Dynamic control of mitochondrial function by the protein unfoldase CLPX

蛋白质解折叠酶 CLPX 对线粒体功能的动态控制

基本信息

批准号：
10717543
负责人：
Julia R. Kardon
金额：
$ 31.47万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-10 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10717543
关键词：
Adaptor Signaling Protein Affect Binding Binding Sites Biochemical Biological Assay Cell Culture Techniques Cells Clinical Trials Coenzyme A Complex Coupled Data Defect Detection Disease Drug Targeting Electrons Environment Enzymes Equilibrium Eukaryotic Cell Family member Fatty Acids Feedback Goals Growth and Development function Hematological Disease Heme Heme Iron Human Human Activities Kinetics Licensing Ligands Ligase Link Malignant Neoplasms Mass Spectrum Analysis Measurement Measures Methods Mitochondria Mitochondrial Proteins Modeling Molecular Molecular Conformation Molecular Target Mus Mutation N-terminal Nature Negative Finding Outcome Oxygen Pathway interactions Peptide Hydrolases Peptides Phenotype Physiology Process Productivity Protein Engineering Protein Family Protein Truncation Proteins Proteome Proteomics Repression Roentgen Rays Role Shapes Signal Transduction Site Spectrum Analysis Structure System Testing Time Variant base cofactor disulfide bond heme a heme biosynthesis in vitro Assay nervous system disorder programs protein function reconstitution response therapeutic target therapy development tool tumor progression unfoldase

项目摘要

Abstract Mitochondria must maintain and regulate their proteome to meet the varied needs of the cell throughout growth and development. Mitochondria employ several AAA+ family protein unfoldases as an important tool in accomplishing this control. We here focus on the AAA+ protein unfoldase CLPX, which can act on its own or unfold proteins for coupled degradation by its partner protease CLPP. Loss of CLPX is lethal in mice, mutations in CLPX and CLPP cause mitochondriopathies, and CLPXP drives cancer progression and is targeted by drugs in clinical trials in a diverse range of cancers. These phenotypes indicate the crucial contributions of CLPX and CLPP to mitochondrial physiology. The overall goal of this project is to reveal the mechanisms by which CLPX selects substrates at the appropriate time, thus tailoring their function to mitochondrial and cellular needs. Proteomic studies have linked CLPX to multiple likely substrates that control core mitochondrial processes, although only a few have been further characterized, and mechanisms for conditional substrate selection remain largely undetermined. One of the best-characterized functions for CLPX is to regulate heme biosynthesis, a crucial mitochondrial function, by acting on the first enzyme in this pathway, ALA synthase (ALAS). CLPX activates ALAS by partial unfolding to facilitate cofactor incorporation. When heme levels are high, CLPX (with its partner protease CLPP) also appears to inactivate ALAS by complete unfolding and degradation, signaled by a heme-binding site in ALAS. In preliminary results, we have biochemically reconstituted heme-induced degradation of ALAS by CLPXP, confirming the direct nature of this activity. We additionally discovered that degradation strongly depends on a heme-sensitive adaptor protein. The project proposed here will (1) elucidate the mechanism by which heme drives assembly and licensing of a degradation complex for ALAS, using our reconstituted system with equilibrium-binding and kinetic analyses of complex assembly and ALAS degradation in parallel with observations in cells. (2) We will determine how heme is directly sensed within the complex using spectroscopic and structural methods and test how the heme-responsiveness of this system is tuned to suit different cellular programs. (3) We will determine how a heme-sensitive adaptor in the CLPXP degradation complex tunes substrate selection by CLPX beyond ALAS, using a candidate-based approach and an unbiased proteomic approach in parallel. This study will reveal fundamental mechanisms for the conditional control of mitochondrial functions and will provide detailed molecular targets for the development of therapy for multiple diseases with a basis in mitochondrial function, including disorders of heme biosynthesis and cancer.

抽象的线粒体必须维持和调节其蛋白质组，以满足细胞在整个生长过程中的各种需求和发展。线粒体使用多种 AAA+ 家族蛋白解折叠酶作为重要工具来完成这个控制。我们在这里重点关注 AAA+ 蛋白解折叠酶 CLPX，它可以单独发挥作用，也可以展开蛋白质并被其伙伴蛋白酶 CLPP 耦合降解。 CLPX 的缺失对小鼠来说是致命的， CLPX 和 CLPP 的突变会导致线粒体病，而 CLPXP 会促进癌症进展，并且在多种癌症的临床试验中被药物靶向。这些表型表明了关键的 CLPX 和 CLPP 对线粒体生理学的贡献。该项目的总体目标是揭示 CLPX 在适当的时间选择底物的机制，从而调整其功能线粒体和细胞的需求。蛋白质组学研究已将 CLPX 与多种可能控制的底物联系起来核心线粒体过程，尽管只有少数得到了进一步的表征，以及其机制条件底物选择在很大程度上仍未确定。 CLPX 最具特色的功能之一是通过作用于该途径中的第一个酶来调节血红素生物合成，这是一种重要的线粒体功能， ALA 合酶 (ALAS)。 CLPX 通过部分解折叠来激活 ALAS，以促进辅因子掺入。什么时候血红素水平很高，CLPX（及其伙伴蛋白酶 CLPP）似乎也会通过完全灭活 ALAS 展开和降解，由 ALAS 中的血红素结合位点发出信号。在初步结果中，我们有 CLPXP 生化重建血红素诱导的 ALAS 降解，证实了这种现象的直接性质活动。我们还发现降解强烈依赖于血红素敏感的衔接蛋白。这里提出的项目将（1）阐明血红素驱动组装和许可的机制 ALAS 的降解复合物，使用我们的重构系统进行平衡结合和动力学分析复杂的组装和 ALAS 降解与细胞中的观察同时进行。 (2) 我们将决定如何使用光谱和结构方法直接感测复合物内的血红素，并测试血红素如何该系统的血红素响应性经过调整以适应不同的细胞程序。 (3) 我们将决定如何 CLPXP 降解复合物中的血红素敏感接头可调节 CLPX 超越 ALAS 的底物选择，并行使用基于候选的方法和公正的蛋白质组学方法。这项研究将揭示线粒体功能条件控制的基本机制，并将提供详细的开发以线粒体功能为基础的多种疾病治疗的分子靶标，包括血红素生物合成障碍和癌症。