Shot-gun proteomic analysis of mitochondrial D-loop DNA binding proteins: identification of mitochondrial histones
Shot-gun proteomic analysis of mitochondrial D-loop DNA binding proteins: identification of mitochondrial histones
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DOI：
10.1039/c0mb00277a
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发表时间：
2011-01-01
影响因子：
--
通讯作者：
Pak, Youngmi Kim
中科院分区：
文献类型：
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作者：
Choi, Yon-Sik;Jeong, Jae Hoon;Pak, Youngmi Kim
线粒体DNA（mtDNA）的转录和复制受靶向线粒体的核DNA编码蛋白的调节。线粒体DNA拷贝数的减少导致线粒体功能障碍，这可能导致胰岛素抵抗和代谢综合征。我们分析了线粒体蛋白质的物理结合人类线粒体D-环DNA使用猎枪蛋白质组学方法后，通过D-环DNA连接的亲和层析蛋白富集。采用超高压毛细管反相液相色谱/串联质谱法进行肽序列测定，共鉴定了152个D-环DNA结合蛋白。生物信息学分析表明，68个是线粒体蛋白，96个是DNA/RNA/蛋白结合蛋白，114个蛋白可能通过蛋白-蛋白相互作用形成复合物。其中H1、H2 A、H2 B、H3和H4组蛋白家族成员含量较高。特别是，组蛋白H2 A和H2 B存在于线粒体膜上作为完整的膜蛋白，而不是直接结合到细胞器内的mtDNA。组蛋白H1.2、H3和H4与线粒体外膜相关。沉默H2 AX表达抑制线粒体蛋白转运。我们的数据表明，许多线粒体蛋白可能存在于多个亚细胞区室，如H2 AX，并发挥多种功能。
Transcription and replication of mitochondrial DNA (mtDNA) are regulated by nuclear DNA-encoded proteins that are targeted into mitochondria. A decrease in mtDNA copy number results in mitochondrial dysfunction, which may lead to insulin resistance and metabolic syndromes. We analyzed mitochondrial proteins that physically bind to human mitochondrial D-loop DNA using a shot-gun proteomics approach following protein enrichment by D-loop DNA-linked affinity chromatography. A total of 152 D-loop DNA binding proteins were identified by peptide sequencing using ultra high pressure capillary reverse-phase liquid chromatography/tandem mass spectrometry. Bioinformatic analysis showed that 68 were mitochondrial proteins, 96 were DNA/RNA/protein binding proteins and 114 proteins might form a complex via protein-protein interactions. Histone family members of H1, H2A, H2B, H3, and H4, were detected in abundance among them. In particular, histones H2A and H2B were present in the mitochondrial membrane as integral membrane proteins and not bound directly to mtDNA inside the organelle. Histones H1.2, H3 and H4 were associated with the outer mitochondrial membrane. Silencing of H2AX expression inhibited mitochondrial protein transport. Our data suggests that many mitochondrial proteins may reside in multiple subcellular compartments like H2AX and exert multiple functions.