Division of Pharmacology/Toxicology, University of Texas at Austin, Austin, Texas 78714, USA.
Antioxid Redox Signal. 2011 Nov 15;15(10):2645-61. doi: 10.1089/ars.2011.3888. Epub 2011 Jul 12.
Uncoupling protein 3 (UCP3) is a member of the mitochondrial solute carrier superfamily that is enriched in skeletal muscle and controls mitochondrial reactive oxygen species (ROS) production, but the mechanisms underlying this function are unclear.
The goal of this work focused on the identification of mechanisms underlying UCP3 functions.
Here we report that the N-terminal, intermembrane space (IMS)-localized hydrophilic domain of mouse UCP3 interacts with the N-terminal mitochondrial targeting signal of thioredoxin 2 (Trx2), a mitochondrial thiol reductase. Cellular immunoprecipitation and in vitro pull-down assays show that the UCP3-Trx2 complex forms directly, and that the Trx2 N-terminus is both necessary and sufficient to confer UCP3 binding. Mutation studies show that neither a catalytically inactivated Trx2 mutant, nor a mutant Trx2 bearing the N-terminal targeting sequence of cytochrome c oxidase (COXMTS-Trx2) bind UCP3. Biochemical analyses using permeabilized mitochondria, and live cell experiments using bimolecular fluorescence complementation show that the UCP3-Trx2 complex forms specifically in the IMS. Finally, studies in C2C12 myocytes stably overexpressing UCP3 (2.5-fold) and subjected to Trx2 knockdown show that Trx2 is required for the UCP3-dependent mitigation of complex III-driven mitochondrial ROS generation. UCP3 expression was increased in mice fed a high fat diet, leading to increased localization of Trx2 to the IMS. UCP3 overexpression also increased expression of the glucose transporter GLUT4 in a Trx2-dependent fashion.
This is the first report of a mitochondrial protein-protein interaction with UCP3 and the first demonstration that UCP3 binds directly, and in cells and tissues with mitochondrial thioredoxin 2.
These studies identify a novel UCP3-Trx2 complex, a novel submitochondrial localization of Trx2, and a mechanism underlying UCP3-regulated mitochondrial ROS production.
解偶联蛋白 3（UCP3）是线粒体溶质载体超家族的成员，在骨骼肌中丰富，控制线粒体活性氧（ROS）的产生，但这种功能的机制尚不清楚。
这项工作的目标是确定 UCP3 功能的基础机制。
在这里，我们报告说，鼠 UCP3 的 N 端、跨膜间隙（IMS）定位的亲水结构域与线粒体硫氧还蛋白 2（Trx2）的 N 端线粒体靶向信号相互作用，Trx2 是一种线粒体硫醇还原剂。细胞免疫沉淀和体外下拉测定表明 UCP3-Trx2 复合物直接形成，并且 Trx2 N 端既是必需的也是充分的赋予 UCP3 结合。突变研究表明，既不是催化失活的 Trx2 突变体，也不是带有细胞色素 c 氧化酶（COXMTS-Trx2）N 端靶向序列的突变 Trx2 不结合 UCP3。使用透化线粒体进行生化分析，以及使用双分子荧光互补进行活细胞实验表明，UCP3-Trx2 复合物特异性地在 IMS 中形成。最后，在 C2C12 肌母细胞中进行的研究，这些细胞稳定过表达 UCP3（2.5 倍）并进行 Trx2 敲低，表明 Trx2 是 UCP3 依赖的减轻复合物 III 驱动的线粒体 ROS 生成所必需的。高脂肪饮食喂养的小鼠中 UCP3 的表达增加，导致 Trx2 向 IMS 的定位增加。UCP3 过表达还以 Trx2 依赖的方式增加葡萄糖转运蛋白 GLUT4 的表达。
这是与 UCP3 进行线粒体蛋白-蛋白相互作用的第一个报告，也是第一个证明 UCP3 直接与线粒体硫氧还蛋白 2 结合并在细胞和组织中结合的报告。
这些研究确定了一种新的 UCP3-Trx2 复合物、Trx2 的新亚线粒体定位以及 UCP3 调节的线粒体 ROS 产生的机制。
