The mechanism of apo-target recognition in cytsolic iron sulfur cluster biosynthesis

胞质铁硫簇生物合成中apo靶标识别机制

• 批准号: 10441415
• 负责人: DEBORAH L PERLSTEIN
• 金额: $ 31.76万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-05 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441415
• 关键词: Address; Affect; Affinity; Anabolism; Attenuated; Automobile Driving; Binding; Binding Sites; Biochemical; Biogenesis; Biological Assay; Cell Nucleus; Cell division; Chromosomal Instability; Code; Complex; Cytosol; DNA Damage; DNA Repair; DNA Repair Gene; DNA biosynthesis; Defect; Disease; Enzymes; Genetic Transcription; Genome; Genome Stability; Goals; Health; Homeostasis; Hot Spot; Human; Hypersensitivity; Impairment; In Vitro; Individual; Iron; Kinetics; Knowledge; Light; Link; Maintenance; Maps; Methods; Molecular; Multiprotein Complexes; Muscle; Mutate; Mutation; Nerve Degeneration; Nervous System Physiology; Nuclear; Pathway interactions; Phenotype; Play; Positioning Attribute; Premature aging syndrome; Process; Proteins; Recombinants; Role; Structure; Sulfur; Surface; System; Testing; Time; Translations; Tube; Work; Yeasts; carcinogenesis; cell growth; cofactor; flexibility; genotoxicity; improved; in vivo; mutant; protein protein interaction; reconstitution; telomere

Project Summary/Abstract The cytosolic iron sulfur (FeS) cluster assembly (CIA) pathway is essential since it supplies FeS clusters to enzymes which are essential for DNA replication and repair, transcription, and translation. Despite CIA's unquestionable importance for cell growth and division, we understand little about its mechanism. In particular, there is a dearth of information regarding how the >20 FeS-enzyme “targets” differing in their sequence, structure and function are all identified as CIA substrates so that their cofactors can be inserted in the final step of the pathway. Recent work has pinpointed a multiprotein complex, termed the CIA targeting complex (CTC), as being essential for this apo-target recognition step. The CTC subunits, called Met18, Cia1 and Cia2 in yeast, are highly conserved across the eukaryotic kingdom and their depletion results in a defect in FeS cofactor maturation in cytosolic and nuclear, but not mitochondrial, enzymes. However, progress to understand the final step of CIA has been slow due, in part, to the inability to access a reconstituted in vitro system for mechanistic analysis. We have recently overcome this barrier and are now poised to begin elucidating the mechanism by which apo-targets are identified. The remarkable ability of the CTC to flexibly yet specifically recognize the diverse pool of CIA targets is likely accomplished by Met18, Cia1 and Cia2 forming multiple distinct complexes, each responsible for recognition of a distinct subset of targets, by individual targets or subsets of targets sharing a common targeting motif sufficient for association with the CTC or by a combination of these two mechanisms. To reveal the molecular details underlying apo-target identification, we will 1) identify residues critical for formation of the targeting complex and evaluate how their mutation affects CIA function in vivo; 2) complete a comprehensive screen to identify functional residues of CTC subunits and pinpoint which are essential for target-binding in vitro and in vivo; and 3) elucidate the targeting motif exploited by the cytosolic FeS protein Leu1 and the nuclear FeS protein Rad3 for their association with the targeting complex. Successful completion of these aims is expected to provide fundamental knowledge about the structure of the targeting complex and the role(s) of its individual subunits, reveal the cryptic code driving CIA target recognition, and yield new information about how different targets or subsets of targets are identified by the CIA system. Since target recognition is not well understood for any cluster biogenesis pathway, this project is expected to shed light on this long-standing black box in the cluster biogenesis field. Finally, since defects CIA function result in sensitivity to DNA damaging agents, chromosomal instabilities, elongated telomeres and other genotoxic phenotypes, elucidation of the fundamental mechanism underlying CIA substrate recognition is expected to provide information essential for understanding the relationship between CIA and genome stability and the impact of CIA deficiencies on human health and disease.

项目摘要/摘要 胞质铁硫（FES）簇组件（CIA）途径是必不可少的 对于DNA复制和修复，转录和翻译至关重要的酶。尽管中央情报局 毫无疑问，对细胞生长和分裂的重要性，我们对其机制一无所知。尤其， 关于20个FES酶“靶标”的信息如何有所不同 结构和功能均被确定为CIA底物，以便可以将其辅助因子插入最后一步 路径。最近的工作指出了多蛋白复合物，称为CIA靶向复合物（CTC）， 对于这个apo-target识别步骤至关重要。 CTC亚基，称为Met18，CIA1和CIA2 酵母在整个真核生物王国中具有很高的保守性，其部署会导致FES缺陷 胞质和核的辅因子成熟，但不是线粒体的酶。但是，进步了解 中央情报局的最后一步部分是由于无法访问重组的体外系统 机械分析。我们最近克服了这一障碍，现在被中毒开始阐明 鉴定出Apo-targets的机制。 CTC具有灵活而专门的非凡能力 认识到CIA目标的多样化池可能是由Met18，Cia1和Cia2形成多个的。 独特的复合物，每个络合物负责识别各个目标或通过个人目标或 目标的子集共享共同的靶向基序，足以与CTC相关或合并 在这两种机制中。为了揭示Apo-Target识别的分子细节，我们将1） 确定残留物对于形成靶向复合物的至关重要并评估其突变如何影响CIA 体内功能； 2）完成一个综合屏幕，以识别CTC亚基的功能残差和 精确点，对于体外和体内目标结合至关重要； 3）阐明利用的靶向基序 通过胞质FES蛋白Leu1和核FES蛋白RAD3与靶向 复杂的。预计这些目标的成功完成将提供有关 靶向复合物的结构及其各个亚基的作用，揭示了驱动CIA的加密货币代码 目标识别，并产生有关如何确定目标的不同目标或子集的新信息 中央情报局系统。由于对于任何集群生物发生途径，目标识别尚未得到很好的了解，因此该项目 预计将在集群生物发生场中的这个长期存在的黑匣子揭示。最后，由于缺陷 CIA功能会对DNA破坏剂，染色体不稳定性，细长端粒和 其他遗传毒性表型，阐明CIA底物识别的基本机制是 预计将提供了解中央情报局与基因组稳定之间关系至关重要的信息 以及中央情报局缺陷对人类健康和疾病的影响。

项目成果

Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide.

胞浆铁硫蛋白组装系统通过 C 端三肽识别客户。

• DOI: 10.1101/2023.05.19.541488
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Marquez,MelissaD;Greth,Carina;Buzuk,Anastasiya;Liu,Yaxi;Blinn,CatharinaM;Beller,Simone;Leiskau,Laura;Hushka,Anthony;Wu,Kassandra;Nur,Kübra;Netz,DailiJ;Perlstein,DeborahL;Pierik,AntonioJ
• 通讯作者: Pierik,AntonioJ