烟酰胺腺嘌呤二核苷酸和Sirtuins在衰老和疾病中的作用

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide, NAD⁺)作为氧化还原反应的重要辅酶,是能量代谢的核心。NAD⁺也是非氧化还原NAD⁺依赖性酶的共底物,包括沉默信息调节因子(Sirtuins)、聚ADP-核糖聚合酶(poly ADP-ribose polymerases, PARPs)、CD38/CD157胞外酶等。NAD⁺已成为细胞信号转导和细胞存活的关键调节剂。最近的研究表明,Sirtuins催化多种NAD⁺依赖性反应,包括去乙酰化、脱酰基化和ADP-核糖基化。Sirtuins催化活性取决于NAD⁺水平的高低。因此,Sirtuins是细胞代谢和氧化还原状态关键传感器。哺乳动物中已经鉴定并表征了7个Sirtuins家族成员(SIRT1-7),其参与炎症、细胞生长、生理节律、能量代谢、神经元功能、应激反应和健康衰老等多种生理过程。本文归纳了NAD⁺的生理浓度及状态、NAD⁺     

低温冷藏提高家蚕滞育卵NAD含量和胞质苹果酸脱氢酶活性

为了调查5℃低温处理是否改变家蚕Bombyx mori卵滞育NAD代谢, 本研究利用HPLC和分光光度法测定了经25℃和5℃分别处理的滞育卵中NADH 含量、 NAD⁺含量、 乳酸脱氢酶（LDH）活性和胞质苹果酸脱氢酶（cMDH）活性。结果表明： 5℃处理的NAD（NADH + NAD⁺）含量和cMDH活性分别增加了106%和53%, 并且显著高于25℃处理（P< 0.01）; 但是两种处理的NADH/NAD⁺比值和LDH活性没有显著差异（P> 0.05）。据此推测, 5℃低温处理加强了家蚕滞育卵NAD⁺合成和再生能力。     

SLC25A51——首次发现的哺乳动物细胞线粒体NAD+转运体

烟酰胺腺嘌呤二核苷酸(NAD⁺)及其还原形式NADH是糖酵解和线粒体呼吸作用中重要的辅因子,在能量代谢中发挥重要作用。当线粒体缺乏NAD⁺细胞因不能持续产生ATP而出现功能异常。以往研究发现酵母与植物的线粒体上均存在NAD+转运体,可以将NAD+转运至线粒体。但哺乳动物线粒体内膜上是否有NAD+转运体,一直存有争议。近来,美国宾夕法尼亚一研究团队首次证明SLC25A51可以在哺乳动物线粒体上发挥NAD+转运蛋白的功能。     

枯草芽孢杆菌氧化还原感应全局调控因子Rex对乙偶姻合成的影响

【背景】枯草芽孢杆菌体内含有一种可响应胞内氧化还原水平的因子,称之为氧化还原感应全局调控因子Rex (由基因ydiH编码)。Rex可通过感知辅酶NADH/NAD+水平的变化来调节胞内氧化还原平衡。【目的】研究Rex对枯草芽孢杆菌乙偶姻合成和辅因子代谢的相关性。【方法】利用比较转录组挖掘乙偶姻和2,3-丁二醇可逆转化过程中显著差异的基因,并通过Cre/lox基因敲除技术敲除ydiH、acuA (乙酰AcsA)和acoC (二氢脂酰胺乙酰转移酶)。随后,利用实时荧光定量PCR (RT-qPCR)技术分析敲除菌株中乙偶姻相关基因的转录水平。【结果】通过发酵实验发现,敲除ydiH会在一定程度上抑制菌体的生长速率,但发酵前期乙偶姻单位细胞产量和底物转化率都得到了显著提高;敲除acuA和acoC后,对乙偶姻合成、菌体生长和糖耗速率均影响不大;敲除ydiH后,与乙偶姻合成相关基因alsR (alsSD的正转录调控因子)、alsS (α-乙酰乳酸合成酶)、alsD (α-乙酰乳酸脱羧酶)和bdhA (2,3-丁二醇脱氢酶)的转录水平显著上调。【结论】枯草芽孢杆菌氧化还原感应全局调控因子Rex通过抑制与乙偶姻相关基因的转录水平影响乙偶姻合成。本研究首次报道了枯草芽孢杆菌中Rex和乙偶姻合成的相关性,为探索Rex如何通过调控相关基因的转录来影响胞内氧化还原稳态奠定了基础,也为提高枯草芽孢杆菌工业化生产强度和底物转化率提供了借鉴。     

工业微生物中NADH的代谢调控

NADH是微生物代谢网络中的一种关键辅因子。调节微生物胞内NADH的形式与浓度是定向改变和优化微生物细胞代谢功能, 实现代谢流最大化、快速化地导向目标代谢产物的重要手段之一。以下在详尽总结了NADH生理功能的基础上, 从生化工程(添加外源电子受体、不同氧化还原态底物及NAD合成前体物, 调节培养环境和氧化还原电势)和代谢工程(过量表达NADH代谢相关酶、缺失NADH竞争途径及引入NADH外源代谢途径)两方面分析、归纳了NADH代谢调控策略, 进而凝练出调控NADH/NAD+比率调节微生物细胞代谢功能研究方面亟待解决的3个科学问题及可能的解决途径。     

辅酶Ⅰ体内代谢调控研究进展

辅酶Ⅰ——烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD⁺）是一种在糖酵解、糖异生、三羧酸循环及呼吸链中发挥重要作用的辅酶,广泛参与DNA修复、组蛋白去乙酰化等生命过程。近年来研究表明NAD⁺合成的前体和中间化合物（具有维生素B3活性的烟酸、烟酰胺、烟酰胺核苷和烟酰胺单核苷酸）在预防糙皮病、延缓衰老,治疗神经和心血管多种疾病、调节胰岛素分泌、调控mRNA的表达等方面具有重要疗效。着重介绍了辅酶Ⅰ体内的合成代谢以及参与的调节衰老进程,以期为利用合成生物学技术在大肠杆菌中富集NAD⁺中间化合物提供理论依据和技术支撑。     

胞内氧化还原水平对嗜热厌氧乙醇菌 发酵代谢的影响

辅酶NADH/NAD+在细胞内氧化还原反应中起着重要的作用,是细胞生长和能量代谢必不可少的辅因子。调节微生物胞内NADH/NAD+的比率是定向改变微生物代谢,高效获得目标代谢产物的有效手段。嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus)是高温厌氧菌中乙醇产量较高的代表性菌株,本文利用不同氧化还原态的碳源改变T.ethanolicus的胞内NADH/NAD+含量和比例,进而研究了其对细胞生长、代谢产物分布的影响。以不同比例的葡萄糖/甘露醇作为混合碳源发酵,胞内氧化还原水平、细胞的生长特性、代谢产物都发生了不同程度的差异,以葡萄糖作为唯一碳源进行培养时,T.ethanolicus生长良好,乙醇产量为0.79g/L,但胞内NADH/NAD+比值和乙醇/乙酸的比值都比较低,分别为0.47和4.82;随着葡萄糖在混合碳源中比例的下降,NADH/NAD+比值增高,发酵产物中乙醇/乙酸比值也呈现上升的趋势。而以甘露醇作为唯一碳源时,发酵产物中乙醇浓度为0.389g/L,NADH/NAD+比值和乙醇/乙酸的比值分别为1.04和16.0。     

SIRT1功能及其对卵泡发育和卵母细胞成熟的调控作用 *

沉默信息调节因子1(silent information regulator1, SIRT1)是NAD⁺ 依赖的去乙酰化酶,通过使底物发生去乙酰化而参与细胞众多生理功能的调节,在糖脂代谢、衰老、细胞凋亡、氧化应激等过程中发挥了重要作用。另外,众多研究表明,SIRT1是调控动物卵巢老化、卵泡发育和卵母细胞成熟的重要因子,SIRT1 表达下降或活性改变将导致卵母细胞老化,降低动物的繁殖力。为了充分理解SIRT1功能,并通过调控SIRT1活性而延缓卵巢和卵母细胞老化,从而提高动物繁殖力,简述了SIRT1的激活及其参与细胞内调控的生物过程,并从能量代谢、抗氧化胁迫、染色质重塑的角度讨论了SIRT1的主要功能,重点阐述了SIRT1对动物卵泡发育和卵母细胞成熟的调控作用。     

NAD~+/NADH代谢机制研究进展

NAD+/NADH是细胞能量代谢所必需的辅酶,小到细胞的各种生命活动,大到整个生命结构的平衡,都需要能量来维持。同时,细胞的氧化还原状态,特别是NAD+/NADH的水平直接影响着细胞的节律、衰老、癌变和死亡等重大生命过程。故而有关细胞内NAD+或NADH代谢的研究近年在国际上形成了一个新的热点。我们以NAD+/NADH代谢为重点,综述国内外关于该机制的研究现状。     

高山被孢霉亚甲基四氢叶酸脱氢酶的克隆、表达和功能鉴定

叶酸代谢途径中的亚甲基四氢叶酸脱氢酶（MTHFD）可将5,10-亚甲基四氢叶酸氧化为5,10-甲炔基四氢叶酸,此过程会生成NADH或NADPH。对高山被孢霉中的MTHFD基因进行克隆、表达和功能鉴定,可进一步阐明脂质合成所需还原力NADPH的来源。首先对MTHFD序列进行分析,并以pET28a（+）质粒为载体构建了MTHFD的表达载体,然后转化至大肠杆菌BL21中进行诱导表达。进一步利用Ni金属螯合层析纯化目的蛋白,采用比色法分析酶反应产物,表明纯化蛋白质具有MTHFD活性。高山被孢霉MTHFD对NAD⁺和NADP⁺均具有催化能力,但更偏好于将NADP⁺转化为NADPH。最后对高山被孢霉进行发酵培养,发现MTHFD的转录水平在脂质开始积累后发生了明显的上调,表明MTHFD在高山被孢霉脂质合成过程中发挥重要作用,很可能是脂质合成所需NADPH的关键来源。这为对高山被孢霉进行分子改造,使之成为高产各种多不饱和脂肪酸的细胞工程提供了理论依据。     

Photocatalyzed Anaerobic Oxidation of Nicotinamide Coenzyme Dimers to NAD+ by Adriamycin

The nicotinamide adenine dinucleotide dimers (NAD)₂ obtained by electrochemical reduction of NAD⁺ are oxidized by adriamycin in anaerobic photocatalyzed reaction yielding NAD⁺ and 7-deoxyadriamyci-none. Under the same conditions NADH is not oxidized.     

Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.     

A versatile colony assay based on NADH fluorescence

Direct visualization of the activity of enzymes expressed by bacterial colonies attached to a solid support, often referred to as “filter assay”, is a powerful strategy for the identification of new or improved biocatalysts. In this work we demonstrate the usefulness of NAD⁺/NADH coupled enzymatic reactions as visualization tool in such experimental setups. Dehydrogenases, capable of oxidizing or reducing the reaction product released from the bacterial colony were supplemented to the screening solution, together with the screening substrate and a sufficient amount of NAD⁺ or NADH, respectively. We also examined the screening of directly NAD⁺/NADH coupled reactions. The release or consumption of NADH in the area of colonies was monitored on behalf of its fluorescence at 450 nm. Excitation was achieved by standard “black-light” UV tubes (340–360 nm). The visible fluorescence signal was recorded using a CCD-camera. We got excellent results for the screening of threonine aldolases and esterases and were able to show the principle utility for amidase, nitrilase, nitrile hydratase, hydroxynitrile lyase and benzaldehyde dehydrogenase active colonies.     

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP⁺ or NAD⁺ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP⁺) is better electron donor (or acceptor), while NADH (or NAD⁺) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP⁺, 2′-AMP and and atebrin.     

Mitochondrial membrane potential, transmembrane difference in the NAD redox potential and the equilibrium of the glutamate-aspartate translocase in the isolated perfused rat heart

The distribution of glutamate and aspartate and the mitochondrial membrane potential (Δψ) were studied in isolated rat heart mitochondria and in the intact perfused rat heart. The diffusion potential imposed by the glutamate-aspartate exchange through mediation of the electrogenic glutamate-aspartate translocator attained a value close to the mitochondrial Δψ measured from the distribution of triphenylmethylphosphonium ion (TPMP⁺) both in isolated mitochondria and in intact myocardium. Distributions of the Δψ probe and metabolites were determined by subcellular fractionation of the heart muscle in a non-aqueous medium. The results indicate that the glutamate-aspartate translocator is in near equilibrium in the myocardium. The diffusion potential of the glutamate-aspartate exchange, and the mitochondrial/cytosolic difference in the redox potentials of the free NAD⁺/NADH pools are equal allowing for experimental error. These data obtained from intact tissue can therefore be interpreted as supporting the notion of the transmembrane uphill transport of reducing equivalent from the cytosolic free NAD⁺/NADH pool being driven by the malate-aspartate cycle energized by the mitochondrial Δψ.     

Redox sensing by a Rex‐family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus

An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox‐sensing regulator Rex of S. aureus binds to this inverted repeat. The binding sequence – TTGTGAAW₄TTCACAA – is highly conserved in S. aureus. Rex binding to this sequence leads to the repression of genes located downstream. The binding activity of Rex is enhanced by NAD⁺ while NADH, which competes with NAD⁺ for Rex binding, decreases the activity of Rex. The impact of Rex on global protein synthesis and on the activity of fermentation pathways under aerobic and anaerobic conditions was analysed by using a rex‐deficient strain. A direct regulatory effect of Rex on the expression of pathways that lead to anaerobic NAD⁺ regeneration, such as lactate, formate and ethanol formation, nitrate respiration, and ATP synthesis, is verified. Rex can be considered a central regulator of anaerobic metabolism in S. aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon.     

Energy-linked electron transfer reactions in Rhodopseudomonas viridis

The particulate fraction of Rhodopseudomonas viridis when supplied with succinate catalyses the reduction of NAD⁺ by light; this reaction is inhibited by uncouplers of oxidative phosphorylation but not by oligomycin. Formation of NADH takes place in the dark when ATP or PP_i is supplied. Both light and dark reactions are inhibited by valinomycin and nigericin, when added together, but not by either separately. NADH formation in R. viridis appears to take place by an energy-dependent reversal of electron flow and energy may be conserved in the form of a membrane potential. The addition of ATP caused the oxidation of both C553 and C558 in chromatophores; carbonylcyanide p-trifluoromethoxyphenylhydrazone and oligomycin abolished this oxidation.

The NAD⁺ and NADH concentrations at equilibrium in the light-dependent reaction were determined and the oxidation-reduction potential of this couple calculated. From this value it was calculated that under these experimental conditions the energy requirement to form NADH from the succinate/fumarate couple at E_h = o V was 9.4 kcal.

Particles of R. viridis contained an active transhydrogenase, driven by either light or ATP, that was sensitive to uncouplers of oxidative phosphorylation; the light-driven reaction was insensitive to oligomycin and was inhibited by antimycin A and 2-heptyl-4-hydroxyquinone-N-oxide.

R. viridis did not grow aerobically but particles contained NADH oxidase activity that was cyanide sensitive. There was no spectroscopic evidence for cytochromes of the b-type in reduced-minus-oxidised spectra of particles or in pyridine haemochrome spectra of whole cells.     

Rapid microfluorimetry of enzyme reactions in single living cells

An optimized photon counting technique allows the microfluorimetric study of NAD⁺ (or NADP⁺) reduction-reoxidation transients in single living cells with a time resolution in the range of 1/50-1/100 sec. The transients resulting from the micro-electrophoretic addition of metabolites (e.g. Glc-6-P or Glc-1-P) can be analyzed in terms of early parameters (e.g. initial lag, rise half time or full rise time) and overall parameters (time of rise and half decay, amplitude, reoxidation time). Both the initial lag and rise half time are considerably longer with Glc-1-P than with Glc-6-P, possibly due to control at the phosphoglucomutase or compartmentation of glycolytic phosphate esters. While glycolytic NAD⁺ (or NADP⁺) reduction proceeds adequately in aerobic EL2 and EAT ascites cells (although ΔNADH/Δt is higher at anaerobiosis), it is critically dependent upon anaerobiosis in L and astrocytoma cells. Thus by rapid microfluorimetry it is possible to resolve the rising phase or other segments of the fluorescence transients into components each corresponding to a particular step in the sequence of intracellular events or control states.     

A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2)

We describe the identification of Rex, a novel redox-sensing repressor that appears to be widespread among Gram-positive bacteria. In Streptomyces coelicolor Rex binds to operator (ROP) sites located upstream of several respiratory genes, including the cydABCD and rex-hemACD operons. The DNA-binding activity of Rex appears to be controlled by the redox poise of the NADH/NAD+ pool. Using electromobility shift and surface plasmon resonance assays we show that NADH, but not NAD+, inhibits the DNA-binding activity of Rex. However, NAD+ competes with NADH for Rex binding, allowing Rex to sense redox poise over a range of NAD(H) concentrations. Rex is predicted to include a pyridine nucleotide-binding domain (Rossmann fold), and residues that might play key structural and nucleotide binding roles are highly conserved. In support of this, the central glycine in the signature motif (GlyXGlyXXGly) is shown to be essential for redox sensing. Rex homologues exist in most Gram-positive bacteria, including human pathogens such as Staphylococcus aureus, Listeria monocytogenes and Streptococcus pneumoniae.