Loss of apoptosis-inducing factor leads to an increase in reactive oxygen species, and an impairment of respiration that can be reversed by antioxidants
Loss of apoptosis-inducing factor leads to an increase in reactive oxygen species, and an impairment of respiration that can be reversed by antioxidants
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DOI：
10.1038/sj.cdd.4401776
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发表时间：
2006-02-01
影响因子：
12.4
通讯作者：
McCreath, KJ
中科院分区：
文献类型：
--
作者：
Apostolova, N;Cervera, AM;McCreath, KJ
自发现以来，细胞凋亡诱导因子（AIF）在细胞凋亡过程中的作用已被详尽地研究。 1-3 目前，人们认识到，在促凋亡刺激后，AIF 从线粒体膜间隙转移到细胞质，然后转移到细胞核，在细胞核中诱导外周染色质浓缩和高分子量 (50kbp) DNA 断裂。 3, 4 AIF 本身没有内在的核酸酶活性，AIF 如何引起这些变化仍不清楚。 AIF 的线粒体外靶向，以及将重组 AIF 蛋白显微注射到细胞中，或将 AIF 添加到分离的细胞核中，通常会导致细胞凋亡表型的产生，例如染色质浓缩、线粒体膜电位耗散和细胞表面的磷脂酰丝氨酸暴露。 4, 5 人 AIF 的晶体结构揭示了两个重要区域，第一个区域表现出氧化还原酶活性，第二个区域代表假定的 DNA 结合位点。 6 氧化还原酶催化区在结构上与二苯基二加氧酶 (bphA4) 同源，二苯基二加氧酶是一种细菌加氧酶偶联的 NAD 依赖性铁氧还蛋白还原酶，与真核谷胱甘肽还原酶家族的酶具有相似的折叠。 7 在功能测定中，AIF 充当 NADH 氧化酶，催化电子从 NADH 到 O2 的净转移并产生超氧化物。 8 由于 AIF 可以稳定结合 FAD，因此属于黄素蛋白类别。重要的是，AIF 的氧化还原酶活性区域对于其凋亡活性并不是必需的。 8 最近有报道称，随着年龄的增长，Harlequin (Hq) 突变小鼠的小脑和视网膜神经元逐渐退化，在 AIF 基因的第一个内含子中插入了前病毒，导致 AIF 表达量下降 80%。 9 有趣的是，Hq 突变小鼠的小脑颗粒细胞比野生型小鼠更容易受到过氧化物诱导的细胞凋亡的影响，这表明 AIF 作为自由基清除剂（而不是超氧化物产生者），因此可以改善过氧化氢诱导的神经元细胞凋亡。 9 这一发现表明，AIF 可以在呼吸中发挥支持作用，因为电子传递链 (ETC) 是活性氧 (ROS) 的主要来源。由此可见，AIF 的突变可能会通过增加 ROS 的产生而导致线粒体呼吸缺陷。 10, 11 体外研究表明，在 NADH 存在的情况下，AIF 可以催化细胞色素 c 的还原，这表明细胞色素 c 是 AIF 可能的电子受体。 8 此外，一项蛋白质组学研究确定了线粒体氧化磷酸化系统复合物 IV 的纯化部分中存在 AIF。 12 在这项工作期间发表的一项最新研究提出，AIF 参与 ETC 的生物发生和/或维护，因为 AIF 突变导致复合物 I 和复合物 III 多蛋白的丢失。 13 本研究中具有特殊意义的是 AIF 缺失会损害氧化磷酸化。在这里，我们在细胞中检查了 AIF 的消耗是否会导致自由基形成的增加。我们首次观察到 AIF 耗尽后 ROS 的增加与 O2 消耗的减少之间存在直接关联。为了研究 AIF 和 ROS 之间可能的关系，我们使用载体驱动的小干扰 RNA (siRNA) 14 来减少 AIF 转录本的表达。两种独特的 AIF 特异性 siRNA（AIF-1、GTACTGATTGTATCTGAA GAT 和 AIF-2、GTAGTACAGCTGGATGTGAGA），但不是对照 siRNA，有效降低了稳态……
Since its discovery, the role of apoptosis-inducing factor (AIF) in the apoptotic process has been studied exhaustively. 1–3 At present, it is recognized that after a proapoptotic stimulus, AIF translocates from the mitochondrial intermembrane space to the cytosol and thence to the nucleus where it induces peripheral chromatin condensation and high molecular weight (50kbp) DNA fragmentation. 3, 4 AIF, itself, has no intrinsic nuclease activity, and how AIF causes these changes remains unclear. Extra-mitochondrial targeting of AIF, as well as microinjection of recombinant AIF protein into cells, or addition of AIF to isolated nuclei, generally leads to the generation of apoptotic phenotypes such as chromatin condensation, mitochondrial membrane potential dissipation and phosphatidylserine exposure on the cell surface. 4, 5 The crystal structure of human AIF reveals two important regions, the first region exhibits an oxidoreductase enzyme activity and the second region represents a putative DNA-binding site. 6 The oxidoreductase catalytic region is structurally homologous to diphenyl dioxygenase (bphA4), a bacterial oxygenase-coupled NAD-dependent ferredoxin reductase that manifests a similar fold to the eucaryotic glutathione reductase family of enzymes. 7 In functional assays, AIF acts as an NADH oxidase, catalyzing the net transfer of electrons from NADH to O2 and generating superoxide. 8 Because it can stably bind FAD, AIF falls into the category of flavoproteins. Importantly, the oxidoreductase-active region of AIF is not essential for its apoptogenic activity. 8 It was recently reported that the Harlequin (Hq) mutant mouse, which displays progressive degeneration of cerebellar and retinal neurons with aging, harbors a proviral insertion in the first intron of the AIF gene, provoking an 80% decrease in AIF expression. 9 Interestingly, cerebellar granule cells from the Hq mutant mice were more susceptible to peroxide-induced apoptosis than wild-type counterparts, suggesting that AIF serves as a free radical scavenger (and not a superoxide producer), and could therefore ameliorate neuronal apoptosis induced by hydrogen peroxide. 9 This finding suggests that AIF could play a supportive role in respiration, as the electron transport chain (ETC) is the main source of reactive oxygen species (ROS). It follows that mutations in AIF might produce a defect in mitochondrial respiration, through increased production of ROS. 10, 11 It has been shown that AIF can catalyze the reduction of cytochrome c in the presence of NADH in vitro, implying that cytochrome c is a possible electron acceptor for AIF. 8 Moreover, a proteomic study identifies the presence of AIF in a purified fraction of the complex IV of the mitochondrial oxidative phosphorylation system. 12 A very recent study published during the course of this work proposed that AIF is involved in the biogenesis and/or maintenance of the ETC, as AIF mutations resulted in a loss of Complex I and Complex III polyproteins. 13 Of special significance in this study was the result that loss of AIF compromised oxidative phosphorylation. Here we examined in cells whether depletion of AIF would lead to increases in free radical formation. We observed, for the first time, that there was a direct association between the increases in ROS, after AIF depletion, and the reduction in O2 consumption. To investigate the possible relationship between AIF and ROS, we used vector-driven small interfering RNA (siRNA) 14 to reduce the expression of the AIF transcript. Two unique AIF-specific siRNAs (AIF-1, GTACTGATTGTATCTGAA GAT, and AIF-2, GTAGTACAGCTGGATGTGAGA), but not control siRNA, effectively reduced the steady-state …