SIRT1 deacetylates a wide range of nuclear and cytoplasmic proteins, its subcellular localization in cancer cells has been controversial. In this study, we uncovered the inconsistent reports about SIRT1 subcellular localization is partially due to different analysis approaches. While immunofluorescence and live cell imaging reveal a predominant nuclear localization of SIRT1, conventional cell fractionation often results in a severe leaking of SIRT1 into the cytoplasm. Such a leakage is mainly caused by loss of cytoplasmic macromolecular crowding effect as well as hypotonic dwelling during the isolation of the nuclei. We also developed an improved cell fractionation procedure which maintains SIRT1 in its original subcellular localization. Analyzing a variety of human cancer cell lines using this approach and other methods demonstrate that SIRT1 predominantly localizes to the nucleus in cancer cells.     

Protein deacetylase SIRT1 in the cytoplasm promotes nerve growth factor-induced neurite outgrowth in PC12 cells

SIRT1, a NAD⁺-dependent protein deacetylase, is known to have neural functions. However, despite its cytoplasmic expression in some neural cells, its cytoplasmic function, if any, is unknown. Here we found that PC12 (pheochromocytoma) cells expressed SIRT1 in the cytoplasm. Nerve growth factor (NGF)-induced neurite outgrowth of these cells was promoted by activators of SIRT1, while inhibitors of SIRT1 or SIRT1-siRNA significantly inhibited it. The overexpression of a mutant SIRT1 that localised to the cytoplasm but not the nucleus enhanced the NGF-dependent neurite outgrowth, and a cytoplasmic dominant-negative SIRT1 suppressed it. Thus, cytoplasmic SIRT1 increases the NGF-induced neurite outgrowth of PC12 cells.     

Function of SIRT1 in physiology

Sirtuins were originally defined as a family of oxidized nicotinamide adenine nucleotide (NAD⁺)-dependent enzymes that deacetylate lysine residues on various proteins. The sirtuins are remarkably conserved throughout evolution from archae to eukaryotes. They were named after their homology to the Saccharomyces cerevisiae gene silent information regulator 2 (Sir2). The mammalian sirtuins, SIRT1-7, are implicated in a variety of cellular functions ranging from gene silencing, control of the cell cycle and apoptosis, and energy homeostasis. As SIRT1 is a nuclear protein and is the mammalian homolog most highly related to Sir2, it has been the focus of a large number of recent studies. Here we review some of the current data related to SIRT1 and discuss its mode of action and biological role in cellular and organismal models. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 869–876.     

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD⁺ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD⁺ cofactor availability is not known. We investigated whether NAD⁺ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H₂O₂ and cigarette smoke (CS) decreased intracellular NAD⁺ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD⁺ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD⁺ cofactor levels by PARP-1 inhibition or NAD⁺ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.     

SIRT1 contains N- and C-terminal regions that potentiate deacetylase activity

SIRT1 is one of seven mammalian sirtuin (silent information regulator 2-related) proteins that harbor NAD(+)-dependent protein deacetylase activity and is implicated in multiple metabolic and age-associated pathways and disorders. The sirtuin proteins contain a central region of high sequence conservation that is required for catalytic activity, but more variable N- and C-terminal regions have been proposed to mediate protein specific activities. Here we show that the conserved catalytic core domain of SIRT1 has very low catalytic activity toward several known protein substrates, but that regions N- and C-terminal to the catalytic core potentiate catalytic efficiency by between 12- and 45-fold, with the N-terminal domain contributing predominantly to catalytic rate, relatively independent of the nature of the acetyl-lysine protein substrate, and the C-terminal domain contributing significantly to the K(m) for NAD(+). We show that the N- and C-terminal regions stimulate SIRT1 deacetylase activity intramolecularly and that the C-terminal region stably associates with the catalytic core domain to form a SIRT1 holoenzyme. We also demonstrate that the C-terminal region of SIRT1 can influence the inhibitory activity of some sirtuin inhibitors that are known to function through the catalytic core domain. Together, these studies highlight the unique properties of the SIRT1 member of the sirtuin proteins and have implications for the development of SIRT1-specific regulatory molecules.     

Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase

The NAD(+)-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that is DBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD(+). We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex.     

Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells

We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD⁺-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.     

Reduced expression of SIRT1 is associated with diminished glucose-induced insulin secretion in islets from calorie-restricted rats

Alterations in food intake such as caloric restriction modulate the expression of SIRT1 and SIRT4 proteins that are involved in pancreatic β-cell function. Here, we search for a possible relationship between insulin secretion and the expression of SIRT1, SIRT4, PKC and PKA in islets from adult rats submitted to CR for 21 days. Rats were fed with an isocaloric diet (CTL) or received 60% (CR) of the food ingested by CTL. The dose-response curve of insulin secretion to glucose was shifted to the right in the CR compared with CTL islets (EC₅₀ of 15.1±0.17 and 10.5±0.11 mmol/L glucose). Insulin release by the depolarizing agents arginine and KCl was reduced in CR compared with CTL islets. Total islet insulin content and glucose oxidation were also reduced in CR islets. Leucine-stimulated secretion was similar in both groups, slightly reduced in CR islets stimulated by leucine plus glutamine but higher in CR islets stimulated by ketoisocaproate (KIC). Insulin secretion was also higher in CR islets stimulated by carbachol, compared with CTL islets. No differences in the rise of cytosolic Ca²⁺ concentrations stimulated by either glucose or KCl were observed between groups of islets. Finally, SIRT1, but not SIRT4, protein expression was lower in CR compared with CTL islets, whereas no differences in the expression of PKC and PKA proteins were observed. In conclusion, the lower insulin secretion in islets from CR rats was, at least in part, due to an imbalance between the expression of SIRT1 and SIRT4.     

SIRT1在宫颈癌细胞耐药中的作用及其机制研究

摘要目的：探讨沉默交配型信息调节因子2同源蛋白1（silentmatingtypeinformationregulation2homologyl,SIRTl）在宫颈癌化疗耐药中的作用及其机制。方法：体外培养人宫颈癌Hela细胞系和宫颈癌Hela／MMC耐药细胞亚系,westernblotting检测MMC对Hela和Hela／MMC细胞内SIRTl蛋白表达的影响;MTT法检测MMC及Nicotinamide对Hela和Hela／MMC细胞增殖的影响;AnnexinV-PI试验检测Hela／MMC细胞凋亡的亡的情况;RT—PCR方法检测耐药相关蛋白P—gP的mRNA表达情况。结果：正常情况下,Hela／MMC细胞中SIRTl的表达显著高于Hela细胞（P〈0．05）,MMC处理的Hela／MMC细胞中SIRTl的表达显著高于未经MMC处理（P〈0．05）。Nicotinamide对Hela和Hela／MMC细胞具有相似的生长抑制作用,Nicotinamide可使MMC诱导的Hela／MMC细胞凋亡增加,同时降低细胞内P-gP的mRNA表达（P〈0．05）。结论：SIRTl表达下调能显著减轻Hela／MMC细胞对MMC的耐药性,其作用可能与P—gp有关。