Esparza-Moltó Pau B, Goswami Arvind V, Bozkurt Süleyman, Münch Christian, Newman Laura E, Moyzis Alexandra G, Rojas Gladys R, Guan Deann, Jones Jeffrey R, Gage Fred H, Shadel Gerald S
Salk Institute for Biological Studies, La Jolla, CA, 92037, USA.
Institute of Molecular Systems Medicine, Faculty of Medicine, Goethe University, Frankfurt Am Main, Germany.
Redox Biol. 2025 Aug 12;86:103812. doi: 10.1016/j.redox.2025.103812.
Mitochondrial reactive oxygen species (mtROS) regulate cellular signaling pathways, but also cause oxidative stress when de-regulated during aging and pathological conditions such as neurodegenerative diseases. The dynamic redistribution of proteins between cellular compartments is a common mechanism to control their stability and biological activities. By targeting the BirA∗ biotin ligase to the outer mitochondrial membrane in HEK293 cells, we identified proteins whose labeling increased or decreased in response to treatment with menadione, consistent with a dynamic change in their mitochondrial localization in response to increased mtROS production. These proteins represent potential candidates for future studies of mitochondrial oxidative stress signaling. A subset of glycolytic enzymes was found in this screen and confirmed, by mitochondrial fractionation and imaging, to increase localization to mitochondria in response to menadione, despite no change in their overall abundance. Submitochondrial fractionation studies are consistent with import of a pool of these enzymes to the mitochondrial intermembrane space. Localization of glycolytic enzymes to mitochondria was also increased in cells grown under hypoxia or that express a mitochondria-targeted d-amino-acid oxidase (conditions that induce increased mtROS production), and inhibited basally under normal growth conditions by the mitochondrial antioxidant MnTBAP. Finally, primary Alzheimer's disease fibroblasts also had glycolytic enzymes associated with mitochondria that was reduced by antioxidants, consistent with increased mtROS altering their relative distribution between the cytoplasm and mitochondria. We speculate that the increased mitochondrial localization of glycolytic enzymes is an adaptive response to mtROS that alters glucose flux toward the antioxidant pentose phosphate pathway, creates distinct regulatory pools of mitochondrial metabolites or new metabolic circuits, and/or provides cytoprotection or other adaptive responses via moonlighting functions unrelated to their enzymatic activity.
线粒体活性氧（mtROS）调节细胞信号通路，但在衰老和神经退行性疾病等病理状况下失调时也会导致氧化应激。蛋白质在细胞区室之间的动态重新分布是控制其稳定性和生物学活性的常见机制。通过将BirA∗生物素连接酶靶向HEK293细胞中的线粒体外膜，我们鉴定出了那些在接受甲萘醌处理后标记增加或减少的蛋白质，这与它们因mtROS产生增加而导致的线粒体定位动态变化一致。这些蛋白质代表了未来线粒体氧化应激信号研究的潜在候选对象。在该筛选中发现了一部分糖酵解酶，并通过线粒体分级分离和成像证实，尽管它们的总体丰度没有变化，但在接受甲萘醌处理后其在线粒体中的定位增加。亚线粒体分级分离研究与这些酶中的一部分导入线粒体膜间隙一致。在缺氧条件下生长的细胞或表达线粒体靶向的d - 氨基酸氧化酶（诱导mtROS产生增加的条件）的细胞中，糖酵解酶在线粒体中的定位也增加，并且在正常生长条件下被线粒体抗氧化剂MnTBAP基本抑制。最后，原发性阿尔茨海默病成纤维细胞中也有线粒体相关的糖酵解酶，其被抗氧化剂减少，这与mtROS增加改变了它们在细胞质和线粒体之间的相对分布一致。我们推测，糖酵解酶在线粒体中定位增加是对mtROS的一种适应性反应，它改变了葡萄糖向抗氧化戊糖磷酸途径的通量，产生了不同的线粒体代谢物调节库或新的代谢回路，和/或通过与其酶活性无关的兼职功能提供细胞保护或其他适应性反应。
