Structure-guided investigation of the mechanisms linking protein import, substrate processing, and respiration in mitochondria

线粒体靶向序列（MTS）-将前体蛋白靶向线粒体的N-末端基序-被线粒体加工肽酶（MPP）切割，MPP是由两个结构相关的α和β亚基组成的异二聚体。然而，人们对这一过程仍然知之甚少。在后生动物中，MTS加工是高度复杂的，并且不仅与生物发生有关，而且与将蛋白质输入耦合到细胞器的能量状态的质量控制途径有关。作为这种偶联的证据，我们特别观察到在脊椎动物中，MPP-β与复合物III的核心1亚基（电子传递链（ETC）的必要组分）具有超过50%的序列同一性。虽然有证据表明在细胞中MPP-A和复合物III的核心2亚基之间存在嵌合复合物，但这种相互作用的意义仍然完全未知。本研究计划的目标是阐明脊椎动物中MPP和相关复合物切割底物的分子机制，并了解该过程如何受ETC的能量状态调节。我们的一般假设是，虽然MPP-a/β切割MTS作为大多数核编码蛋白正常成熟的一部分，嵌合的核心-2/MPP-B1以氧化还原依赖性方式结合并切割MTS的不同亚类。为了开展这项研究计划，我们将主要使用结构和蛋白质组学方法实现以下目标。具体目的：1.阐明MPP-a/MPP-a和Core-2/MPP-a切割底物的结构密码。我们已经建立了这些异源二聚体蛋白酶的细菌共表达系统，我们将使用该系统来分析它们的底物选择性，并通过X射线晶体学确定它们的结构。2.开发选择性的细胞渗透性MPP-a/β 2和Core-2/MPP-a抑制剂。我们发现，PINK 1的MTS，一种参与线粒体质量控制的激酶，以高亲和力结合MPP-a/MPP 2。我们将使用这种肽来设计选择性拟肽抑制剂，其使用基于FRET的测定来测量切割抑制而筛选。3.表征MPP复合物在哺乳动物细胞中的分布和活性。定量蛋白质组学方法将用于表征基础和氧化还原应激条件下两种异源二聚体蛋白酶的分布和活性。结合底物的MPP的结构，结合它们的切割动力学，将提供关于这种蛋白酶如何选择性地识别各种肽序列的关键线索，这将有助于预测对其他底物的切割效率。由于这些复合物对于存活是必需的，因此难以在细胞中进行功能丧失研究;因此，选择性抑制剂将是表征其功能并确定Core-2/MPP-B1嵌合体的作用的强大工具。最后，该项目将使学生成为结构生物学和蛋白质组学方法的高素质。

Mitochondrial targeting sequences (MTS) - N-terminal motifs which target precursor proteins to the mitochondria - are cleaved by the mitochondrial processing peptidase (MPP), a heterodimer consisting of two structurally related a and ß subunits. However, the process is still poorly understood. In metazoans, MTS processing is highly complex and is linked not only to biogenesis, but also to quality control pathways coupling protein import to the energetic status of the organelle. As evidence for this coupling, we notably observed that in vertebrates, MPP-ß shares more than 50% sequence identity with the Core 1 subunit of complex III, an essential component of the electron transport chain (ETC). While there is evidence of a chimeric complex between MPP-ß and the Core 2 subunit of complex III in cells, the significance of this interaction remains completely unknown. The goals of this research program are to elucidate the molecular mechanisms of substrate cleavage by MPP and related complexes in vertebrates and understand how the process is regulated by the energetic status of the ETC. Our general hypothesis is that while MPP-a/ß cleaves MTS from most nuclear-encoded proteins as part of their normal maturation, chimeric Core-2/MPP-ß binds and cleaves a different subset of MTS in a redox-dependent manner. To carry out this research program, we will pursue the following objectives using primarily structural and proteomics approaches. Specific objectives: 1.Elucidate the structural code of substrate cleavage by MPP-a/ß and Core-2/MPP-ß. We have established a bacterial co-expression system for these heterodimeric proteases that we will use to profile their substrate selectivity, and determine their structure by X-ray crystallography 2.Develop selective cell-permeable MPP-a/ß and Core-2/MPP-ß inhibitors. We discovered that the MTS of PINK1, a kinase involved in mitochondrial quality control, binds with high affinity to MPP-a/ß. We will use this peptide to design selective peptidomimetic inhibitors screened using a FRET-based assay to measure cleavage inhibition. 3.Characterize the distribution and activity of MPP complexes in mammalian cells. Quantitative proteomics methods will be used to characterize the distribution and activity of the two heterodimeric proteases, under basal and redox stress conditions. The structure of MPP bound to substrates, combined with their kinetics of cleavage, will provide critical clues on how this protease recognizes selectively a variety of peptide sequences, which will help predict the efficiency of cleavage for other substrates. Because these complexes are essential for survival, it is difficult to perform loss-of-function study in cells; thus, selective inhibitors will be formidable tools to characterize their function and ascertain the role of the Core-2/MPP-ß chimera. Finally, the project will enable students to become highly qualified in structural biology and proteomics methods.