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不同需求。 和发展。线粒体利用几种AAA+家族蛋白解折叠酶作为重要工具 完成这一控制。这里我们关注的是AAA+蛋白展开酶CLPX，它可以自己起作用或 通过其伙伴蛋白酶CLPP展开蛋白质以进行偶联降解。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疾病，而ClpXP会推动癌症的进展，并 在各种癌症的临床试验中被药物靶向。这些表型表明关键的 CLPX和CLPP对线粒体生理学的贡献。这个项目的总体目标是揭示 CLPX在适当的时间选择底物的机制，从而使它们的功能适合 线粒体和细胞的需求。蛋白质组学研究已将CLPX与多种可能的底物联系起来，这些底物控制着 核心线粒体过程，尽管只有几个得到了进一步的表征，以及 有条件的底物选择在很大程度上仍未确定。CLPX最具特征的函数之一 是通过作用于这一途径中的第一个酶来调节血红素的生物合成，这是线粒体的一项关键功能， 丙氨酸合成酶(ALA)。CLPX通过部分解离激活ALAS，促进辅因子掺入。什么时候 血红素水平很高，CLPX(与其伙伴蛋白酶CLPP)似乎也通过完全灭活ALAS 展开和降解，通过ALAS中的一个血红素结合部位发出信号。在初步结果中，我们有 生化重组血红素诱导ClpXP对ALAs的降解，证实了其直接性质 活动。我们还发现，降解强烈依赖于一种对血红素敏感的适配蛋白。 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的降解复合体，使用我们的重组系统进行平衡结合和动力学分析 复杂的组装和碱性磷酸酶的降解与细胞内的观察平行。(2)我们将决定如何 使用光谱和结构方法在复合体中直接感觉到血红素，并测试如何 这个系统对血红素的反应能力经过调整，以适应不同的细胞程序。(3)我们将确定如何 ClpXP降解复合体中的血红素敏感适配器调节CLPX Beyond Alas的底物选择， 同时使用基于候选的方法和无偏见的蛋白质组学方法。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 以线粒体功能为基础的多种疾病治疗发展的分子靶点， 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.