烟酰胺腺嘌呤二核苷酸在心血管疾病中的研究进展

心血管疾病（cardiovascular disease, CVD）是造成全球性死亡率升高的主要原因。烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide, NAD⁺）是一种多功能的辅酶,参与多种生物学过程,如细胞能量代谢、细胞信号传导、DNA修复和蛋白质修饰等。许多研究表明,NAD⁺水平随着年龄、肥胖和高血压等CVD的风险因素而降低。此外,提高机体NAD⁺水平还可以减轻慢性炎症反应、重新激活自噬和线粒体的生物功能。在患有血管疾病的人类中,提高NAD⁺水平还能增强细胞的抗氧化和代谢能力。动物模型研究发现,NAD⁺水平的提高还能降低血压、增加寿命、预防代谢相关疾病。最近有研究揭示,通过基因手段、药物或日常饮食提升NAD⁺水平,能有效改善动物和人类心血管健康的病理生理状况。在本综述中,我们首先详细阐述了NAD⁺在维持血管健康方面的作用,然后总结了其与血管功能和疾病相关的最新研究进展,包括高血压、动脉粥样硬化和冠状动脉疾病等。此外,本综述还评估了各种NAD⁺前体在提升NAD⁺水平方面的效率,以及这些NAD⁺前体对预防或治疗CVD的临床效果,旨在为新的治疗策略提供潜在参考。     

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⁺     

烟酰胺腺嘌呤二核苷酸合成相关酶有助于1,4-丁二醇转化为4-羟基丁酸

4-羟基丁酸(4-HB)不仅具有医学应用价值,而且是合成生物材料P3HB4HB的重要前体.在烟酰胺腺嘌呤二核苷酸(NAD)参与情况下,大肠杆菌Escherichia coli S17-1(pZL-dhaT-aldD)可以把1,4-丁二醇(1,4-BD)转化为4HB.为提高4HB产率,通过过表达烟酸磷酸核糖转移酶(PncB)和烟酰胺腺嘌呤二核苷酸合成酶(NadE)增加胞内NAD含量,从而加速1,4-BD转化反应的进行.结果表明,PncB-NadE的表达使1,4-BD转化率比对照组增加13.03％,由10g/L的1,4-BD得到4.87 g/L的4HB,单位细胞的4HB产量由1.32 g/g提高40.91％至1.86 g/g.因此PncB和NadE可用于促进1,4-BD转化为4HB.     

腺嘌呤核苷酸及代谢产物在调节糖脂代谢稳态中的作用

糖脂代谢是高等生命体最基本的代谢活动,其过程受到环境和遗传的影响,并在器官、细胞和分子等不同水平上得到精确调控。脂质的过度摄取和积累导致肥胖,诱导糖代谢调节的失控,从而形成胰岛素抵抗和高血糖等代谢综合征。本文综述了游离脂肪酸诱导靶细胞释放腺嘌呤核苷酸的相关研究,回顾了过去数十年关于正常和特殊生理状态下腺嘌呤核苷酸信号发生、转导和作用方式的研究成果,提出了腺嘌呤核苷酸及其代谢产物是脂质代谢和糖代谢相互调节过程中的介导因子,其在2型糖尿病发生、发展中可能承担新角色。     

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。     

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。     

腺嘌呤核苷酸化合物对多型核白细胞内[Ca~(2+)]_i的影响

为了探讨腺嘌呤核苷酸化合物对多型核白细胞内［Ｃａ２＋］ｉ的影响,本文采用ＡＴＰ和ＡＤＰ激活此类白细胞,并用Ｆｕｒａ－２双波长荧光法测定细胞内游离钙浓度的变化。结果表明,ＡＴＰ和ＡＤＰ能激活多型核白细胞,引起细胞内［Ｃａ２＋］ｉ的明显升高。多型核白细胞对ＡＴＰ和ＡＤＰ具有不同的敏感性。在较低的激活剂浓度（１０μｍｏｌ／Ｌ）下,此类白细胞对ＡＤＰ更敏感;而在较高的激活剂浓度（１００μｍｏｌ／Ｌ）下,此类白细胞对ＡＴＰ更敏感。另外,多型核白细胞被ＡＤＰ激活后能再度被ＡＴＰ所激活,而被ＡＴＰ激活后不能再被ＡＤＰ激活,表明ＡＴＰ和ＡＤＰ对此类白细胞的作用机制不同。     

β-烟酰胺单核苷酸功能与合成研究进展

β-烟酰胺单核苷酸(nicotinamide mononucleotide,NMN)是辅酶I——NAD~+(nicotinamide adenine dinucleotide)合成的关键中间体,存在于各种生物体内。NAD~+广泛参与体内多种反应,对人体健康起着非常重要的作用。服用烟酰胺单核苷酸后可以快速提升体内NAD~+水平,从而在体内起到多种关键功能。近年来,研究NMN为年龄相关性功能衰退和疾病的发病机制提供了许多重要的见解。研究发现NMN具有多种功能作用,例如抗衰老,促进心脑健康等。NMN已经成为保健品、食品原料等领域研究的热点,其市场容量增长迅速,目前已有多种以NMN为主要成分的保健品上市销售。基于NMN持续火热的研究态势以及未来巨大的市场预期,本文较为系统地综述了NMN的研究背景、作用机理、保健功能、全球品牌产品、主要的化学方法与生物学方法的合成路线等,旨在为普及以及推动NMN在人类健康领域的研究和应用提供参考。     

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...     

Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.     

Deficiency of Nicotinamide Mononucleotide Adenylyltransferase 3 (Nmnat3) Causes Hemolytic Anemia by Altering the Glycolytic Flow in Mature Erythrocytes

NAD biosynthesis is of substantial interest because of its important roles in regulating various biological processes. Nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) is considered a mitochondria-localized NAD synthesis enzyme involved in de novo and salvage pathways. Although the biochemical properties of Nmnat3 are well documented, its physiological function in vivo remains unclear. In this study, we demonstrated that Nmnat3 was localized in the cytoplasm of mature erythrocytes and critically regulated their NAD pool. Deficiency of Nmnat3 in mice caused splenomegaly and hemolytic anemia, which was associated with the findings that Nmnat3-deficient erythrocytes had markedly lower ATP levels and shortened lifespans. However, the NAD level in other tissues were not apparently affected by the deficiency of Nmnat3. LC-MS/MS-based metabolomics revealed that the glycolysis pathway in Nmnat3-deficient erythrocytes was blocked at a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step because of the shortage of the coenzyme NAD. Stable isotope tracer analysis further demonstrated that deficiency of Nmnat3 resulted in glycolysis stall and a shift to the pentose phosphate pathway. Our findings indicate the critical roles of Nmnat3 in maintenance of the NAD pool in mature erythrocytes and the physiological impacts at its absence in mice.     

Structure and Mechanism of the Bifunctional CinA Enzyme from Thermus thermophilus

CinA is a widely distributed protein in Gram-positive and Gram-negative bacteria. It is associated with natural competence and is proposed to have a function as an enzyme participating in the pyridine nucleotide cycle, which recycles products formed by non-redox uses of NAD. Here we report the determination of the crystal structure of CinA from Thermus thermophilus, in complex with several ligands. CinA was shown to have both nicotinamide mononucleotide deamidase and ADP-ribose pyrophosphatase activities. The crystal structure shows an unusual asymmetric dimer, with three domains for each chain; the C-terminal domain harbors the nicotinamide mononucleotide deamidase activity, and the structure of a complex with the product nicotinate mononucleotide suggests a mechanism for deamidation. The N-terminal domain belongs to the COG1058 family and is associated with the ADP-ribose pyrophosphatase activity. The asymmetry in the CinA dimer arises from two alternative orientations of the COG1058 domains, only one of which forms a contact with the KH-type domain from the other chain, effectively closing the active site into, we propose, a catalytically competent state. Structures of complexes with Mg²⁺/ADP-ribose, Mg²⁺/ATP, and Mg²⁺/AMP suggest a mechanism for the ADP-ribose pyrophosphatase reaction that involves a rotation of the COG1058 domain dimer as part of the reaction cycle, so that each active site oscillates between open and closed forms, thus promoting catalysis.     

Critical Role for NAD Glycohydrolase in Regulation of Erythropoiesis by Hematopoietic Stem Cells through Control of Intracellular NAD Content

NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD.     

Structure-based Conversion of the Coenzyme Requirement of a Short-chain Dehydrogenase/Reductase Involved in Bacterial Alginate Metabolism

The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades the polysaccharides to monosaccharides through four alginate lyase reactions. The resultant monosaccharide, which is nonenzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronate (DEH), is further metabolized to 2-keto-3-deoxy-d-gluconate by NADPH-dependent reductase A1-R in the short-chain dehydrogenase/reductase (SDR) family. A1-R-deficient cells produced another DEH reductase, designated A1-R′, with a preference for NADH. Here, we show the identification of a novel NADH-dependent DEH reductase A1-R′ in strain A1, structural determination of A1-R′ by x-ray crystallography, and structure-based conversion of a coenzyme requirement in SDR enzymes, A1-R and A1-R′. A1-R′ was purified from strain A1 cells and enzymatically characterized. Except for the coenzyme requirement, there was no significant difference in enzyme characteristics between A1-R and A1-R′. Crystal structures of A1-R′ and A1-R′·NAD⁺ complex were determined at 1.8 and 2.7 Å resolutions, respectively. Because of a 64% sequence identity, overall structures of A1-R′ and A1-R were similar, although a difference in the coenzyme-binding site (particularly the nucleoside ribose 2′ region) was observed. Distinct from A1-R, A1-R′ included a negatively charged, shallower binding site. These differences were caused by amino acid residues on the two loops around the site. The A1-R′ mutant with the two A1-R-typed loops maintained potent enzyme activity with specificity for NADPH rather than NADH, demonstrating that the two loops determine the coenzyme requirement, and loop exchange is a promising method for conversion of coenzyme requirement in the SDR family.     

Depletion of the Central Metabolite NAD Leads to Oncosis-mediated Cell Death

Depletion of the central metabolite NAD in cells results in broad metabolic defects leading to cell death and is a proposed novel therapeutic strategy in oncology. There is, however, a limited understanding of the underlying mechanisms that connect disruption of this central metabolite with cell death. Here we utilize GNE-617, a small molecule inhibitor of NAMPT, a rate-limiting enzyme required for NAD generation, to probe the pathways leading to cell death following NAD depletion. In all cell lines examined, NAD was rapidly depleted (average t_½ of 8.1 h) following NAMPT inhibition. Concurrent with NAD depletion, there was a decrease in both cell proliferation and motility, which we attribute to reduced activity of NAD-dependent deacetylases because cells fail to deacetylate α-tubulin-K40 and histone H3-K9. Following depletion of NAD by >95%, cells lose the ability to regenerate ATP. Cell lines with a slower rate of ATP depletion (average t_½ of 45 h) activate caspase-3 and show evidence of apoptosis and autophagy, whereas cell lines with rapid depletion ATP (average t_½ of 32 h) do not activate caspase-3 or show signs of apoptosis or autophagy. However, the predominant form of cell death in all lines is oncosis, which is driven by the loss of plasma membrane homeostasis once ATP levels are depleted by >20-fold. Thus, our work illustrates the sequence of events that occurs in cells following depletion of a key metabolite and reveals that cell death caused by a loss of NAD is primarily driven by the inability of cells to regenerate ATP.     

Radicals in Berkeley?

In a previous autobiographical sketch for DNA Repair (Linn, S. (2012) Life in the serendipitous lane: excitement and gratification in studying DNA repair. DNA Repair 11, 595–605), I wrote about my involvement in research on mechanisms of DNA repair. In this Reflections, I look back at how I became interested in free radical chemistry and biology and outline some of our bizarre (at the time) observations. Of course, these studies could never have succeeded without the exceptional aid of my mentors: my teachers; the undergraduate and graduate students, postdoctoral fellows, and senior lab visitors in my laboratory; and my faculty and staff colleagues here at Berkeley. I am so indebted to each and every one of these individuals for their efforts to overcome my ignorance and set me on the straight and narrow path to success in research. I regret that I cannot mention and thank each of these mentors individually.     

Design and discovery of thioether and nicotinamide containing sorafenib analogues as multikinase inhibitors targeting B-Raf,B-RafV600E and VEGFR-2

New sorafenib derivatives containing thioether and nicotinamide moiety were designed and synthesized as B-Raf, B-Raf^V600E and VEGFR-2 multikinase inhibitors. Their in vitro enzymatic inhibitory activities against B-Raf, B-Raf^V600E and VEGFR-2 and their antiproliferative activities against HCT-116 and B16BL6 cell lines were evaluated and described. Most of the compounds showed potent activities against both cell lines and specific kinases. Compounds a1, b1 and c4, which exhibited the most potent inhibitory activities against B-Raf with IC₅₀ of 21?nM, 27?nM and 17?nM, B-Raf^V600E with IC₅₀ of 29?nM, 28?nM and 16?nM, VEGFR-2 with IC₅₀ of 84?nM, 46?nM and 63?nM, respectively, and good antiproliferative activities, also demonstrated competitive antiangiogenic activities to sorafenib in in vitro HUVEC tube formation assay.