Mitochondria-cytoplasm interactions for cytosolic Fe-S cluster assembly

细胞质 Fe-S 簇组装的线粒体-细胞质相互作用

• 批准号: 8692127
• 负责人: ANDREW B. DANCIS
• 金额: $ 57.82万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8692127
• 关键词: ATP Hydrolysis; Apoptosis; Apoptotic; Bacteria; Biogenesis; Biological Assay; Bypass; Catalysis; Cell Survival; Cell physiology; Cells; Complex; Cysteine; Cytoplasm; Cytosol; DNA Repair; Disease; Electron Transport; Erythrocytes; Etiology; Eukaryota; Eukaryotic Cell; Eye; Functional disorder; Generations; Health; Homeostasis; Human; Inner mitochondrial membrane; Interleukin-3; Iron; Iron-Sulfur Proteins; Label; Mammalian Cell; Mass Spectrum Analysis; Mediating; Mitochondria; Modification; Molecular Chaperones; Myopathy; NADP; Nerve Degeneration; Nucleotides; Organelles; Organic solvent product; Orthologous Gene; Oxidoreductase; Pancytopenia; Process; Protein S; Proteins; Respiration; Ribosomes; Role; Sideroblastic Anemia; Source; Sulfur; System; Testing; Transfer RNA; Withdrawal; Work; Yeasts; base; biophysical techniques; cell type; cofactor; cytokine; human BIRC2 protein; human disease; mitochondrial membrane; mutant; novel; nucleotide analog; overexpression; research study; solvent extraction

DESCRIPTION (provided by applicant): Iron-sulfur (Fe-S) clusters are essential protein cofactors required for numerous cellular processes including tRNA thiolation. In yeast and humans, Fe-S proteins are found in mitochondria and cytoplasm. A specialized system in mitochondria, called the Iron-Sulfur Cluster (ISC) machinery, catalyzes Fe-S cluster synthesis, and isolated mitochondria by themselves can form clusters. Fe-S cluster assembly in cytosol requires the Cytoplasmic Iron-Sulfur Protein Assembly (CIA) machinery. However, the CIA system does not work by itself. We hypothesize that the ISC machinery in mitochondria generates a sulfur-containing intermediate (Sint), which is exported by the ATP-dependent transporter Atm1 and used by the CIA in the cytosol for Fe-S cluster synthesis and tRNA thiolation. In novel assays using 35S-cysteine as the sulfur donor, we find that isolated cytoplasm cannot synthesize Fe-S clusters or thiolate tRNAs. However, addition of mitochondria or a mitochondrial generated sulfur species to the cytoplasm allows these processes to occur. Aim 1 is to define the requirements for formation and use of Sint in yeast - mitochondria produce it and cytoplasm uses it for Fe-S cluster assembly and tRNA thiolation. Experiments will involve manipulations of mitochondrial Fe-S cluster synthesis, and separate manipulations of nucleotides in mitochondria or cytosol. Aim 2 is to define the function of Atm1 and its export substrate. Intact atm1 mutant mitochondria did not support cytoplasmic Fe-S cluster assembly or tRNA thiolation in our assays. Experiments will determine if these processes can be restored by intact atm1 mitochondria with newly imported Atm1, or by bypassing the export block in atm1 through disruption of mitochondrial membranes. The active Sint species exported by Atm1 will be identified by chromatographic purifications, mass spectrometry, and other biophysical methods. Aim 3 is to define the source of iron and the role of Dre2 in Fe-S cluster synthesis in yeast cytoplasm. Experiments will determine the origin of iron for cytosolic cluster assembly - mitochondria or cytoplasm. Dre2 is an essential CIA component, and it forms a reductase complex with Tah18. The ability of purified Dre2�Tah18 complex to restore Fe-S cluster assembly in cytoplasm lacking Dre2 (or Tah18) will be examined. Aim 4 is to define the functions of ABCB7 (human Atm1) and CIAPIN1 (human Dre2) in cytoplasmic Fe-S cluster assembly and apoptosis in mammalian cells. These proteins might perform their anti-apoptotic functions via effects on cytosolic Fe-S cluster assembly, and this hypothesis will be tested. Fe-S cluster biogenesis is conserved, and all of the proteins mentioned above have orthologs in humans and yeast. Perturbed Fe-S cluster assembly results in disease manifestations such as bone marrow failure, neurodegeneration and myopathy. In sideroblastic anemia associated with ABCB7 dysfunction, red cell precursors accumulate toxic amounts of mitochondrial iron, and they undergo apoptosis. Mitochondria- cytoplasm interactions are central to causation of this and other human diseases.

描述：铁硫（Fe-s）簇是众多过程中的必不可少的蛋白质辅助因子，在INT和人类中，fe-s蛋白在线粒体和细胞质中被称为铁硫酸盐（ISC）机械，催化剂。 Fe-S簇和分离的线粒体簇在细胞溶胶中的TER组装需要细胞质的铁硫蛋白组装（CIA）机制。 ATP依赖性转运蛋白的ATM1并由Fe-S簇合成和硫醇化的新测定法中使用35s甲基化的硫酸盐供体。发生。在线粒体或细胞质中。由ATM1导出的SINT物种将通过色谱净化e来鉴定铁的来源，而DRE2在Fe -S簇合成中的作用确定了胞质簇的铁的铁质 - 丝状细胞质或细胞质。与TAH18形成的还原酶复合物，将检查纯化的DRE2TAH18复合物恢复缺乏DRE2的Fe -S细胞质（或TAH18）的能力。哺乳动物细胞的凋亡。和东方的Fe-s簇会导致疾病表现，例如骨骼和肌病，与ABCB7有关这种因果关系和OTER人类疾病。