Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, USA.
J Neurosci. 2012 Aug 15;32(33):11250-8. doi: 10.1523/JNEUROSCI.0907-12.2012.
Mitochondrial oxidative stress and damage have been implicated in the etiology of temporal lobe epilepsy, but whether or not they have a functional impact on mitochondrial processes during epilepsy development (epileptogenesis) is unknown. One consequence of increased steady-state mitochondrial reactive oxygen species levels is protein post-translational modification (PTM). We hypothesize that complex I (CI), a protein complex of the mitochondrial electron transport chain, is a target for oxidant-induced PTMs, such as carbonylation, leading to impaired function during epileptogenesis. The goal of this study was to determine whether oxidative modifications occur and what impact they have on CI enzymatic activity in the rat hippocampus in response to kainate (KA)-induced epileptogenesis. Rats were injected with a single high dose of KA or vehicle and evidence for CI modifications was measured during the acute, latent, and chronic stages of epilepsy. Mitochondrial-specific carbonylation was increased acutely (48 h) and chronically (6 week), coincident with decreased CI activity. Mass spectrometry analysis of immunocaptured CI identified specific metal catalyzed carbonylation to Arg76 within the 75 kDa subunit concomitant with inhibition of CI activity during epileptogenesis. Computational-based molecular modeling studies revealed that Arg76 is in close proximity to the active site of CI and carbonylation of the residue is predicted to induce substantial structural alterations to the protein complex. These data provide evidence for the occurrence of a specific and irreversible oxidative modification of an important mitochondrial enzyme complex critical for cellular bioenergetics during the process of epileptogenesis.
线粒体氧化应激和损伤与颞叶癫痫的病因有关，但它们在癫痫发展（癫痫发生）过程中是否对线粒体过程产生功能影响尚不清楚。线粒体活性氧稳态水平升高的一个后果是蛋白质翻译后修饰（PTM）。我们假设线粒体电子传递链的蛋白复合物复合体I（CI）是氧化诱导的PTM（如羰基化）的靶点，导致癫痫发生过程中功能受损。本研究的目的是确定在大鼠海马体中，氧化修饰是否发生以及它们对CI酶活性在 kainate（KA）诱导的癫痫发生中的影响。给大鼠注射单次高剂量的KA或赋形剂，并在癫痫的急性、潜伏期和慢性期测量CI修饰的证据。线粒体特异性羰基化在急性（48小时）和慢性（6周）时增加，与CI活性降低同时出现。对免疫捕获的CI进行质谱分析，发现在癫痫发生过程中，75 kDa亚基内的Arg76发生了特定的金属催化羰基化，同时伴随着CI活性的抑制。基于计算的分子模拟研究表明，Arg76靠近CI的活性位点，该残基的羰基化预计会导致蛋白质复合物发生实质性的结构改变。这些数据为在癫痫发生过程中，对细胞生物能量学至关重要的重要线粒体酶复合物发生特定且不可逆的氧化修饰提供了证据。
