Ubiquitin-dependent mitochondrial quality control

总结 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 疾病随着目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。 需要的保护策略，主要是针对中和有毒的活性氧（ROS），只有 部分成功。因此，需要具有独立作用模式的新的治疗选择来改善患者的健康状况。 目前治疗的成功率。拟议的工作直接回应了这一需要，并将提供关键的 深入了解线粒体质量控制途径的调节和功能。真核细胞具有 致力于线粒体质量控制的多层系统。最初的防御包括一个网络， 蛋白水解系统降解受损、错误折叠或错误定位的蛋白质，或控制 应激反应线粒体因子。最近的研究结果，包括PI实验室的工作，表明 泛素（Ub）/蛋白酶体系统（UPS），通过Ub依赖性降解/控制OMM， 相关蛋白对于线粒体质量控制至关重要。线粒体融合和分裂的调节 线粒体动力学（mitochondrial dynamics）是另一种主要的线粒体质量控制机制。最近我们提议 线粒体外膜（OMM）蛋白的稳定性，包括线粒体融合因子Mfn 1和 Mfn 2由线粒体分裂蛋白Drp 1和Mff控制。与此相一致，我们的新数据表明， 先前未被怀疑的Drp 1和Mff的Ub依赖性信号传导功能是这一过程所必需的。 我们还发现，线粒体蛋白泛素化是焦点，这一过程在Mff-/-和 Drp 1-/-细胞。这些和其他初步结果表明，不受重视的机制调节， 协调线粒体质量控制途径，包括新线粒体蛋白的参与 降解中间体和线粒体动力学和线粒体自噬之间的相互依赖。我们也 鉴定了OMM相关的E3 Ub连接酶Rnf 179，根据我们的初步发现， 控制OMM蛋白质的稳定性，包括Mfn 1和Mfn 2，但也影响ESTA特异性自噬 线粒体自噬（mitophagy），一种关键的UPS依赖性线粒体质量控制途径。本提案的主要目标是 通过Ub依赖性信号传导，Drp 1和Mff控制和协调不同的细胞， 线粒体质量途径，包括线粒体分裂和融合率以及线粒体自噬的激活。 我们还期望通过线粒体蛋白泛素化来确定Rnf 179在细胞凋亡中的作用和机制。 控制线粒体功能和整合线粒体质量控制途径，包括帕金- 依赖性和非依赖性线粒体自噬。为了实现这些目标，我们将联合收割机 成像，以及最近开发的基因编辑方法来解决以下问题：（1）Ub- 介导线粒体分裂和融合机器之间串扰的依赖机制，以及如何 它是否在这两个过程之间保持平衡？(2)Rnf 179在体内的作用机制是什么？ 与Mff和Drp 1共同控制线粒体稳态？(3)什么是Rnf 179的机制？ 与Mff和Drp 1复合体控制线粒体自噬的协同作用？

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?