Oxaloacetic acid mediates ADP-dependent inhibition of mitochondrial complex II-driven respiration
Oxaloacetic acid mediates ADP-dependent inhibition of mitochondrial complex II-driven respiration
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DOI：
10.1074/jbc.ra118.005144
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发表时间：
2018-12-21
影响因子：
4.8
通讯作者：
Sivitz, William I.
中科院分区：
文献类型：
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作者：
Fink, Brian D.;Bai, Fan;Sivitz, William I.
我们最近报道了一种以前未被识别的线粒体呼吸现象。在没有鱼藤酮（通常用于阻断复合物 I）的情况下，当[ADP]在琥珀酸供能的肌肉线粒体中以连续增加的浓度保持恒定（钳制）时，我们观察到双相的呼吸反应，先增加然后减少。在这里我们研究了其中的机制。我们证实了数十年前的报道，即草酰乙酸 (OAA) 会抑制琥珀酸脱氢酶 (SDH)。然后，我们使用 NMR 方法评估分离的琥珀酸呼吸线粒体中的 OAA 浓度（已知很难通过 MS 测量）以及苹果酸、富马酸和柠檬酸的浓度。当这些线粒体在不同的钳位 ADP 浓度下孵育时，考虑到膜电位同时降低，呼吸作用在低 [ADP] 下增加，正如预期的那样。随着 [ADP] 的进一步增加，与 OAA 积累相关的呼吸减少。此外，低丙酮酸浓度（单独不足以驱动呼吸）足以将 OAA 代谢为柠檬酸，并完全逆转高 [ADP] 时琥珀酸支持的呼吸的损失。此外，丙酮酸摄取的化学或遗传抑制阻止了 OAA 清除并保留了呼吸。此外，我们还测量了增量 [ADP] 对 NADH、超氧化物和 H2O2（从复合物 II 到 I 的反向电子传输的标记）的影响。总之，我们的研究结果综合起来支持一种机制（详见内），其中琥珀酸供能呼吸作为增加 [ADP] 的函数，最初通过对膜电位的 [ADP] 依赖性效应而增加，但随后通过 OAA 抑制琥珀酸脱氢酶在较高 [ADP] 时减少。讨论了生理相关性。
We recently reported a previously unrecognized mitochondrial respiratory phenomenon. When [ADP] was held constant (clamped) at sequentially increasing concentrations in succinate-energized muscle mitochondria in the absence of rotenone (commonly used to block complex I), we observed a biphasic, increasing then decreasing, respiratory response. Here we investigated the mechanism. We confirmed decades-old reports that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH). We then used an NMR method to assess OAA concentrations (known as difficult to measure by MS) as well as those of malate, fumarate, and citrate in isolated succinate-respiring mitochondria. When these mitochondria were incubated at varying clamped ADP concentrations, respiration increased at low [ADP] as expected given the concurrent reduction in membrane potential. With further increments in [ADP], respiration decreased associated with accumulation of OAA. Moreover, a low pyruvate concentration, that alone was not enough to drive respiration, was sufficient to metabolize OAA to citrate and completely reverse the loss of succinate-supported respiration at high [ADP]. Further, chemical or genetic inhibition of pyruvate uptake prevented OAA clearance and preserved respiration. In addition, we measured the effects of incremental [ADP] on NADH, superoxide, and H2O2 (a marker of reverse electron transport from complex II to I). In summary, our findings, taken together, support a mechanism (detailed within) wherein succinate-energized respiration as a function of increasing [ADP] is initially increased by [ADP]-dependent effects on membrane potential but subsequently decreased at higher [ADP] by inhibition of succinate dehydrogenase by OAA. The physiologic relevance is discussed.