Sirtuin在热量限制延长寿命机制中的研究进展

热量限制(caloric restriction, CR)可以引起细胞、生物体寿命延长和降低衰老相关疾病的发生,其中Sirtuin起着关键作用．Sirtuin将机体能量代谢和基因表达调控相偶联,通过赖氨酸去乙酰化改变蛋白质的活性和稳定性,从而调节衰老进程．酵母中度CR影响其复制寿命和时序寿命,主要依赖于激活Sir2,增加细胞内NAD+/NADH的比例和调节尼克酰胺浓度来实现．类似的机制也存在于秀丽线虫和果蝇中．哺乳动物在CR条件下SIRT1蛋白表达应答性上升,细胞中NAM磷酸基转移酶能够直接影响NAM和NAD+浓度,并影响SIRT1活性．NO表达增加能导致SIRT1上调和线粒体合成增加．SIRT1可能通过改变组蛋白、p53、NES1、FOXO等底物蛋白的乙酰化影响到细胞和个体的衰老．表明不同生物体中的Sirtuin及其同源类似物在CR条件下对衰老进程和寿命都起着非常重要的作用．     

NAD+/NADH homeostasis affects metabolic adaptation to hypoxia and secondary metabolite production in filamentous fungi*

Filamentous fungi are used to produce fermented foods, organic acids, beneficial secondary metabolites and various enzymes. During such processes, these fungi balance cellular NAD⁺:NADH ratios to adapt to environmental redox stimuli. Cellular NAD(H) status in fungal cells is a trigger of changes in metabolic pathways including those of glycolysis, fermentation, and the production of organic acids, amino acids and secondary metabolites. Under hypoxic conditions, high NADH:NAD⁺ ratios lead to the inactivation of various dehydrogenases, and the metabolic flow involving NAD⁺ is down-regulated compared with normoxic conditions. This review provides an overview of the metabolic mechanisms of filamentous fungi under hypoxic conditions that alter the cellular NADH:NAD⁺ balance. We also discuss the relationship between the intracellular redox balance (NAD/NADH ratio) and the production of beneficial secondary metabolites that arise from repressing the HDAC activity of sirtuin A via Nudix hydrolase A (NdxA)-dependent NAD⁺ degradation.     

Modulating NAD+ metabolism,from bench to bedside

Discovered in the beginning of the 20^th century, nicotinamide adenine dinucleotide (NAD⁺) has evolved from a simple oxidoreductase cofactor to being an essential cosubstrate for a wide range of regulatory proteins that include the sirtuin family of NAD⁺‐dependent protein deacylases, widely recognized regulators of metabolic function and longevity. Altered NAD⁺ metabolism is associated with aging and many pathological conditions, such as metabolic diseases and disorders of the muscular and neuronal systems. Conversely, increased NAD⁺ levels have shown to be beneficial in a broad spectrum of diseases. Here, we review the fundamental aspects of NAD⁺ biochemistry and metabolism and discuss how boosting NAD⁺ content can help ameliorate mitochondrial homeostasis and as such improve healthspan and lifespan.     

Dietary restriction involves NAD+‐dependent mechanisms and a shift toward oxidative metabolism

Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD⁺‐dependent deacylases known as sirtuins, and increases in NAD⁺ that derive from higher levels of respiration. Here, we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens [DAF‐16 (FOXO), SKN‐1 (Nrf1/2/3), PHA‐4 (FOXA), AAK‐2 (AMPK)]. Complete DR lifespan extension required the sirtuin SIR‐2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC‐1, a key NAD⁺ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc‐1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD⁺‐dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD⁺ salvage. Under DR conditions, NAD⁺‐dependent processes may be supported by a DR‐induced shift toward oxidative metabolism rather than an increase in total respiration.     

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O₂ consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD⁺ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD⁺ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD⁺ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD⁺ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD⁺-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Comparison of the Kinetic Behavior toward Pyridine Nucleotides of NAD-Linked Dehydrogenases from Plant Mitochondria

In this article we compare the kinetic behavior toward pyridine nucleotides (NAD⁺, NADH) of NAD⁺-malic enzyme, pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine decarboxylase extracted from pea (Pisum sativum) leaf and potato (Solanum tuberosum) tuber mitochondria. NADH competitively inhibited all the studied dehydrogenases when NAD⁺ was the varied substrate. However, the NAD⁺-linked malic enzyme exhibited the weakest affinity for NAD⁺ and the lowest sensitivity for NADH. It is suggested that NAD⁺-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I.     

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD⁺]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD⁺]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD⁺]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD⁺]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD⁺]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.     

Potential role of sirtuins in livestock production

Sirtuins are NAD⁺-dependent histone and protein deacetylases, which have been studied during the last decade with a focus on their role in lifespan extension and age-related diseases under normal and calorie-restricted or pathological conditions. However, sirtuins also have the ability to regulate energy homeostasis as they can sense the metabolic state of the cell through the NAD⁺/NADH ratio; hence, changes in the diet can modify the expression of these enzymes. Dietary manipulations are a common practice currently being used in livestock production with favorable results, probably due in part to the enhanced activity of sirtuins. Nevertheless, sirtuin expression in livestock species has not been a research target. For these reasons, the goal of this review is to awaken interest in these enzymes for future detailed characterization in livestock species by presenting a general introduction to what sirtuins are, how they work and what is known about their role in livestock.     

Hexose metabolism in pancreatic islets: Effect of D-glucose on the mitochondrial redox state

The mitochondrial NADH/NAD⁺ ratio for free nucleotides in rat pancreatic islets was judged from the cell content in L-glutamate and L-alanine, 2-ketoglutarate and pyruvate, and NH ₄ ⁺ . At a physiological concentration of D-glucose, such a ratio averaged 9.6±1.1%. A rise in hexose concentrations, above a threshold value in excess of 5.6 mM, caused a rapid, sustained and rapidly reversible decrease in the mitochondrial NADH/NAD⁺ ratio. It is speculated that in the process of glucose-stimulated insulin release, the latter change participates in the coupling between metabolic and secretory events by favouring both the activity of key mitochondrial dehydrogenases and the translocation of Ca²⁺ from the mitochondria into the cytosol.     

Fasting promotes the expression of SIRT1, an NAD+-dependent protein deacetylase, via activation of PPARα in mice

Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD⁺/NADH ratio in cells and results in activation of SIRT1, an NAD⁺-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARα, PPARγ, and PPARβ/δ, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARγ by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARα correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARα in the livers, where the NAD⁺ levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD⁺ salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD⁺ or NADH showed that the mRNA expression of both SIRT1 and PPARα can be enhanced by addition of NAD⁺, and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARα agonist suggested that PPARα is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.     

Elimination of glycerol and replacement with alternative products in ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2–3% of the sugar fermented is converted to glycerol. Replacing the NAD⁺-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD⁺ by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD⁺ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.     

Augmentation of cellular NAD+ by NQO1 enzymatic action improves age‐related hearing impairment

Age‐related hearing loss (ARHL) is a major neurodegenerative disorder and the leading cause of communication deficit in the elderly population, which remains largely untreated. The development of ARHL is a multifactorial event that includes both intrinsic and extrinsic factors. Recent studies suggest that NAD⁺/NADH ratio may play a critical role in cellular senescence by regulating sirtuins, PARP‐1, and PGC‐1α. Nonetheless, the beneficial effect of direct modulation of cellular NAD⁺ levels on aging and age‐related diseases has not been studied, and the underlying mechanisms remain obscure. Herein, we investigated the effect of β‐lapachone (β‐lap), a known plant‐derived metabolite that modulates cellular NAD⁺ by conversion of NADH to NAD⁺ via the enzymatic action of NADH: quinone oxidoreductase 1 (NQO1) on ARHL in C57BL/6 mice. We elucidated that the reduction of cellular NAD⁺ during the aging process was an important contributor for ARHL; it facilitated oxidative stress and pro‐inflammatory responses in the cochlear tissue through regulating sirtuins that alter various signaling pathways, such as NF‐κB, p53, and IDH2. However, augmentation of NAD⁺ by β‐lap effectively prevented ARHL and accompanying deleterious effects through reducing inflammation and oxidative stress, sustaining mitochondrial function, and promoting mitochondrial biogenesis in rodents. These results suggest that direct regulation of cellular NAD⁺ levels by pharmacological agents may be a tangible therapeutic option for treating various age‐related diseases, including ARHL.     

Analysis and identification of ADP-ribosylated proteins of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> M145

Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD⁺ to acceptor proteins catalyzed by ADP-ribosyltransferases. Using m-aminophenylboronate affinity chromatography, 2D-gel electrophoresis, in-gel digestion and MALDI-TOF analysis we have identified eight in vitro ADP-ribosylated proteins in Streptomyces coelicolor, which can be classified into three categories: (i) secreted proteins; (ii) metabolic enzymes using NAD⁺/NADH or NADP⁺/NADPH as coenzymes; and (iii) other proteins. The secreted proteins could be classified into two functional categories: SCO2008 and SC05477 encode members of the family of periplasmic extracellular solute-binding proteins, and SCO6108 and SC01968 are secreted hydrolases. Dehydrogenases are encoded by SC04824 and SC04771. The other targets are GlnA (glutamine synthetase I., SC02198) and SpaA (starvation-sensing protein encoded by SC07629). SCO2008 protein and GlnA had been identified as ADP-ribosylated proteins in previous studies. With these results we provided experimental support for a previous suggestion that ADP-ribosylation may regulate membrane transport and localization of periplasmic proteins. Since ADP-ribosylation results in inactivation of the target protein, ADP-ribosylation of dehydrogenases might modulate crucial primary metabolic pathways in Streptomyces. Several of the proteins identified here could provide a strong connection between protein ADP-ribosylation and the regulation of morphological differentiation in S. coelicolor.     

Glutamate Dehydrogenases: The Why and How of Coenzyme Specificity

NAD⁺ and NADP⁺, chemically similar and with almost identical standard oxidation–reduction potentials, nevertheless have distinct roles, NAD⁺ serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD⁺-dependent for glutamate oxidation, NADP⁺-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD⁺ reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD⁺ but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP⁺ reduction by NADH, maintaining the coenzyme pools at different oxidation–reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD⁺-dependent, NADP⁺-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD⁺ or for NADP⁺ has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2′- and 3′-hydroxyls, dictating NAD⁺ specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD⁺ only, NADP⁺ only, or in higher animals both.     

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

Calorie restriction effects on silencing and recombination at the yeast rDNA

Aging research has developed rapidly over the past decade, identifying individual genes and molecular mechanisms of the aging process through the use of model organisms and high throughput technologies. Calorie restriction (CR) is the most widely researched environmental manipulation that extends lifespan. Activation of the NAD⁺‐dependent protein deacetylase Sir2 (S ilent I nformation R egulator 2) has been proposed to mediate the beneficial effects of CR in the budding yeast Saccharomyces cerevisiae, as well as other organisms. Here, we show that in contrast to previous reports, Sir2 is not stimulated by CR to strengthen silencing of multiple reporter genes in the rDNA of S. cerevisiae. CR does modestly reduce the frequency of rDNA recombination, although in a SIR2‐independent manner. CR‐mediated repression of rDNA recombination also does not correlate with the silencing of Pol II‐transcribed noncoding RNAs derived from the rDNA intergenic spacer, suggesting that additional silencing‐independent pathways function in lifespan regulation.     

Polyol Dehydrogenases in the Soluble Fraction of Acetobacter melanogenum

Polyol dehydrogenases of Acetobacter melanogenum were investigated. Three polyol dehydrogenases, i. e. NAD⁺-linked d-mannitol dehydrogenase, NAD⁺-linked sorbitol dehydrogenase and NADP⁺-linked d-mannitol dehydrogenase, in the soluble fraction of the organism were purified 12-fold, 8-fold and 88-fold, respectively, by fractionation with ammonium sulfate and DEAE-cellulose column chromatography. NAD⁺-linked sorbitol dehydrogenase reduced 5-keto-d-fructose (5KF) to l-sorbose in the presence of NADH, whereas NADP⁺-linked d-mannitol dehydrogenase reduced the same substrate to d-fructose in the presence of NADPH. It was also shown that NAD⁺-linked d-mannitol dehydrogenase was specific for the interconversion between d-mannitol and d-fructose and that this enzyme was very unstable in alkaline conditions.     

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1^H/H) live shorter and show signs of accelerated aging. As wild‐type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age‐related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1‐7) are a family of NAD⁺‐dependent deacetylases that can delay age‐related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD⁺ and the ability of SIRT2 to maintain lysine‐668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD⁺ precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1^H/H animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD⁺ to delay diseases of aging in mammals is warranted.