Ocular Proteomics, Singapore Eye Research Institute, Singapore 169856, Singapore.
Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore.
Int J Mol Sci. 2020 Apr 24;21(8):3027. doi: 10.3390/ijms21083027.
Leber's hereditary optic neuropathy (LHON, MIM#535000) is the most common form of inherited optic neuropathies and mitochondrial DNA-related diseases. The pathogenicity of mutations in genes encoding components of mitochondrial Complex I is well established, but the underlying pathomechanisms of the disease are still unclear. Hypothesizing that oxidative stress related to Complex I deficiency may increase protein -glutathionylation, we investigated the proteome-wide -glutathionylation profiles in LHON ( 11) and control ( 7) fibroblasts, using the GluICAT platform that we recently developed. Glutathionylation was also studied in healthy fibroblasts ( 6) after experimental Complex I inhibition. The significantly increased reactive oxygen species (ROS) production in the LHON group by Complex I was shown experimentally. Among the 540 proteins which were globally identified as glutathionylated, 79 showed a significantly increased glutathionylation ( < 0.05) in LHON and 94 in Complex I-inhibited fibroblasts. Approximately 42% (33/79) of the altered proteins were shared by the two groups, suggesting that Complex I deficiency was the main cause of increased glutathionylation. Among the 79 affected proteins in LHON fibroblasts, 23% (18/79) were involved in energetic metabolism, 31% (24/79) exhibited catalytic activity, 73% (58/79) showed various non-mitochondrial localizations, and 38% (30/79) affected the cell protein quality control. Integrated proteo-metabolomic analysis using our previous metabolomic study of LHON fibroblasts also revealed similar alterations of protein metabolism and, in particular, of aminoacyl-tRNA synthetases. -glutathionylation is mainly known to be responsible for protein loss of function, and molecular dynamics simulations and 3D structure predictions confirmed such deleterious impacts on adenine nucleotide translocator 2 (ANT2), by weakening its affinity to ATP/ADP. Our study reveals a broad impact throughout the cell of Complex I-related LHON pathogenesis, involving a generalized protein stress response, and provides a therapeutic rationale for targeting -glutathionylation by antioxidative strategies.
Leber 遗传性视神经病变（LHON，MIM#535000）是最常见的遗传性视神经病变和线粒体 DNA 相关疾病形式。编码线粒体复合物 I 组成成分的基因突变的致病性已得到充分证实，但该疾病的潜在发病机制仍不清楚。我们假设与复合物 I 缺陷相关的氧化应激可能会增加蛋白质的谷胱甘肽化，因此使用我们最近开发的 GluICAT 平台研究了 LHON（11 个）和对照（7 个）成纤维细胞的全蛋白质组谷胱甘肽化谱。还研究了实验性复合物 I 抑制后健康成纤维细胞（6 个）中的谷胱甘肽化。实验表明，LHON 组的复合物 I 导致活性氧（ROS）产生显著增加。在全球鉴定为谷胱甘肽化的 540 种蛋白质中，79 种在 LHON 中谷胱甘肽化显著增加（<0.05），94 种在复合物 I 抑制的成纤维细胞中增加。两组共有约 42%（33/79）的改变蛋白，表明复合物 I 缺陷是谷胱甘肽化增加的主要原因。在 LHON 成纤维细胞中受影响的 79 种蛋白质中，23%（18/79）参与能量代谢，31%（24/79）具有催化活性，73%（58/79）显示出各种非线粒体定位，38%（30/79）影响细胞蛋白质质量控制。使用我们之前对 LHON 成纤维细胞的代谢组学研究进行的综合蛋白质组代谢组学分析也揭示了蛋白质代谢的类似变化，特别是氨酰基-tRNA 合成酶的变化。谷胱甘肽化主要负责蛋白质失去功能，分子动力学模拟和 3D 结构预测证实了对腺嘌呤核苷酸转运蛋白 2（ANT2）的这种有害影响，削弱了其对 ATP/ADP 的亲和力。我们的研究揭示了复合物 I 相关 LHON 发病机制在整个细胞中的广泛影响，涉及广泛的蛋白质应激反应，并为通过抗氧化策略靶向谷胱甘肽化提供了治疗依据。
