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（由应用提供）：铁硫（Fe-S）簇是许多细胞过程所需的必需蛋白质辅助因子，包括tRNA硫醇化。在酵母和人类中，在线粒体和细胞质中发现了Fe-S蛋白。线粒体中的专门系统，称为铁硫簇（ISC）机械，催化Fe-S簇合成和孤立的线粒体本身可以形成簇。 Fe-S簇组装中的细胞质需要细胞质铁硫蛋白组件（CIA）机械。但是，中央情报局系统本身不起作用。我们假设线粒体中的ISC机械会产生含硫的中间体（SINT），该中级（SINT）由ATP依赖性转运蛋白ATM1导出，并由CIA在胞质醇中用于Fe-S群集合成和TRNA Thiolation。在新的使用35-cysteine作为硫供体的新颖测定中，我们发现分离的细胞质无法合成Fe-S簇或硫醇酸盐TRNA。但是，将线粒体或线粒体产生的硫物种添加到细胞质中可以使这些过程发生。目的1是定义在酵母中形成和使用SINT的要求 - 线粒体产生它，细胞质将其用于Fe -S群集组装和TRNA硫醇化。实验将涉及线粒体Fe-S簇合成的操作，以及线粒体或细胞质中核动肽的单独操作。 AIM 2是定义ATM1及其导出基板的功能。完整的ATM1突变体线粒体不支持我们的测定中的细胞质Fe-S簇组件或tRNA硫醇化。实验将确定这些过程是否可以通过新进口的ATM1进行完整的ATM1线粒体恢复，或者通过破坏线粒体膜的破坏来绕过ATM1中的导出块。 ATM1导出的活性SINT物种将通过色谱纯化，质谱和其他生物物理方法来识别。目标3是定义铁的来源和DRE2在酵母菌细胞质中Fe-S簇合成中的作用。实验将确定胞质簇组装铁的铁的起源 - 线粒体或细胞质。 DRE2是必不可少的CIA组件，它与TAH18形成了简化的复合物。纯化DRE2的能力。将检查缺乏DRE2（或TAH18）的Fe-S簇组件的TAH18复合物。 AIM 4是定义ABCB7（人ATM1）和CIAPIN1（人DRE2）在哺乳动物细胞中细胞质Fe-S簇组装和凋亡中的功能。这些蛋白质可能会通过对胞质Fe-S簇组装的影响执行其抗凋亡功能，并将检验该假设。 Fe-S簇生物发生是保守的，上面提到的所有蛋白质都有人类和酵母中的直系同源物。扰动的Fe-S簇组装导致疾病表现，例如骨髓衰竭，神经退行性变性和肌病。在与ABCB7功能障碍相关的Sideroblastic贫血中，红细胞前体积累了线粒体铁的有毒量，并且会发生凋亡。线粒体 - 细胞质相互作用是这种和其他人类疾病的核心。