Molecular mechanisms of Parkin-directed mitochondrial quality control

描述(由申请人提供)：帕金森氏病(PD)是一种破坏性的疾病，到目前为止只有对症治疗。病因仍然是个谜，因此没有阻止或预防帕金森病的治疗方法。在过去的几年里，由于我们的实验室和其他实验室发现了一种新的线粒体质量控制(MtQC)途径，这是非常令人兴奋的。到目前为止，这条通路连接了三个帕金森病相关基因PINK1 Parkin和Fbxo7，以及参与疾病发病机制的两个主要细胞功能障碍：MT功能障碍和降解途径障碍。MtQC途径被认为有助于消除功能障碍的细胞器，否则将导致进一步的细胞损伤。然而，这一途径的(病理)生理相关触发因素，特别是在与疾病相关的细胞和体内，尚不清楚。当PINK1激酶在受损的线粒体上聚集时，Parkin被招募来催化线粒体底物的不同泛素化。然而，Parkin的酶功能、它的E2辅助因子、形成的泛素链的拓扑结构及其生物学作用仍然是个谜。考虑到Parkin最近被分解的结构及其‘封闭’的自抑制构象，我们认为Parkin被顺序激活以释放其泛素连接酶功能。我们已经确定了一些E2酶，它们通过不同和相反的机制兴奋地调节Parkin的激活和酶功能。鉴于帕金氏激活级联过程中可能存在几种治疗机会，我们将对这种神经保护蛋白进行结构-功能分析。此外，错误折叠的蛋白质在线粒体中的积累可能对PINK1和Parkin的激活起到生理刺激作用。引人注目的是，诱导的mt特异性未折叠蛋白反应(MtUPR)最近被描述为一种保守的长寿机制。我们建议在结构、分子、细胞和生物水平上阐明Parkin功能的(In-)激活机制。因此，我们将 使用尖端技术，将计算、功能生化和细胞生物学与遗传方法相结合，在人类IPSC来源的神经元和线虫体内进行研究。根据初步数据，我们假设Parkin是通过mtUPR激活的，受生物能量学的调节，并与保守的衰老途径整合。在分子和结构水平上，Parkin受翻译后修饰、构象重排和特定的E2辅酶控制。具体地说，我们将1)揭示在健康和疾病中激活Parkin的生物学和分子机制；2)确定Parkin的生理E2辅酶，它们的调节作用和对PD的贡献；3)确定Parkin的活性及其与生物能量学和衰老途径的相互作用。建议的研究是为了充分认识Parkin指导的线粒体质量控制在疾病干预中的生物学意义和潜力，并揭示将为合理的药物设计提供基础的重要机制见解。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a devastating disorder for which to date only symptomatic treatments exist. The causes remain enigmatic and thus therapeutics that halt or prevent PD are not available. The last few years have been extremely exciting due to the discovery of a novel mitochondrial quality control (mtQC) pathway by our laboratory and others. By now, this pathway links three parkinsonism associated genes, PINK1 PARKIN, and FBXO7, as well as the two major cellular dysfunctions involved in disease pathogenesis: mt dysfunction and impairment of degradation pathways. The mtQC pathway is thought to facilitate the elimination of dysfunctional organelles that would otherwise cause further cellular damage. However, (patho-) physiological relevant triggers of this pathway, particular in disease-relevant cells and in vivo are unclear. Upon accumulation of the kinase PINK1 specifically on damaged mitochondria, Parkin is recruited to catalyze differential ubiquitinations of mitochondrial substrates. However, Parkin's enzymatic functions, its E2 co-factors, the topologies of formed ubiquitin chains and their biological roles remain enigmatic. Given the recently resolved structure of Parkin and its 'closed' auto-inhibited conformation, we suggest that Parkin is sequentially activated to unleash its ubiquitin ligase functions. We have identified select E2 enzymes that regulate Parkin's activation and its enzymatic functions, excitingly through different and opposing mechanisms. Given that several therapeutic opportunities may exist along Parkin's activation cascade, we will perform structure-function analyses of this neuroprotective protein. Further, the accumulation of misfolded proteins in mitochondria may act as a physiological stimulus for PINK1 and Parkin activation. Strikingly, the induced mt- specific unfolded protein response (mtUPR) has very recently been described as a conserved longevity mechanism. We propose to elucidate the (in-) activation mechanisms of Parkin's functions on the structural, molecular, cellular, and organismal level. Therefore, we will use cutting-edge technologies and combine computational, functional biochemical and cell-biological with genetic methods in human iPSC-derived neurons and in vivo in C. elegans. Based on preliminary data, we hypothesize that Parkin is activated through the mtUPR, is regulated by bioenergetics and integrates with conserved aging pathways. On the molecular and structural level, Parkin is controlled by post-translational modifications, conformational rearrangements and by select E2 co-enzymes. Specifically, we will 1) unravel biological and molecular mechanism that (in)-activate Parkin in health and disease; 2) determine Parkin's physiological E2 co-enzymes, their regulatory roles and contribution to PD; 3) determine Parkin's activity(ies) and their interplay with bioenergetics and aging pathways. The proposed studies are relevant to fully appreciate the biological significance and potential of Parkin-directed mitochondrial quality control for disease intervention and to uncover important mechanistic insights that will provide the basis for rationale drug design.