控制 NAD +/NADH 比例对线粒体电子传递链的互补作用

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Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio

Taking control of cellular NAD+ concentrations

控制细胞内 NAD + 浓度

Cellular concentrations of the nicotinamide adenine dinucleotide (NAD+) are critical for proper metabolism and are often altered in aging and disease. To enable better understanding of these processes, Titov et al. altered the concentration of NAD+ in particular cellular compartments. They did this through expression of a bacterial enzyme targeted to specific compartments of human cells in culture. Their experiments emphasize the important role of the electron transport chain in redox transfer of electrons to NADH, rather than proton pumping, in mitochondrial pathogenesis.

烟酰胺腺嘌呤二核苷酸的细胞浓度(NAD +)对于正常的新陈代谢是至关重要的,并且在衰老和疾病中经常发生改变。为了更好地理解这些过程,Titov 等人改变了 NAD + 在特定细胞间隙中的浓度。他们通过表达一种针对培养中的人类细胞特定区域的细菌酶来达到这一目的。他们的实验强调电子传递链在线粒体发病机制中的重要作用是将电子转移到 NADH,而不是质子泵。

Science, this issue p. 231

科学》 ,本期第231页

Abstract

摘要

A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD+) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD+/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD+/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.

电子传递链(ETC)活性的下降与许多人类疾病有关。虽然线粒体三磷酸腺苷生成的减少被认为是病理学的一个来源,但是相关的氧化烟酰胺腺嘌呤二核苷酸(NAD +)与还原形式(NADH)的比值减少的贡献还不太清楚。本研究利用 Lactobacillus 脱氧核糖核酸氧化酶(LbNOX)作为诱导人细胞 NAD +/NADH 比率增加的分室特异性遗传工具。我们使用 LbNOX 来证明关键的代谢通量、糖异生和信号对胞浆或线粒体 NAD +/NADH 比率的依赖性。在细胞质或线粒体中表达 LbNOX 可以改善由于机能受损引起的增殖和代谢缺陷。这些结果强调了还原应激在线粒体发病机制中的作用,并证明了靶向 LbNOX 对氧化还原状态的直接、隔室特异操纵的效用。

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A decline in electron transport chain (ETC) activity has been linked to numerous human disorders, ranging from rare genetic syndromes to common diseases, such as neurodegeneration, cancer, and diabetes, as well as the aging process itself (12). How a decline in ETC activity gives rise to the spectrum of observed pathology cannot be readily explained by a simple deficiency in adenosine triphosphate (ATP) production (1). A key challenge in deciphering mitochondrial pathogenesis stems from the fact that the ETC performs at least two coupled functions: redox transfer of electrons from NADH [the reduced form of nicotinamide adenine dinucleotide (NAD+)] to oxygen and a simultaneous conversion of the free energy of the electromotive force into a proton gradient across the mitochondrial inner membrane. In principle, pathology could stem from an excess of reducing equivalents (termed reductive stress or pseudohypoxia, which includes stalling of NAD+-coupled reactions) or a reduced proton gradient (impairing pH and voltage-coupled processes, including ATP synthesis by the F1Fo-ATP synthase). Currently, there are no methods for dissecting the redox function of the ETC from its proton pumping function.

电子传递链(ETC)活性的下降与许多人类疾病有关,从罕见的遗传综合症到常见的疾病,如神经退行性疾病、癌症、糖尿病,以及衰老过程本身。ETC 活性的下降是如何引起所观察到的病理谱的,并不能简单地用三磷酸腺苷(ATP)产生的缺陷来解释。解释线粒体发病机制的一个关键挑战源于这样一个事实,即 ETC 至少执行两个耦合功能: 电子从 NADH [还原形式的烟酰胺腺嘌呤二核苷酸(NAD +)]到氧气的氧化还原转移,以及同时将电压的自由能转化为跨越线粒体内膜的质子梯度。原则上,病理学可能源于当量减少过多(称为还原应激或假缺氧,包括 NAD + 耦合反应的停滞)或质子梯度减少(削弱 pH 值和电压耦合过程,包括 f1fo-ATP 合酶的 ATP 合成)。目前尚无从质子泵功能分析 ETC 氧化还原功能的方法。

Here, we report the application of a genetically encoded tool for compartment-specific manipulation of the NAD+/NADH ratio. Our tool is based on the flavin adenine dinucleotide (FAD)–dependent H2O-forming NADH oxidases, which catalyze the four-electron reduction of O2 to two molecules of H2O (Fig. 1A). We focused on bacterial oxidases with specificity for NADH over reduced nicotinamide adenine dinucleotide phosphate (NADPH) (37), whose natural function is protection of redox balance and defense against oxygen toxicity (8). Such oxidases have been successfully expressed in bacteria and yeast for biotechnological applications (911). We screened several H2O-forming NADH oxidases by expressing their human codon–optimized, epitope-tagged versions in cultured human cancer–derived epithelial (HeLa) cells. The enzyme from Lactobacillus brevis (LbNOX) was most highly expressed and had the highest oxidase activity when targeted to mitochondria (fig. S1).

在这里,我们报告的应用遗传编码工具为隔室特定的操作 NAD +/NADH 比例。我们的工具是基于黄素腺嘌呤二核苷酸依赖的 H2O 形成 NADH 氧化酶,它催化 O2的四电子还原为 H2O 的两个分子(图1A)。我们重点研究了 NADH 特异性的细菌氧化酶,其中还原型烟酰胺腺嘌呤二核苷酸磷酸 NADPH (3-7)具有保护氧化还原平衡和抗氧化毒性的天然功能。这种氧化酶已成功地在细菌和酵母中表达,用于生物技术应用(9-11)。我们通过在培养的人癌源性上皮细胞(HeLa)中表达人密码子优化的表位标记型 NADH,筛选了几种产生 h2o 的 NADH 氧化酶。以线粒体为靶点,短乳杆菌(Lactobacillus brevis,LbNOX)表达量最高,氧化酶活性最高。中一)。

Fig. 1 图一 H2O-forming NADH oxidase from O 形成 NADH 氧化酶L. brevis 短毛乳杆菌 (LbNOX). 氮氧化物)(A) Reaction catalyzed by LbNOX. (B) UV-visible spectrum of purified LbNOX. Protein (83 μM FAD active sites) in oxidized form (solid line) and after addition of excess of sodium dithionite, reduced form (dashed line). (Inset) SDS–polyacrylamide gel electrophoresis of purified LbNOX. (C) Simultaneous measurement of NADH and oxygen consumption by LbNOX. NADH and LbNOX were added as indicated by arrows. (D) Dependence of the specific activity (S.A.) of recombinant LbNOX on the concentration of NADH and NADPH. Reported values for Vmaxkcat, and Km for NADH are means ± SD from n = 4 independent experiments. (E) Crystal structure of the catalytic dimer of LbNOX. Each of the two-fold symmetry–related monomers (cyan and green ribbons) contains bound FAD, shown here in spherical (CPK) representation. Details of the catalytic center on the Si-face of FAD and of the substrate selectivity loop are shown in fig. S3, A to C.(a) LbNOX 催化反应。(b)纯化 LbNOX 的紫外-可见光谱。蛋白质(83微米 FAD 活性位点)在氧化形式(实线)和添加过量的连二亚硫酸钠后,还原形式(虚线)。(插图) SDS-纯化的 LbNOX 的聚丙烯酰胺凝胶电泳。(c) LbNOX 同时测定 NADH 和耗氧量。用箭头表示 NADH 和 LbNOX。(d)重组 LbNOX 比活力(s.a.)与 NADH 和 NADPH 浓度的关系。报道的 Vmax、 kcat 和 Km 的 NADH 值均为 n = 4个独立实验的 ± SD。(e)催化型二聚体的晶体结构。两重对称相关单体(青色和绿色带)中的每一个都包含束缚 FAD,如图所示为球形(CPK)表示。详细的催化中心的硅面的基板选择性环显示在图。3,a to c.

We evaluated the biochemical properties of recombinant LbNOX modified to contain a C-terminal FLAG tag and a cleavable N-terminal hexahistidine tag. Purified LbNOX-FLAG has a yellow color in solution and a characteristic ultraviolet (UV)–visible absorption spectrum (major absorption peaks at 371 and 444 nm) consistent with the presence of FAD, which can be reduced upon the addition of sodium dithionite (Fig. 1B). Our recombinant enzyme consumes oxygen and is strictly specific for NADH rather than NADPH with the Michaelis constant(Km) for NADH of 69 ± 3 μM, the maximum velocity(Vmax) of 758 ± 33 μmol·min−1·mg−1, and the turnover number(kcat) of 648 ± 28 s−1, which is more active than previously reported (312) (Fig. 1, C and D). The molecular size of LbNOX-FLAG was determined to be 197 ± 4 kD, which indicates that the protein is a tetramer in solution. Although enzymes in this family often produce H2O2, LbNOX-FLAG produces amounts of H2O2 that constitute only 1 to 2% of the amount of H2O produced during its catalytic cycle (fig. S2A) (467). The apparent Km for O2 of LbNOX-FLAG was below 2 μM (~0.17% O2), as estimated from enzyme-monitored turnover experiments (fig. S2B), which is less than 1/10th of the concentration of oxygen in human venous blood (13). Thus, we expect LbNOX to be active in most animal tissues. The enzymatic properties of LbNOX-FLAG in solution were well founded in the 2.4 Å resolution x-ray structure of this protein that we determined (Fig. 1E, fig. S3, and table S1). Our structure is generally similar to the reported structures of H2O-forming the reduced form of nicotinamide adenine dinucleotide phosphate NAD(P)H oxidases from L. sanfranciscensis (PDB ID 2CDU) and Streptococcus pyogenes (PDB ID 2BC0) (1415). However, our structure captures LbNOX in a new state with molecular oxygen (O2) bound and the redox active Cys42 in a reduced form (Cys42-SH) (fig. S3). [See supplementary materials (SM) for a detailed discussion of the x-ray structure.] In conclusion, the high selectivity for NADH over NADPH, negligible H2O2 production, and very low Km for O2 made LbNOX attractive for additional studies in human cells.

我们评估了包含 c 末端 FLAG 标签和 n 末端可剪切的六亚甲基四氢吡啶标签的重组 LbNOX 的生化特性。纯化的 lbnox 旗在溶液中呈现黄色,并具有特征的紫外-可见吸收吸收光谱(主要吸收峰在371和444 nm) ,这与 FAD 的存在相一致,加入连二亚硫酸钠可以降低 FAD 的含量(图1B)。我们的重组酶对 NADH 具有严格的特异性,对 NADH 的米氏常数(Km)为69 ± 3μM,最大流速(Vmax)为758 ± 33μmol min-1mg-1,周转数(kcat)为648 ± 28s-1,比以前报道的(3,12)更有活性(图1,c 和 d)。分子大小为197 ± 4kd,表明该蛋白为四聚体。虽然这个家族中的酶经常产生 H2O2,但 lbnox 旗帜产生的 H2O2数量只占其催化循环中产生的 H2O 数量的1-2% (图)。S2A)(4,6,7).根据酶监测周转试验结果,LbNOX-FLAG 的 O2表观 Km 小于2μM (~ 0.17% O2)。S2B) ,低于人体静脉血氧浓度的十分之一(13)。因此,我们认为 LbNOX 在大多数动物组织中是活跃的。在我们测定的这种蛋白质的2.4 nm 分辨率 x 射线结构中,发现了溶液中 lbnox 旗的酶学性质(图1E,图)。表 S3及表 S1)。我们的结构与报道的 h2o 结构基本相似,形成了烟酰胺腺嘌呤二核苷酸磷酸和产脓链球菌的还原型 NAD (p) h 氧化酶(14,15)。然而,我们的结构捕获 LbNOX 在一个新的状态与分子氧(O2)结合和氧化还原活性 Cys42在一个还原形式(Cys42-SH)(图。S3).[参见补充材料(SM)的 x 射线结构的详细讨论。]总之,NADH 对 NADPH 的高选择性,可忽略的 H2O2产生,以及极低的 O2 Km,使 LbNOX 对于人体细胞的进一步研究具有吸引力。

To determine whether we could express LbNOX-FLAG safely and efficaciously in various compartments of human cells, we used lentiviral infection to generate HeLa cells that expressed untargeted or mitochondria-targeted human codon–optimized LbNOX-FLAG (referred to as LbNOX and mitoLbNOX henceforth) under the control of a doxycycline-inducible promoter (TRE3G) (Fig. 2A and fig. S1A). We used fluorescence microscopy and cell fractionation to confirm diffuse localization of LbNOX and mitochondrial localization of mitoLbNOX (Fig. 2, B and C). Cells appeared grossly normal without any impact on cell proliferation or production of reactive oxygen species (ROS) (fig. S4, A and B). As expected, expression of LbNOX and mitoLbNOX in HeLa cells increased oxygen consumption by 1.6- and 2.4-fold, respectively (Fig. 2D and fig. S4C). The increase was resistant to ETC inhibitors, which indicates that it resulted from LbNOX oxidase activity and not from the increased ETC activity. Despite similar expression levels (Fig. 2A), mitoLbNOX induced a larger increase in oxygen consumption than LbNOX (Fig. 2D), likely because of the higher concentration of NADH within mitochondria (1618). It is important to remember that in converting NADH to NAD+LbNOX also consumes protons and oxygen and, therefore, could affect cellular pH or oxygen levels, depending on experimental context.

为了确定我们是否能够在人类细胞的不同区域安全有效地表达 LbNOX 旗,我们利用慢病毒感染产生 HeLa 细胞,在强力霉素诱导的启动子(TRE3G)的控制下,表达非靶向或靶向线粒体的人密码子优化 LbNOX 旗(简称 LbNOX 和 mitoLbNOX)。S1A).我们使用荧光显微镜和细胞分离来确认 LbNOX 的扩散定位和 mitoLbNOX 的线粒体定位(图2,b 和 c)。细胞基本正常,不影响细胞增殖或产生活性氧类(ROS)。中四、甲及乙)。正如预期的那样,HeLa 细胞中 LbNOX 和 mitoLbNOX 的表达分别使氧消耗增加了1.6倍和2.4倍(图2 d 和图2)。S4C).这种增加对 ETC 抑制剂具有抗性,说明这是由于 LbNOX 氧化酶的活性增加,而不是由于增加了 ETC 活性。尽管有相似的表达水平(图2A) ,mitoLbNOX 比 LbNOX 引起更大的氧消耗增加(图2D) ,可能是因为线粒体内较高浓度的 NADH (16-18)。重要的是要记住,在将 NADH 转化为 NAD + 的过程中,LbNOX 也会消耗质子和氧气,因此,根据实验环境,LbNOX 可能会影响细胞的 pH 值或氧气水平。

Fig. 2 图二 Expression and activity of 表达和活性LbNOX in human cells. 人体细胞中的氮氧化物(A) Western blot of LbNOX and mitoLbNOX in HeLa cells after 24-hour induction with water or doxycycline (300 ng/ml) (Dox). Representative gel from one of three independent experiments. (B) Subcellular localization of LbNOX and mitoLbNOX in HeLa cells determined by cell fractionation. LRPPRC is a mitochondrial marker and actin is a cytosolic marker. Representative gel from one of three independent experiments. (C) Subcellular localization of LbNOX and mitoLbNOX in HeLa cells determined by using fluorescence microscopy. Tomm20 is a marker of mitochondria. (D) Effect of LbNOX and mitoLbNOX expression in HeLa cells on basal, piericidin-resistant, and antimycin-resistant oxygen consumption measured with an XF24 extracellular flux analyzer. Means ± SEM, n = 3 independent experiments.(a)水或强力霉素(300ng/ml)24小时诱导 HeLa 细胞产生 LbNOX 和 mitoLbNOX 蛋白印迹。来自三个独立实验之一的代表性凝胶。(b)细胞分级法测定 HeLa 细胞中 LbNOX 和 mitoLbNOX 的亚细胞定位。LRPPRC 是一种线粒体标记物而肌动蛋白是一种细胞溶液标记物。来自三个独立实验之一的代表性凝胶。(c)荧光显微技术检测 HeLa 细胞中 LbNOX 和 mitoLbNOX 的亚细胞定位。Tomm20是线粒体的标志物。(d) XF24型细胞外通量分析仪测定 HeLa 细胞 LbNOX 和 mitoLbNOX 表达对基础细胞、皮粉蝶素抗性细胞和抗霉素抗性细胞耗氧量的影响。方法 ± SEM,n = 3个独立实验。

We determined the impact of expressing LbNOX or mitoLbNOX on cellular concentrations of NAD+and NADH (Fig. 3 and fig. S5). We used a genetic sensor, SoNar (19), to measure cytosolic NADH. SoNar is a fusion of circularly permuted yellow fluorescent protein and a modified NADH-binding protein, Rex, from Thermus aquaticus. Binding of NADH to SoNar leads to an increase in fluorescence. Expression of LbNOX or mitoLbNOX decreased the fluorescence signal from SoNar, which indicated that both LbNOX and mitoLbNOX decrease cytosolic NADH (Fig. 3A and fig. S5B). Consistent with this result, intracellular and secreted lactate/pyruvate ratios, traditionally used as proxies for the cytosolic NADH/NAD+ ratio (17), decreased in cells expressing LbNOX or mitoLbNOX (Fig. 3B and fig. S5D). The ratio of total cellular NAD+ to total NADH, based on high-performance liquid chromatography (HPLC) measurements, was increased twofold by mitoLbNOX, whereas LbNOX did not have a significant effect (Fig. 3C and fig. S5C). Perturbation of the total NAD+/NADH ratio likely reflects changes in amounts of mitochondrial NADH, because most of the effect on the ratio resulted from changes in NADH concentration (fig. S5A), and most of the NADH inside the cell is present in mitochondria. The latter effect is supported by fractionation experiments (20) and by the observation that the majority of NAD(P)H autofluorescence in cells comes from mitochondrial NADH (21). In summary, LbNOX and mitoLbNOX can be used to perturb the NAD+/NADH ratio, and our compartment-specific measurements of HeLa cells (Fig. 3, A to C) indicate that, although perturbation of the mitochondrial NAD+/NADH ratio leads to changes in the cytosolic NAD+/NADH ratio, the converse is not true.

我们测定了表达 LbNOX 或 mitoLbNOX 对细胞内 NAD + 和 NADH 浓度的影响。中五)。我们使用基因传感器 SoNar (19)来测量细胞质 NADH。SoNar 是一种环状排列的黄色荧光蛋白和一种修饰过的 nadh 结合蛋白 Rex 的融合物,来自水生栖热菌。NADH 与 SoNar 的结合导致荧光增强。LbNOX 和 mitoLbNOX 的表达降低了来自 SoNar 的荧光信号,表明 LbNOX 和 mitoLbNOX 都降低了细胞内 NADH (图3A 和图3A)。S5B).与这一结果相一致的是,传统上用作衡量细胞内 NADH/NAD + 比值(17)的细胞内和分泌乳酸/丙酮酸比值,在表达 LbNOX 或 mitoLbNOX 的细胞中降低了(图3B 和图3B)。S5D).根据高效液相色谱法(HPLC)的测量,细胞 NAD + 总数与 NADH 总数的比值被 mitoLbNOX 提高了两倍,而 LbNOX 没有显著的影响(图3 c 和图2)。S5C).整个 NAD +/NADH 比值的变化可能反映了线粒体 NADH 数量的变化,因为 NADH 浓度的变化对这一比值的影响最大(图)。细胞内的 NADH 大部分存在于线粒体中。后一种效应得到了细胞分离实验(20)和细胞中大部分 NAD (p) h 自体荧光来自线粒体 NADH (21)的观察的支持。总之,LbNOX 和 mitoLbNOX 可以用来扰乱 NAD +/NADH 比值,我们对 HeLa 细胞的室内特异性测量表明,虽然线粒体 NAD +/NADH 比值的扰动导致细胞内 NAD +/NADH 比值的变化,反之则不正确。

Fig. 3 图3 Effect of 的影响LbNOX and mito 氮氧化物和水户LbNOX on NAD 氮氧化物对 NAD 的影响+/NADH ratios, metabolic fluxes, PDH phosphorylation, and gluconeogenesis. NADH 比值、代谢通量、 PDH 磷酸化和糖异生(A to C) Effect of LbNOX and mitoLbNOX expression in HeLa cells on (A) cytoplasmic NADH concentrations determined with fluorescence microscopy using SoNar-expressing cells (n = 7), (B) intracellular and secreted lactate/pyruvate ratio determined by liquid chromatography–mass spectrometry (LC-MS) (n = 4), and (C) intracellular NAD+/NADH ratios determined by HPLC (n = 4). Student’s t test. ns, P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Means ± SEM. (D) Effect of LbNOX and mitoLbNOX expression in HeLa cells on release rate of pyruvate, aspartate, and succinate, determined by comparing concentrations in spent versus fresh media. Student’s t test. ns, P > 0.05; **P < 0.01; ***P< 0.001. Means ± SEM, n = 3 replicates from one experiment. (E) Effect of LbNOX and mitoLbNOX expression in HeLa cells on PDH phosphorylation. Representative gel from one of three independent experiments. DCA, dichloroacetate. (F) Effect of adenoviral transduction of green fluorescent protein (GFP), LbNOX, or mitoLbNOX on primary rat hepatocyte gluconeogenesis in Dulbecco’s modified Eagle’s medium containing no glucose, no glutamine, and no pyruvate and by using either no substrate, 5 mM pyruvate, or 5 mM lactate. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. ns, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001. Means ± SEM, n = 3 (no substrate, pyruvate) or n = 7 (lactate) independent experiments. (G) Effect of LbNOX and mitoLbNOX on the secreted β-hydroxybutyrate/acetoacetate ratio in rat hepatocytes performing gluconeogenesis from lactate as a substrate. Metabolite levels determined by using LC-MS. One-way ANOVA followed by Tukey’s multiple comparisons test. ns, P > 0.05; *P < 0.05; **P < 0.01. Means ± SEM, n = 10 independent experiments.(a 至 c) LbNOX 和 mitoLbNOX 在 HeLa 细胞中的表达对(a)细胞质 NADH 浓度的影响: 荧光显微镜法(SoNar-expressing cell,n = 7) ,液相色谱-质谱法质谱法(LC-MS,n = 4)测定细胞内和分泌的乳酸/丙酮酸比值,高效液相色谱法(c)测定细胞内 NAD +/NADH 比值。学生 t 测验。P < 0.001; p < 0.001; p < 0.001.平均值 ± 扫描电镜。(d)在 HeLa 细胞中 LbNOX 和 mitoLbNOX 的表达对丙酮酸、天冬氨酸和琥珀酸释放速率的影响,通过比较废培养基和新鲜培养基中的浓度来确定。学生 t 测验。纳米粒子,p > 0.05; * * p < 0.01; * * p < 0.001。方法 ± 扫描电镜,n = 3重复一个实验。(e) HeLa 细胞 LbNOX 和 mitoLbNOX 表达对 PDH 磷酸化的影响。来自三个独立实验之一的代表性凝胶。二氯乙酸。(f)在 Dulbecco 改良的 Eagle’ s 培养基中,不含葡萄糖、谷氨酰胺和丙酮酸,不用底物,5mm 丙酮酸或5mm 乳酸盐,转导绿色荧光蛋白(GFP)、 LbNOX 或 mitoLbNOX 对大鼠原代肝细胞糖异生的影响。单因素方差分析变异数分析(ANOVA) ,然后是 Tukey 的多重比较测试。P < 0.05; * * p < 0.01; * * * p < 0.001.方法 ± SEM,n = 3(无底物,丙酮酸盐)或 n = 7(乳酸盐)独立实验。(g) LbNOX 和 mitoLbNOX 对以乳酸为底物进行糖异生的大鼠肝细胞分泌 β- 羟丁酸酯/乙酰乙酸酯比值的影响。液相色谱-质谱联用法测定代谢产物含量。单因素方差分析,再进行多重比较测验。纳米粒子,p > 0.05; * p < 0.05; * * p < 0.01。平均值 ± SEM,n = 10个独立实验。

We performed metabolic profiling on medium in which cells expressing LbNOX or mitoLbNOX had been grown (Fig. 3D and fig. S6, A and B). We identified pyruvate, aspartate, and succinate as three metabolites whose consumption or release was changed more than twofold (Student’s t test; P < 0.01) by either enzyme. These changes are attributable to compartment-specific changes of NAD+/NADH by LbNOX or mitoLbNOX (see SM for discussion). It is notable that LbNOX and mitoLbNOX did not have a significant effect on the uptake of glucose and release of lactate (fig. S6C).

我们在表达 LbNOX 或 mitoLbNOX 的细胞培养基上进行代谢轮廓分析(图3D 和图3D)。中六、甲及乙)。我们鉴定丙酮酸、天冬氨酸和琥珀酸为三种代谢产物,它们的消耗或释放被两种酶改变了两倍以上(学生 t 检验; p < 0.01)。这些变化可归因于由 LbNOX 或 mitoLbNOX 引起的 NAD +/NADH 的分室特异性变化(参见 SM 的讨论)。值得注意的是,LbNOX 和 mitoLbNOX 对葡萄糖的摄取和乳酸的释放没有显著影响(图2。S6C).

In vitro phosphorylation of mitochondrial pyruvate dehydrogenase (PDH) is regulated by the NAD+/NADH ratio (22), but this has not been shown directly in intact cells. Treatment of HeLa cells with dichloroacetate, an inhibitor of pyruvate dehydrogenase kinases (PDKs), inhibits phosphorylation of PDH, and antimycin treatment, which blocks the ETC and decreases the mitochondrial NAD+/NADH ratio, increases PDH phosphorylation. In agreement with in vitro studies, PDH was almost completely dephosphorylated in the presence of mitoLbNOX but not LbNOX (Fig. 3E and fig. S6D). The data on PDH phosphorylation are consistent with our observation that mitoLbNOX, but not LbNOX, increases the mitochondrial NAD+/NADH ratio in HeLa cells (Fig. 3C).

在体外,线粒体丙酮酸脱氢酶(PDH)的磷酸化受 NAD +/NADH 比值(22)的调节,但是在完整的细胞中还没有直接显示出来。丙酮酸脱氢酶(PDKs)抑制剂二氯醋酸酯(dichloroacetate)处理 HeLa 细胞可抑制 PDH 的磷酸化,而抗霉素处理可阻断 ETC,降低线粒体 NAD +/NADH 比值,增加 PDH 的磷酸化。与体外研究一致的是,PDH 在 mitoLbNOX 存在下几乎完全去磷酸化,但 LbNOX 不能(图3E 和图。S6D).PDH 磷酸化的数据与我们观察到的提高 HeLa 细胞线粒体 NAD +/NADH 比值的是 mitoLbNOX,而不是 LbNOX 相一致(图3C)。

We expressed LbNOX and mitoLbNOX in primary rat hepatocytes to study gluconeogenesis. The cytosolic NAD+/NADH ratio has been reported to affect gluconeogenesis, although classical approaches relied on indirect methods for manipulating the redox state (2324). In our hepatocyte system, rates of gluconeogenesis were significantly higher if pyruvate rather than lactate was used as a substrate, which we attribute to the NAD+/NADH ratio-dependent inhibition of lactate-to-pyruvate conversion (23). Consistent with this hypothesis, rates of gluconeogenesis from lactate were increased to those seen with pyruvate when primary hepatocytes expressed LbNOX, whereas gluconeogenesis from pyruvate was not affected (Fig. 3F). Gluconeogenesis from lactate was also increased by mitoLbNOX. Gluconeogenesis from pyruvate, however, was inhibited by mitoLbNOX, perhaps because strong oxidation of mitochondrial NADH prevents formation of malate (25). In assays of gluconeogenesis using lactate as a precursor, expression of either LbNOX or mitoLbNOX decreased the ratio of secreted β-hydroxybutyrate/acetoacetate (Fig. 3G), the classical proxy for the mitochondrial NADH/NAD+ ratio (17). Although the NAD+/NADH ratio appears to be increased, we cannot exclude the possibility that LbNOX or mitoLbNOX induce hypoxia. LbNOX evidently increased the mitochondrial NAD+/NADH ratio in rat hepatocytes (Fig. 3G) but not in HeLa cells (Fig. 3C), which might reflect differences in cell type or media conditions.

我们在原代大鼠肝细胞中表达 LbNOX 和 mitoLbNOX 来研究糖异生。细胞内 NAD +/NADH 比值影响糖异生,虽然传统的方法依赖于间接操纵氧化还原状态的方法(23,24)。在我们的肝细胞系统中,如果以丙酮酸而不是乳酸为底物,糖异生率明显增加,这归因于 NAD +/NADH 比率依赖性抑制乳酸到丙酮酸的转化(23)。与这一假设相一致的是,当初级肝细胞表达 LbNOX 时,乳酸的糖异生率增加到丙酮酸的糖异生率,而丙酮酸的糖异生率则没有受到影响(图3F)。来自乳酸的糖异生作用也增加了 mitoLbNOX。然而,丙酮酸的糖异生作用被 mitoLbNOX 所抑制,可能是因为线粒体 NADH 的强烈氧化阻止了苹果酸的形成(25)。在以乳酸为前体的糖异生实验中,LbNOX 或 mitoLbNOX 的表达降低了线粒体 NADH/NAD + 比值的经典代表—— β- 羟丁酸酯/乙酰乙酸酯的分泌比值(图3G)。虽然 NAD +/NADH 比值增加,但我们不能排除 LbNOX 或 mitoLbNOX 诱发缺氧的可能性。LbNOX 明显增加了大鼠肝细胞线粒体 NAD +/NADH 比值(图3 g) ,但不增加 HeLa 细胞线粒体 NAD +/NADH 比值(图3 c) ,这可能反映了细胞类型或培养基条件的差异。

Mammalian cells lacking a functional ETC require the addition of exogenous pyruvate and uridine for cell proliferation (2628). Uridine is required because one of the enzymes in de novo uridine biosynthesis (dihydroorotate dehydrogenase) is coupled to the ETC through coenzyme Q. The requirement for pyruvate, however, has been less clear because it participates in many reactions but has been proposed to rescue cell growth by recycling NAD+ from NADH through cytosolic lactate dehydrogenase (2629). If the NAD+ recycling hypothesis is correct, then supplementation with oxaloacetate should have the same effect as pyruvate, because it can be reduced by malate dehydrogenase while recycling NAD+. Oxaloacetate, like pyruvate, rescued the proliferation defect induced by piericidin, whereas malate and lactate did not (Fig. 4A). Alpha-ketobutyrate also rescues the proliferative defect induced by ETC inhibition (30). Furthermore, a large number of α-keto acids can rescue the pyruvate dependence of proliferation in cells with intact ETC (31). These findings support the NAD+-recycling hypothesis, although they are still indirect, as α-keto acids have many metabolic roles.

缺乏功能性 ETC 的哺乳动物细胞需要添加外源性丙酮酸和尿苷来促进细胞增殖(26-28)。尿苷是必需的,因为新尿苷生物合成中的一个酶(二氢乳酸脱氢酶)通过辅酶 q 与 ETC 结合。然而,丙酮酸的需求还不是很清楚,因为它参与了许多反应,但是已经有人提出通过细胞溶液乳酸脱氢酶(26,29)从 NADH 中回收 NAD + 来拯救细胞生长。如果 NAD + 循环假说是正确的,那么补充草酰乙酸应该和丙酮酸有同样的效果,因为在回收 NAD + 的同时,苹果酸脱氢酶可以降低 NAD + 的含量。草酰乙酸和丙酮酸一样,能够有效地挽救皮粉蝶苷诱导的细胞增殖缺陷,而苹果酸和乳酸盐则不能。Α-丁酮酸还可以挽救 ETC 抑制剂诱导的增殖性缺损(30)。此外,大量的 α- 酮酸可以挽救完整 ETC (31)细胞对丙酮酸的增殖依赖性。这些发现支持 NAD +-循环假说,尽管它们仍然是间接的,因为 α- 酮酸有许多新陈代谢作用。

Fig. 4 图4 NAD 美国国家广播公司+ recycling rescues proliferation in cells with impaired ETC. 循环拯救受损等细胞的增殖(A) Effect of pyruvate, oxaloacetate, lactate, and malate addition on proliferation of HeLa Tet3G luciferase cells in the presence of 200 μM uridine and in the presence or absence of 1 μM piericidin. Means ± SEM, n = 5 independent experiments. (B) Effect of LbNOX and mitoLbNOX expression in HeLa cells on inhibition of cell proliferation by 1 μM piericidin, 1 μM antimycin, 10 μg/ml chloramphenicol, and 30 ng/ml ethidium bromide (EtBr) in the presence of 200 μM uridine. DMSO, dimethyl sulfoxide. Means ± SEM, n = 3 independent experiments.(a)丙酮酸、草酰乙酸、乳酸和苹果酸对200μM 尿嘧啶核苷存在和1μM 粉蝶啶存在时 HeLa Tet3G 荧光素酶细胞增殖的影响。方法 ± SEM,n = 5个独立实验。(b)在200μM 尿嘧啶核苷存在下,LbNOX 和 mitoLbNOX 在 HeLa 细胞中的表达对1μM 粉蝶啶、1μM 抗霉素、10μg/ml 氯霉素和30ng/ml 溴化乙锭抑制细胞增殖的影响。二甲基亚砜。方法 ± SEM,n = 3个独立实验。

We used LbNOX to directly test whether NAD+ recycling is an essential function of the ETC that is required for cell proliferation. We inhibited ETC function, with piericidin (a complex I inhibitor), antimycin (a complex III inhibitor), ethidium bromide (a mitochondrial DNA replication inhibitor), and chloramphenicol (an inhibitor of mitochondrial translation) in HeLa cells supplemented with uridine but lacking pyruvate. HeLa cells cannot proliferate in these conditions (Fig. 4B and fig. S7). Expression of either LbNOX or mitoLbNOX rescued cell proliferation in the presence of these ETC inhibitors, which indicated that regeneration of NAD+ in either cytosol or mitochondria is sufficient to complement ETC activity that is required for cell proliferation (Fig. 4B). Metabolic profiling showed that of the nine metabolites whose uptake or release is affected greater than twofold by antimycin (Student’s t test; P < 0.01), all could be reversed by either LbNOX or mitoLbNOX, which reflects a metabolic rescue (fig. S8) (see SM for discussion). Our metabolic profiling data are complementary to recent studies demonstrating an inhibition of aspartate biosynthesis in cells with dysfunctional ETC (303233). As a control, we also showed that the rescue by LbNOX or mitoLbNOX was not attributable to an effect on mitochondrial membrane potential (fig. S9, A to C), nor was it due to a rescue of ETC-derived ATP synthesis (fig. S9, D to G).

我们使用 LbNOX 直接测试 NAD + 循环是否是 ETC 的基本功能,这是细胞增殖所必需的。我们在补充了尿苷但缺乏丙酮酸的 HeLa 细胞中抑制了 ETC 功能,包括粉蝶苷(一种复合 i 抑制剂)、抗霉素(一种复合 III 抑制剂)、溴化乙锭(一种线粒体脱氧核糖核酸复制抑制剂)和氯霉素(一种线粒体翻译抑制剂)。细胞在这些条件下不能增殖。中七)。在存在这些 ETC 抑制剂的情况下,LbNOX 或 mitoLbNOX 的表达促进了细胞增殖,这表明无论是在胞浆还是线粒体中,NAD + 的再生都足以补充细胞增殖所需的 ETC 活性(图4B)。代谢分析表明,9种代谢物的摄取或释放受到 antimycin 的影响大于2倍(Student’s t test; p < 0.01) ,所有这些代谢物都可以被 LbNOX 或 mitoLbNOX 逆转,这反映了一种代谢救助(metaborbnox)。S8)(有关讨论请参阅 SM)。我们的代谢谱数据补充了最近的研究,证明了在功能失调的 ETC (30,32,33)细胞中抑制天冬氨酸的生物合成。作为对照,我们还发现 LbNOX 或 mitoLbNOX 的拯救作用并不归因于对线粒体膜电位的影响(图1)。S9,a 到 c) ,也不是由于 etc 来源的 ATP 合成的抢救(图。S9,d 至 g)。

Collectively, these studies (Fig. 4 and figs. S7 to S9) underscore the importance of NAD+ recycling by the ETC to support proliferation. In healthy cells, the ETC produces ATP and simultaneously recycles mitochondrial NADH to NAD+, with a secondary oxidation of cytosolic NADH via shuttles. In the absence of a functional ETC, glycolysis is capable of compensating for the lack of ATP production, but it is net redox-neutral. NAD+ recycling is likely key for cell proliferation, because many biosynthetic pathways produce NADH as a byproduct (34). These insights confirm the long-standing hypothesis (2629) that pyruvate supplementation rescues proliferation in cells with disrupted ETC by restoring NAD+/NADH balance via the LDH reaction.

总的来说,这些研究(图4和无花果。S7到 S9)强调了由 ETC 进行 NAD + 回收以支持扩散的重要性。在健康细胞中,ETC 产生 ATP,同时将线粒体 NADH 转化为 NAD + ,并通过梭子二次氧化细胞质 NADH。在缺乏功能性 ETC 的情况下,糖酵解能够弥补 ATP 生成的不足,但它是净氧化还原中性的。NAD + 循环可能是细胞增殖的关键,因为许多生物合成途径都会产生副产物 NADH (34)。这些发现证实了一个长期存在的假说(26,29) ,即丙酮酸补充剂通过 LDH 反应恢复 NAD +/NADH 平衡,从而挽救 ETC 中断细胞的增殖。

In the future, LbNOX and engineered or naturally occurring variants may become valuable tools for studying compartmentalization of redox metabolism. These constructs will allow for a dissection of the relative contributions of redox imbalance and ATP insufficiency to mitochondrial disease pathogenesis. If a substantial amount of the organ pathology of mitochondrial disease stems from reductive stress or pseudohypoxia, then expression of this single polypeptide holds promise as a “protein prosthesis” for the large number of disorders characterized by ETC dysfunction.

在未来,LbNOX 和工程或自然产生的变种可能成为研究氧化还原代谢防火分区的有价值的工具。这些构造将允许剖析氧化还原失衡和 ATP 不足的相对贡献线粒体疾病的发病机制。如果线粒体疾病的大量器官病变源于还原性应激或假性缺氧,那么这种单一多肽的表达有望成为大量拥有属性 ETC 功能障碍的“蛋白质修复体”。

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