Towards an Atomistic Understanding of Mitochondrial Protein Biogenesis

对线粒体蛋白质生物发生的原子理解

• 批准号: 10810236
• 负责人: Mark Anthony Herzik
• 金额: $ 1.4万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10810236
• 关键词: 3-Dimensional; Architecture; Award; Biochemical; Bioenergetics; Biogenesis; Biological Assay; Cells; Communities; Complex; Cryoelectron Microscopy; Data Collection; Dedications; Disease; Disease Progression; Electrons; Functional disorder; Genes; Human; In Vitro; Malignant Neoplasms; Membrane; Metabolic; Methodology; Mitochondria; Mitochondrial Proteins; Modeling; Modern Medicine; Molecular; Molecular Conformation; Motion; Natural Source; Nerve Degeneration; Neurodegenerative Disorders; Nuclear; Outer Mitochondrial Membrane; Oxidative Phosphorylation; Pathologic; Physiology; Preparation; Process; Protein Import; Protein Precursors; Proteins; Proteome; Research; Resolution; Ribosomes; Risk Factors; Role; Sampling; Signal Transduction; Sorting; Structure; Technology; Testing; Translating; combat; computerized data processing; insight; light microscopy; mitochondrial dysfunction; novel; technology development; translocase

PROJECT SUMMARY Maintaining mitochondrial integrity is necessary for normal eukaryotic physiology and, not surprisingly, mitochondrial dysfunction is a pathological hallmark of diseases and has been implicated as a primary risk factor for many cancers and neurodegenerative disorders. Critical to mitochondrial function is its dual-membrane architecture which provides appropriate microenvironments that facilitate specific metabolic functions – such as oxidative phosphorylation – and allow for otherwise incompatible processes to occur simultaneously inside the cell. Traditionally, research on mitochondria have focused on bioenergetics, but recent studies have begun to shed light on the intricacies and complexities of the mitochondrial proteome and the biogenesis machineries. This is particularly important as >99% of the mitochondrial proteome (~1500 proteins in humans) are encoded by nuclear genes and synthesized by cytosolic ribosomes as precursor proteins (preproteins). These preproteins contain endogenous signals that target them to mitochondria, where they are subsequently translocated across the outer membrane, sorted, compartmentalized, and properly folded by three main protein import machineries: the translocase of the outer mitochondrial membrane (TOM) complex, the mitochondrial translocase of the inner membrane (TIM)-23 complex (TIM23), and the TIM22 complex. These protein import complexes are required for the biogenesis of nearly all mitochondrial proteins and dysregulation poses a significant challenge to maintaining normal mitochondrial physiology. However, a dearth of structural information has precluded a molecular understanding of these processes and the mechanisms by which they perform their critical functions. Under this award, I will develop groundbreaking three-dimensional (3D) electron cryomicroscopy (cryoEM) technologies to pioneer studies of these critically important mitochondrial protein import complexes, providing critical insights into their function and their roles in the disease state. I will utilize targeted biochemical approaches to isolate the TOM, TIM23, and TIM22 complexes from natural sources for high-resolution cryoEM studies. I will then develop novel EM sample preparation, data collection and data processing strategies to yield a suite of high-resolution structures of each of these import machines during active preprotein import. I will then quantify the degree of local and global dynamics within these states through novel atomic modeling strategies as a means to define the conformational landscape. I will then establish in vitro functional assays to test key molecular steps during these processes. Lastly, I will then develop correlated light microscopy and high- resolution electron cryotomography (cryoET) methodologies to determine structure of these import complexes in their native membranes. Through these combined efforts, I will answer fundamental questions pertaining to the overall 3D architecture of these complexes and fully describe the molecular motions necessary for these import machines to import and fold mitochondrial preproteins. Importantly, these methodologies can be extended beyond the immediate scope of this award and be applied ubiquitously across the cryoEM community.

项目摘要 维持线粒体的完整性对于正常的真核生物生理是必要的， 线粒体功能障碍是疾病的病理学标志，并被认为是主要的危险因素 许多癌症和神经退行性疾病。线粒体功能的关键是它的双膜 提供适当的微环境以促进特定代谢功能的结构-例如 氧化磷酸化-并允许其他不相容的过程同时发生在细胞内。 cell.传统上，对线粒体的研究主要集中在生物能量学上，但最近的研究已经开始 揭示了线粒体蛋白质组和生物发生机制的复杂性。 这一点特别重要，因为>99%的线粒体蛋白质组（人类约1500种蛋白质）是编码的 由核基因和由胞质核糖体合成的前体蛋白（前蛋白）。这些前蛋白 含有内源性信号，将其靶向线粒体，随后在线粒体中转运， 外膜，通过三种主要的蛋白质输入机制进行分选、区室化和适当折叠： 线粒体外膜（TOM）复合物的易位酶，线粒体内膜（TOM）复合物的线粒体易位酶， 膜（TIM）-23复合物（TIM 23）和TIM 22复合物。这些蛋白质输入复合物是必需的， 几乎所有线粒体蛋白质的生物发生和失调对维持线粒体蛋白质的稳定性构成了重大挑战。 正常的线粒体生理然而，由于缺乏结构信息， 了解这些过程及其执行关键功能的机制。 在这个奖项下，我将开发突破性的三维（3D）电子低温显微镜 （cryoEM）技术，以开拓这些至关重要的线粒体蛋白质输入复合物的研究， 提供关键的洞察他们的功能和他们在疾病状态中的作用。我会利用有针对性的生化武器 从天然来源中分离TOM、TIM 23和TIM 22复合物用于高分辨率cryoEM的方法 问题研究然后，我将开发新的EM样品制备，数据收集和数据处理策略， 在活性前蛋白导入期间，这些导入机器中的每一个的一套高分辨率结构。然后我将 通过新颖的原子建模策略，量化这些状态中局部和全局动态的程度 作为定义构象景观的一种手段。然后，我将建立体外功能测定来测试关键的 在这些过程中的分子步骤。最后，我将开发相关的光学显微镜和高- 分辨率电子冷冻断层扫描（cryoET）方法，以确定这些进口复合物的结构 在它们的细胞膜上。通过这些共同努力，我将回答有关以下方面的基本问题： 这些复合物的整体三维结构，并充分描述了这些所需的分子运动， 进口机器来进口和折叠线粒体前蛋白。重要的是，这些方法可以扩展 超出了该奖项的直接范围，并在cryoEM社区中普遍应用。

项目成果

Electron counting takes microED to the next level.

电子计数将 microED 提升到了一个新的水平。

• DOI: 10.1038/s41592-022-01518-y
• 发表时间: 2022
• 期刊: Nature methods
• 影响因子: 48
• 作者: Corbett,KevinD;HerzikJr,MarkA
• 通讯作者: HerzikJr,MarkA

The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA.

• DOI: 10.1016/j.molcel.2022.09.008
• 发表时间: 2022-11-03
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Deep, Amar;Gu, Yajie;Gao, Yong-Qi;Ego, Kaori M.;Herzik Jr, Mark A.;Zhou, Huilin;Corbett, Kevin D.
• 通讯作者: Corbett, Kevin D.