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

线粒体靶向序列(MTS) -将线粒体前体蛋白靶向的n端基序-由线粒体加工肽酶（MPP）切割，MPP是一种由两个结构相关的a和β亚基组成的异源二聚体。然而，人们对这一过程仍然知之甚少。在后生动物中，MTS加工非常复杂，不仅与生物发生有关，还与将蛋白质输入与细胞器的能量状态耦合在一起的质量控制途径有关。作为这种耦合的证据，我们特别观察到，在脊椎动物中，MPP-ß与电子传递链（ETC）的重要组成部分复合物III的核心1亚基具有超过50%的序列一致性。虽然有证据表明细胞中MPP-ß与复合物III的Core 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。本研究的目的是阐明MPP和相关复合物在脊椎动物中切割底物的分子机制，并了解ETC的能量状态如何调节这一过程。我们的一般假设是，当MPP-a/ß在大多数核编码蛋白的正常成熟过程中切割MTS时，嵌合的Core-2/MPP-ß以氧化还原依赖的方式结合和切割MTS的不同子集。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来实现以下目标。具体目标：1；阐明MPP-a/ß和Core-2/MPP-ß裂解底物的结构代码。我们已经建立了这些异二聚体蛋白酶的细菌共表达系统，我们将用它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。开发选择性细胞渗透性MPP-a/ß和Core-2/MPP-ß抑制剂。我们发现，参与线粒体质量控制的激酶PINK1的MTS与MPP-a/ß具有高亲和力。我们将使用这种肽来设计选择性拟肽抑制剂，使用基于fret的测定来测量切割抑制。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这两种异二聚体蛋白酶在基础和氧化还原应激条件下的分布和活性。结合底物的MPP结构及其裂解动力学，将为该蛋白酶如何选择性识别各种肽序列提供关键线索，这将有助于预测其他底物的裂解效率。由于这些复合物对细胞的生存至关重要，因此很难在细胞中进行功能丧失的研究；因此，选择性抑制剂将是表征其功能和确定Core-2/MPP-ß嵌合体作用的强大工具。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人才。

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.