Dynamic control of mitochondrial function by the protein unfoldase CLPX

摘要 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要 发展先行者的要求线粒体利用几种AAA+家族蛋白解折叠酶作为重要工具， 实现这种控制。我们在这里关注AAA+蛋白解折叠酶CLPX，它可以单独作用， 解折叠蛋白质，用于由其伴侣蛋白酶CLPP偶联降解。CLPX的缺失在小鼠中是致命的， CLPX和CLPP中的突变引起乳腺癌，CLPXP驱动癌症进展， 在各种癌症的临床试验中被药物靶向。这些表型表明， 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， 线粒体和细胞的需要。蛋白质组学研究已经将CLPX与多种可能的底物联系起来， 核心线粒体过程，虽然只有少数已被进一步表征，和机制， 有条件的底物选择在很大程度上仍不确定。CLPX最具特色的功能之一 是通过作用于该途径中的第一种酶来调节血红素生物合成，这是一种至关重要的线粒体功能， ALA合酶（ALAS）。CLPX通过部分解折叠激活ALAS以促进辅因子掺入。当 血红素水平高，CLPX（与其伴侣蛋白酶CLPP）也似乎通过完全抑制ALAS， 解折叠和降解，由ALAS中的血红素结合位点发出信号。初步结果显示， CLPXP生物化学重建血红素诱导的ALAS降解，证实了这一直接性质。 活动我们还发现，降解强烈依赖于血红素敏感的衔接蛋白。 本文提出的项目将（1）阐明血红素驱动装配和许可的机制， 降解复合物的ALAS，使用我们的重建系统与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平行。(2)我们将决定如何 血红素是直接感觉到复杂的使用光谱和结构的方法，并测试如何 调节该系统的血红素响应性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衔接子调节CLPX对底物的选择超过ALAS， 同时使用基于候选人的方法和无偏的蛋白质组学方法。这项研究将揭示 线粒体功能的条件控制的基本机制，并将提供详细的 用于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。

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.