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（由申请人提供）：铁硫 (Fe-S) 簇是许多细胞过程（包括 tRNA 硫醇化）所需的重要蛋白质辅因子。在酵母和人类中，Fe-S 蛋白存在于线粒体和细胞质中。线粒体中有一个特殊的系统，称为铁硫簇（ISC）机制，催化铁硫簇的合成，分离的线粒体本身可以形成簇。细胞质中的 Fe-S 簇组装需要细胞质铁硫蛋白组装 (CIA) 机器。然而，中央情报局系统本身并不能运作。我们假设线粒体中的 ISC 机制产生一种含硫中间体 (Sint)，该中间体由 ATP 依赖性转运蛋白 Atm1 输出，并由 CIA 在细胞质中用于 Fe-S 簇合成和 tRNA 硫醇化。在使用 35S-半胱氨酸作为硫供体的新测定中，我们发现分离的细胞质不能合成 Fe-S 簇或硫醇盐 tRNA。然而，将线粒体或线粒体产生的硫物质添加到细胞质中可以使这些过程发生。目标 1 是确定酵母中 Sint 形成和使用的要求 - 线粒体产生它，细胞质使用它进行 Fe-S 簇组装和 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苷酸的单独操作。目标 2 是定义 Atm1 及其输出底物的功能。在我们的分析中，完整的 atm1 突变体线粒体不支持细胞质 Fe-S 簇组装或 tRNA 硫醇化。实验将确定这些过程是否可以通过使用新导入的 Atm1 的完整 atm1 线粒体来恢复，或者通过破坏线粒体膜绕过 atm1 中的导出块来恢复。 Atm1 输出的活性 Sint 物种将通过色谱纯化、质谱和其他生物物理方法进行鉴定。目标 3 是确定铁的来源以及 Dre2 在酵母细胞质中 Fe-S 簇合成中的作用。实验将确定细胞质簇组装的铁来源——线粒体或细胞质。 Dre2 是 CIA 的重要组成部分，它与 Tah18 形成还原酶复合物。将检查纯化的 Dre2�Tah18 复合物在缺乏 Dre2（或 Tah18）的细胞质中恢复 Fe-S 簇组装的能力。目标 4 是确定 ABCB7（人 Atm1）和 CIAPIN1（人 Dre2）在哺乳动物细胞细胞质 Fe-S 簇组装和细胞凋亡中的功能。这些蛋白质可能通过影响胞质 Fe-S 簇组装来发挥其抗凋亡功能，这一假设将得到检验。 Fe-S簇的生物发生是保守的，并且上述所有蛋白质在人类和酵母中都有直向同源物。 Fe-S 簇组装受到干扰会导致骨髓衰竭、神经变性和肌病等疾病表现。在与 ABCB7 功能障碍相关的铁粒幼细胞性贫血中，红细胞前体积累有毒量的线粒体铁，并且它们会发生凋亡。线粒体-细胞质相互作用是导致这种疾病和其他人类疾病的核心。