Identification of human mitochondrial metabolome proteins

（目的）线粒体 DNA (mtDNA) 编码线粒体电子传递系统必需的基因。最近我们提出 mtDNA 呈现出一种称为类核的大型蛋白质-DNA 复合物，类核结构对于 mtDNA 的维持至关重要。线粒体转录因子 A (TFAM) 是类核的主要成分。在这项研究中，我的目标是通过 LC/MS/MS 鉴定类核的蛋白质成分。（方法）用交联剂处理分离的人线粒体。溶解后，用抗 TEAM 抗体对类核进行免疫沉淀。沉淀的蛋白质在一维 SDS-PAGE 上分离、提取、胰酶消化并通过 LC/MS/MS 进行分析。（结果）1) 潜在的 DNA 或 RNA 结合蛋白 2) 热休克蛋白 3) 核糖体蛋白 4) TCA 循环相关蛋白 5) 脂肪酸 β 氧化酶 所有鉴定的蛋白质仅分为上述 5 组。这些结果表明线粒体核仁为翻译、TCA 循环和脂肪酸 β 氧化的有效代谢提供了支架。

(Aim) Mitochondrial DNA (mtDNA) encodes genes essential for the mitochondrial electron transfer system. Recently we proposed that mtDNA takes on a large protein-DNA complex called nucleoid and the nucleoid structure is critical for maintenance of mtDNA. Mitochondrial transcription factor A (TFAM) is a main component of the nucleoid. In this study, I aim to identify protein components of the nucleoid by LC/MS/MS.(Methods) Isolated human mitochondria were treated with a c ross-linker. After solubilization, the nucleoid was immunoprecipitated with anti-TEAM antibody. The precipitated proteins were separated on one dimensional SDS-PAGE, extracted, tripsinized, and and analyzed by LC/MS/MS.(Results)1)Potential DNA- or RNA binding proteins2)Heat shock proteins3)Ribosomal proteins4)TCA cycle related proteins5)Enzymes for β oxidation of fatty acidsAll proteins identified were classified into only 5 groups as above. These results suggest that the mitochondrial nucleoid provides a scaffold for efficient metabolism of translation, TCA cycles, and β oxidation of fatty acids.