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）簇是多种细胞过程（包括tRNA巯基化）所需的必需蛋白质辅因子。在酵母和人类中，Fe-S蛋白存在于线粒体和细胞质中。线粒体中的一个专门系统，称为铁硫簇（ISC）机制，催化Fe-S簇的合成，并且分离的线粒体本身可以形成簇。细胞质中Fe-S簇的组装需要细胞质铁硫蛋白组装（CIA）机制。然而，中央情报局的系统本身并不起作用。我们假设，ISC机器在线粒体中产生含硫的中间体（Sint），这是出口的ATP依赖性转运蛋白Atm 1和使用的CIA在细胞质中的Fe-S簇合成和tRNA巯基化。在使用35 S-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簇或硫醇化tRNA。然而，向细胞质中添加线粒体或线粒体产生的硫物质允许这些过程发生。目的1是确定在酵母中形成和使用Sint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，以及线粒体或胞质溶胶中核苷酸的单独操作。目的二是明确Atm 1及其输出底物的功能。完整的atm 1突变体线粒体不支持细胞质Fe-S簇组装或tRNA巯基化在我们的测定。实验将确定这些过程是否可以通过完整的atm 1线粒体与新输入的atm 1恢复，或者通过破坏线粒体膜绕过atm 1中的输出块。Atm 1输出的活性Sint物质将通过色谱纯化、质谱和其他生物物理方法进行鉴定。目的3：确定酵母细胞质中铁的来源及Dre 2在Fe-S簇合成中的作用。实验将确定铁的起源细胞质簇组装-线粒体或细胞质。Dre 2是一种重要的CIA组分，它与Tah 18形成还原酶复合物。将检测纯化的Dre 2-Tah 18复合物在缺乏Dre 2（或Tah 18）的细胞质中恢复Fe-S簇组装的能力。目的4：研究ABCB 7（人Atm 1）和CIAPIN 1（人Dre 2）在哺乳动物细胞胞质Fe-S簇组装和细胞凋亡中的作用。这些蛋白质可能通过影响胞质Fe-S簇组装来执行其抗凋亡功能，并且将测试这一假设。Fe-S簇生物发生是保守的，并且上述所有蛋白质在人类和酵母中具有直系同源物。扰乱的Fe-S簇组装导致疾病表现，如骨髓衰竭、神经变性和肌病。在与ABCB 7功能障碍相关的铁粒幼细胞性贫血中，红细胞前体积累了毒性量的线粒体铁，并且它们进行细胞凋亡。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。