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(申请人提供)：铁-硫(铁-S)簇是许多细胞过程所必需的蛋白质辅助因子，包括tRNA硫基化。在酵母和人类中，铁-S蛋白存在于线粒体和细胞质中。线粒体中有一个专门的系统，称为铁-硫簇机制，催化铁-S簇的合成，单独的线粒体本身就可以形成簇。胞质中铁-S簇的组装需要细胞质中铁-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。我们假设线粒体中的ISC机制产生一种含硫中间体(SINT)，该中间体由依赖三磷酸腺苷的转运体Atm1输出，并被中央情报局用于胞浆中的Fe-S簇合成和tRNA硫基化。在以35S-半胱氨酸为硫供体的新实验中，我们发现分离的细胞质不能合成铁-S簇或硫代tRNA。然而，将线粒体或线粒体产生的硫物种添加到细胞质中，就可以发生这些过程。目的1是确定酵母线粒体中Sint的形成和使用条件，线粒体产生Sint，细胞质将Sint用于Fe-S簇组装和tRNA硫基化。实验将涉及线粒体铁-S簇合成的操作，以及线粒体或胞浆中核苷酸的单独操作。目的2是确定ATM1及其输出底物的功能。在我们的检测中，完整的atm1突变线粒体不支持细胞质Fe-S簇组装或tRNA硫基化。实验将确定，是否可以通过新进口的完整atm1线粒体恢复这些过程，或者通过破坏线粒体膜绕过atm1的出口障碍。ATM1出口的活性SINT物种将通过层析纯化、质谱分析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以及DRE2在Fe-S簇合成中的作用。实验将确定细胞质组装所需铁的来源--线粒体或细胞质。Dre2是CIA的重要组成部分，它与Tah18形成还原酶复合体。将检测纯化的DRE2�Tah18复合体在缺乏DRE2(或Tah18)的细胞质中恢复Fe-S簇组装的能力。目的4确定ABCB7(人ATM1)和CIAPIN1(人DRE2)在哺乳动物细胞Fe-S簇组装和细胞凋亡中的作用。这些蛋白可能通过影响胞内Fe-S簇的组装来发挥其抗凋亡功能，这一假说将得到验证。Fe-S簇的生物发生是保守的，上述蛋白在人和酵母中都有同源蛋白。铁-S簇聚乱会导致骨髓衰竭、神经变性和肌病等疾病表现。在与ABCB7功能障碍相关的铁粒细胞性贫血中，红细胞前体积累了有毒数量的线粒体铁，并经历了细胞凋亡。线粒体-细胞质的相互作用是导致这种疾病和其他人类疾病的主要原因。