Ubiquitin-dependent mitochondrial quality control

摘要 线粒体是与年龄相关的人类疾病的中心，如帕金森氏症、阿尔茨海默氏症、亨廷顿氏症 疾病。随着当前人口老龄化的趋势，开发这些疾病的治疗方法是至关重要的 需要。保护策略，主要旨在中和有毒的活性氧物种(ROS)，一直只是 部分成功。因此，需要具有独立行动模式的新的治疗选择来改善 目前治疗的成功率。拟议的工作直接回应了这一需求，并将提供关键的 对线粒体质量控制途径的调节和功能的洞察。真核细胞有一种 致力于线粒体质量控制的多层系统。最初的防御由一个网络组成 降解受损、错误折叠或错位的蛋白质或控制其活性的蛋白质分解系统 应激反应线粒体因子。最近的发现，包括在PI实验室的工作，证明了 泛素(Ub)/蛋白酶体系统(UPS)，通过Ub依赖于OMM的降解/控制 相关蛋白是线粒体质量控制的关键。线粒体融合与分裂的调控 (线粒体动力学)是另一种主要的线粒体质量控制机制。最近我们提出了 线粒体膜外膜(OMM)蛋白的稳定性，包括线粒体融合因子Mfn1和 Mfn2由线粒体分裂蛋白Drp1和Mff控制。与此一致的是，我们的新数据表明 这一过程需要DRp1和MFf先前未被怀疑的依赖于Ub的信令功能。 我们还发现线粒体蛋白泛素化是局部的，这一过程在MFF-/-和MFF-/-中加速 Drp1-/-细胞。这些和其他初步结果表明，不受欢迎的机制调节和 协调线粒体质量控制途径，包括新的线粒体蛋白的参与 降解中间体以及线粒体动力学和有丝分裂之间的相互依赖。我们也 鉴定出一种与OMM相关的E3 Ub连接酶Rnf179，根据我们的初步发现，它不仅 控制OMM蛋白的稳定性，包括Mfn1和Mfn2，但也影响线粒体特异性自噬 (有丝分裂)，一个关键的UPS依赖的线粒体质量控制途径。这项提案的主要目标是 是为了检验假设，通过依赖于Ub的信号，Drp1和Mff控制和协调不同的 线粒体质量途径，包括线粒体分裂和融合率以及有丝分裂激活。 我们还期望确定Rnf179在线粒体蛋白泛素化中的作用和机制。 线粒体功能的控制和线粒体质量控制途径的整合，包括Parkin- 依赖的和独立的有丝分裂。为了实现这些目标，我们结合了生物化学、最先进的 成像，以及最近开发的基因编辑方法来解决以下问题：(1)什么是Ub- 介导线粒体分裂和融合机制之间串扰的依赖机制，以及如何 它是否保持这两个过程之间的平衡？(2)RNF179是通过什么机制进入 与MFF和Drp1协同控制线粒体内稳态？(3)Rnf179通过什么机制参与 与MFF和Drp1复合体的音乐会控制有丝分裂？

Summary Mitochondria are at the center of age-related human diseases, such as Parkinson’s, Alzheimer’s, Huntington’s diseases. With current trends toward the aging population, developing therapies for these diseases is a critical need. Protective strategies, mostly aimed at neutralizing toxic reactive oxygen species (ROS), have only been partially successful. Thus, new treatment options with independent modes of action are needed to improve the success rate of current therapies. The proposed work directly responds to this need and will provide critical insights into the regulation and function of mitochondrial quality control pathways. Eukaryotic cells have a multi-layered system dedicated to mitochondrial quality control. The initial defense consists of a network of proteolytic systems that degrade proteins that are damaged, misfolded or mislocalized, or control activities of stress responsive mitochondrial factors. Recent findings, including work in the PI’s laboratory, demonstrated that the ubiquitin (Ub)/proteasome system (UPS), through Ub-dependent degradation/control of the OMM associated proteins is critical for mitochondrial quality control. Regulation of mitochondrial fusion and fission (mitochondrial dynamics) is another principal mitochondrial quality control mechanism. Recently we proposed that stability of outer mitochondrial membrane (OMM) proteins, including mitochondrial fusion factors Mfn1 and Mfn2 is controlled by mitochondrial fission proteins Drp1 and Mff. Consistent with this, our new data indicate that previously unsuspected Ub-dependent signaling functions of Drp1 and Mff are required for this process. We also found that mitochondrial protein ubiquitination is focal and that this process is accelerated in Mff-/- and Drp1-/- cells. These and other preliminary results, suggest unappreciated mechanisms regulating and coordinating mitochondrial quality control pathways, including participation of the novel mitochondrial protein degradation intermediates and mutual dependence between mitochondrial dynamics and mitophagy. We also identified an OMM-associated E3 Ub ligase Rnf179, which according to our preliminary findings not only controls stability of OMM proteins, including Mfn1 and Mfn2, but also affects mitochondria-specific autophagy (mitophagy), a critical UPS-dependent mitochondrial quality control pathway. The principle goal of this proposal is to test the hypothesis that through Ub-dependent signaling, Drp1 and Mff control and coordinate distinct mitochondrial quality pathways, including mitochondrial fission and fusion rates and activation of mitophagy. We also anticipate defining the role and mechanism of Rnf179 with mitochondrial protein ubiquitination in the control of mitochondrial function and integration of mitochondrial quality control pathways, including Parkin- dependent and -independent mitophagy. To achieve these goals we combine biochemical, state-of-the-art imaging, and recently developed gene editing methods to address the following questions: (1) What is the Ub- dependent mechanism that mediates crosstalk between mitochondrial fission and fusion machineries, and how does it maintain balance between these two processes? (2) What is the mechanism by which Rnf179 in concert with Mff and Drp1 controls mitochondrial homeostasis? (3) What is the mechanism by which Rnf179 in concert with Mff and Drp1 complex controls mitophagy?