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。     

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。     

转录调控因子Rex的功能及调节机制研究进展

转录因子Rex是一种广泛存在于革兰氏阳性菌,能够与NADH或者NAD⁺直接结合响应胞内NADH/NAD⁺的氧化还原传感器,与靶基因的结合可调节细胞内的多种生理代谢。NAD（H）是调节细胞能量代谢的必需辅酶,显示微生物细胞内的氧化还原状态。研究发现Rex的调节活性与细胞内NADH/NAD⁺比率相关。需氧和厌氧菌属中Rex单体和复合物晶体结构的解析揭示了Rex、NADH/NAD⁺和靶基因间的作用关系及调控机制。通过比较分析了不同菌株中Rex单体和复合物的晶体蛋白结构,并揭示了NADH/NAD⁺对Rex调控活性的影响,进一步解析了Rex与碳和能量代谢、厌氧代谢、发酵、生物膜等之间的联系,并展望了Rex的研究和应用方向。     

关键酶基因转录水平对重组大肠杆菌合成NAD+的影响

【目的】烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide)是生物体内一种重要的辅因子,其细胞内含量对于NAD⁺依赖型氧化还原反应和有关生化合成代谢具有重要意义。为强化辅因子合成,本文通过不同诱导条件下关键酶基因转录水平与产物合成水平的相关性分析,利用增加正向关键酶基因拷贝数策略提高胞内氧化型辅酶NAD+的浓度。【方法】以实验室前期构建的大肠杆菌NA016为出发菌株,分别从诱导温度、诱导剂浓度、诱导时机三个方面进行了诱导条件的优化,并利用实时荧光定量PCR(RT-qPCR)技术对代谢改造中的有关过表达基因进行了转录水平分析,确定了NAD+~含量与这些过表达基因转录水平之间的相关性,增加对NAD⁺合成代谢具有正向作用的基因拷贝数以进一步提高胞内NAD+水平。【结果】通过诱导条件优化实验确定菌株NA016的最适诱导温度,在诱导时机为OD600达到0.6时加入0.8mmol/L的IPTG可使胞内NAD+含量提高35.37%;转录水平方面分析发现基因nadE和pncB的表达对NAD+的合成具有正向调节作用。进一步...     

β-烟酰胺单核苷酸合成研究进展

衰老过程中辅酶I-烟酰胺腺嘌呤二核苷酸(NAD⁺)水平下降被认为是导致疾病和残疾的主要原因。β-烟酰胺单核苷酸(NMN)是NAD⁺的直接前体,是提高细胞内NAD⁺水平的关键成分。口服补充NMN可以延缓、改善、防止与衰老相关的多种表型,改善代谢紊乱和老年疾病等。详细介绍了NMN的主要生理作用以及目前合成NMN的方法,包括化学法、微生物发酵法以及基于各种生物酶的体外合成方法。其中重点介绍了生物酶法的各种合成路线,并指出以烟酰胺核糖(NR)为基础的NMN的合成是最为理想的工艺路线,但转化工艺中涉及到的关键酶如烟酰胺核糖激酶(NRK)等的活性以及酶的稳定性需要进一步提高,才能达到降低生产成本的目的。     

NAD^+与细胞衰老

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD^+)作为糖酵解、三羧酸循环和氧化磷酸化中关键酶的辅助因子,参与了细胞的物质代谢、能量合成、损伤DNA的修复等多种生理病理过程。近年来越来越多的研究发现,细胞内NAD^+水平在机体或细胞衰老过程中呈明显下降趋势,而补充NAD^+能延缓细胞/机体的衰老,使NAD^+及其前体物质在细胞衰老中的作用受到广泛关注。该文就NAD^+及其前体物质与细胞代谢、衰老的关系及相关分子机制研究的最新进展进行综述,以期深入认识NAD^+与细胞衰老的内在联系,为细胞衰老相关的基础及应用研究提供理论参考。     

槲皮素对节律钟基因的调控研究*

目的:探讨槲皮素对节律钟基因表达的影响。方法:通过50%的马血清刺激诱导人骨肉瘤U2OS细胞同步化,利用槲皮素处理同步化后的U2OS细胞。进一步利用荧光定量PCR检测节律钟关键基因的变化。利用western blot检测槲皮素对组蛋白乙酰化的影响,并且通过试剂盒测定槲皮素对细胞内氧化型烟酰胺腺嘌呤二核苷酸(NAD+)的影响。通过尼克酰胺和槲皮素同时处理U2OS细胞,利用荧光定量PCR检测节律钟关键基因的变化。结果:U2OS细胞经过槲皮素处理后节律钟关键基因芳烃受体核转位蛋白3(brain and muscle Arnt-like protein-1,BMAL1),节律周期蛋白2(Period 2, PER2),孤儿核受体alpha(REV-ERBα)和蓝光受体蛋白1(Cryptochrome 1,CRY1)在转录水平的表达水平有明显的升高。槲皮素处理可以显著降低U2OS细胞的组蛋白乙酰化的水平,并且显著升高U2OS细胞内的氧化型烟酰胺腺嘌呤二核苷酸的水平。进一步研究发现,尼克酰胺(Nicotinamide,NAM)处理完全抑制了槲皮素对节律基因的影响。结论:槲皮素显著地激活了节律钟关键基因的mRNA表达水平,槲皮素对于节律基因的调控依赖于Sirtuins的活性,其机制可能是由于槲皮素增加了细胞内的氧化型烟酰胺腺嘌呤二核苷酸的水平所导致。     

含有多种酶活性的SIRT5蛋白在细胞代谢中的功能

Sirtuins作为Ⅲ型蛋白质去乙酰化酶调控机体多种生理进程,包括DNA修复、基因组稳定性、能量代谢、衰老以及癌症发生.目前已鉴定出7种人类Sirtuins家族的蛋白(SIRT1–SIRT7),其组织分布、亚细胞定位以及酶作用的底物都不尽相同.本文将着重描述Sirtuins家族的一个成员—SIRT5以及其在调控细胞代谢中的多种酶活性.     

加速康复外科对腹腔镜胃癌根治术患者营养状态,免疫功能及炎性因子水平的影响

目的:探讨加速康复外科(ERAS)对腹腔镜胃癌根治术患者营养状态,免疫功能及炎性因子水平的影响。方法:将2016年1月~2020年1月于我院行腹腔镜胃癌根治术的胃癌患者166例纳入本研究,按照随机数字表法分为ERAS组(n=83)与对照组(n=83),对照组行常规处理,ERAS组以ERAS处理。观察两组术后1 d、7 d血红蛋白(Hb)、视黄醇结合蛋白(RbP)、转铁蛋白(TRF)、前白蛋白(PRE)、白蛋白(ALB)等营养指标,免疫球蛋白A(Ig A)、免疫球蛋白G(Ig G)、免疫球蛋白M(Ig M)等免疫球蛋白,CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺等T细胞亚群,白介素-6(IL-6)、白介素-8(IL-8)、白介素-10(IL-10)等炎性因子及术后并发症等指标。结果:与术后1 d比较,两组术后7 d Hb、RbP、TRF、PRE、ALB、Ig A、Ig G、Ig M,CD3⁺、CD4⁺、CD4⁺/CD8⁺水平均升高,CD8⁺、IL-6、IL-8、IL-10水平均降低(P<0.05)。术后7 d,ERAS组Hb、RbP、TRF、PRE、ALB、Ig A、Ig G、Ig M、CD3⁺、CD4⁺、CD4⁺/CD8⁺水平均高于对照组,CD8⁺、IL-6、IL-8、IL-10低于对照组(P<0.05)。ERAS组术后并发症发病率(8.43%)低于对照组(22.89%),差异有统计学意义(P<0.05)。结论:ERAS应用于腹腔镜胃癌根治术中可有效降低患者机体应激反应,改善营养状态,解除免疫抑制,清除炎性因子,减少术后并发症,有助于患者康复。     

构建可合成非天然辅酶的圆红冬孢酵母工程菌*

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD)及其还原态是生物体通用的氧化还原辅酶和重要小分子,参与胞内众多代谢反应,因此调控NAD水平不仅难以选择性作用于代谢途径,还常常产生意外的生物学效应。最近研究发现利用非天然辅酶烟酰胺胞嘧啶二核苷酸(nicotinamide cytosine dinucleotide,NCD),可构建正交的氧化还原催化体系,为调控胞内代谢提供了新机遇。为实现在产油酵母圆红冬孢酵母中建立NCD介导的氧化还原代谢,采用农杆菌介导转化方法,在基因组整合表达密码子优化的NCD合酶(NcdS)编码基因NCDS,获得系列有效表达NcdS的工程菌株。酶偶联法分析发现,工程菌细胞裂解液NcdS酶活达8.1×10^-3 U/OD_{600 nm}。通过高效液相色谱法(HPLC)和超高分辨率质谱检测,确定细胞裂解液可催化合成NCD。在培养基内补加5.0 mmol/L烟酰胺核糖后,工程菌胞内合成NCD达41.6 μmol/L。对工程菌进行发酵和油脂提取,发现胞内表达NCD合酶未导致细胞产油性能降低,后续可通过表达其他NCD偏好性酶,有望在圆红冬孢酵母中建立受NCD调控的油脂合成代谢体系。     

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.     

SIRTain regulators of premature senescence and accelerated aging

The sirtuin proteins constitute class III histone deacetylases (HDACs). These evolutionarily conserved NAD⁺-dependent enzymes form an important component in a variety of cellular and biological processes with highly divergent as well as convergent roles in maintaining metabolic homeostasis, safeguarding genomic integrity, regulating cancer metabolism and also inflammatory responses. Amongst the seven known mammalian sirtuin proteins, SIRT1 has gained much attention due to its widely acknowledged roles in promoting longevity and ameliorating age-associated pathologies. The contributions of other sirtuins in the field of aging are also gradually emerging. Here, we summarize some of the recent discoveries in sirtuins biology which clearly implicate the functions of sirtuin proteins in the regulation of premature cellular senescence and accelerated aging. The roles of sirtuins in various cellular processes have been extrapolated to draw inter-linkage with anti-aging mechanisms. Also, the latest findings on sirtuins which might have potential effects in the process of aging have been reviewed.     

去乙酰化酶Sirtuin研究进展

依赖于NAD 的去乙酰化酶Sirtuin对细胞的存活、衰老、凋亡等生理活动的调节起到十分重要的作用。Sirtuin系统中的ySir2和SIRT1就目前来说是研究得较为透彻的两个成员。ySir2参与了酵母的交配型基因沉默、端粒的沉默、rDNA重复序列的沉默以及细胞寿命等生理功能。人类SIRT1在细胞存活与代谢等过程中也起到调节作用。本文对Sirtuin的结构、作用机制、底物特异性、影响因子及其功能作了综述。     

Coenzyme production using immobilized enzymes. I. Preparation, characterization, and laboratory-scale application of an immobilized NAD kinase

NAD⁺ kinase (ATP: NAD⁺ 2-phosphotransferase, EC2.7.1.23) isolated from chicken liver was immobilized on a silica-based support possessing aldehyde functional groups. The highest catalytic activity achieved was 16 U g⁻¹ solid. The optimal pH for the catalytic activity of the immobilized NAD⁺ kinase was pH 7.1–7.3. The apparent optimum temperature for the immobilized enzyme was about 5°C higher than that of the soluble enzyme. There were no significant differences in the K_{m app} values. The immobilization improved the conformational stability of the enzyme. In preliminary experiments, a 95% conversion of NAD⁺ to NADP⁺ was achieved with use of the immobilized NAD⁺ kinase, which preserved its starting activity practically unchanged up to 36 days.     

核糖核酸5'端NAD+帽子修饰研究进展

m7G帽子具有保护RNA不被降解以及招募相关蛋白参与内含子剪切、poly(A)加尾、出核和翻译等功能。一直以来,它被认为是真核生物mRNA所特有的修饰类型。然而近年来,在包括原核生物在内的多个物种中均检测到一种新的RNA 5’端修饰,即核酸代谢物烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD⁺)帽子。目前NAD⁺修饰RNA(NAD-RNA)的生物学功能研究仍处于起始阶段。本文概述了NAD-RNA的发现及其检测和鉴定技术的发展;探讨了NAD⁺帽子对RNA的调控功能,以及NAD-RNA脱帽和加帽的影响因素;并进一步推测NAD-RNA在生物的生长、发育和环境响应中发挥的潜在功能。最后,展望了未来NAD-RNA的研究方向和主题。     

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.     

SIRT2-mediated protein deacetylation: An emerging key regulator in brain physiology and pathology

Protein function is considerably altered by posttranslational modification. In recent years, cycles of acetylation/deacetylation emerged as fundamental regulators adjusting biological activity of many proteins. Particularly, protein deacetylation by Sirtuins, a family of atypical histone deacetylases (HDACs), was demonstrated to regulate fundamental cell biological processes including gene expression, genome stability, mitosis, nutrient metabolism, aging, mitochondrial function and cell motility. Given this wealth of biological functions, perhaps not unexpectedly then, pharmacological compounds targeting Sirtuin activity are now prime therapeutic agents for alleviating severity of major diseases encompassing diabetes, cancer, cardiovascular and neurodegenerative disorders in many organs. In this review, we will focus on the brain and its physiological and pathological processes governed by Sirtuin-mediated deacetylation. Besides discussing Sirtuin function in neurodegenerative diseases, emphasis will be given on the mounting evidence deciphering key developmental brain functions for Sirtuins in neuronal motility, neuroprotection and oligodendrocyte differentiation. In this respect, we will particularly highlight functions of the unconventional family member SIRT2 in post-mitotic neurons and glial cells.     

Emerging roles of SIRT6 on telomere maintenance,DNA repair,metabolism and mammalian aging

With the characterization of Sir2 gene in yeast aging, its mammalian homologs Sirtuins 1–7 have been attracting attention from scientists with various research backgrounds. Among Sirtuins, SIRT1 is the most extensively studied. Recent progress on mammalian Sirtuins has shown that SIRT6 as a histone deacetylase may also play a critical role in regulating mammalian aging. This review summarizes recent advances on SIRT6 as a key modulator of telomere structure, DNA repair, metabolism, and NF-kappa B pathway in aging. In addition, we discuss the challenges that remain to be studied in SIRT6 biology.