Characterization of the Rnf185/Membralin ubiquitin ligase complex and identification of its endogenous substrates

蛋白质几乎参与了所有的细胞活动，它们的正确功能由过多的蛋白质质量控制系统确保。它们监测单个蛋白质的折叠、组装、定位和水平，并促进缺陷和多余分子的清除。质量控制上的缺陷往往会导致有毒蛋白质和蛋白质聚集体的积累，这是许多神经退行性疾病的特征。在内质网相关蛋白降解(ERAD)的监控下，膜蛋白和分泌蛋白的生物发生发生在内质网中。该质量控制过程由针对不同底物类别的不同分支组成。虽然ERAD分支在像发芽酵母这样的简单真核生物中有很好的特征，但在哺乳动物中只有几个分支被详细研究过。我们努力扩大对哺乳动物ERAD的了解，最近发现了一个由RNF185/膜泛素连接酶复合体定义的新的ERAD分支。我们发现该复合体由内质网膜蛋白RNF185、膜蛋白和TMUB1/2组成，并促进膜蛋白的降解。这些蛋白质如何组装成复合体并处理它们的底物尚不清楚。同样，RNF185/膜蛋白复合体的全部底物及其降解如何影响细胞生理仍然是需要解决的重要问题。虽然对RNF185或TMUB1/2了解不多，但缺乏膜蛋白的小鼠出生后不久就会死于与星形胶质细胞功能障碍有关的严重中枢神经系统缺陷。重要的是，在肌萎缩侧索硬化症(ALS)患者分离的脊髓中也观察到了类似的缺陷，将星形胶质细胞的膜功能与神经变性联系起来。膜蛋白在星形胶质细胞中的这些基本功能是否也依赖于其伴侣RNF185和TMUB1/2目前尚不清楚。这项建议结合了培养细胞和髋关节来源的星形胶质细胞的遗传学、生化和蛋白质组学方法，以系统地解决这些悬而未决的问题。我们的工作将提供对RNF185/膜ERAD分支的全面见解，并阐明其在相关细胞系统中的生理作用。这些发现可能会揭示在ALS等早期起病的神经退行性疾病中被破坏的机制，并最终暗示治疗干预的新途径。

Proteins participate in virtually all cellular events and their correct functions are ensured by a plethora of protein quality control systems. These monitor the folding, assembly, localization and levels of individual proteins, and promote the clearance of defective and superfluous molecules. Defects in quality control often result in the accumulation of toxic proteins and protein aggregates, hallmarks of many neurodegenerative diseases. The biogenesis of membrane and secreted proteins takes place in the endoplasmic reticulum (ER) under the surveillance of the ER-associated protein degradation (ERAD). This quality control process consists of various branches with specificity for different substrate classes. While ERAD branches are well-characterized in simple eukaryotes like budding yeast, in mammals only a few branches have been studied in detail. Our efforts to expand the knowledge on mammalian ERAD, recently led to the identification of a new ERAD branch defined by the RNF185/Membralin ubiquitin ligase complex. We showed that this complex consists of the ER membrane proteins RNF185, Membralin and TMUB1/2, and promotes the degradation of a subset of membrane proteins. How these proteins assemble into a complex and process their substrates is unknown. Similarly, the full repertoire of substrates of the RNF185/Membralin complex and how their degradation impacts cellular physiology remain important issues to address. While not much is known about RNF185 or TMUB1/2, mice lacking Membralin die soon after birth due to severe central nervous system defects linked to astrocyte dysfunction. Importantly, similar defects were observed in spinal cords isolated from amyotrophic lateral sclerosis (ALS) patients, linking Membralin function in astrocytes to neurodegeneration. Whether these essential functions of Membralin in astrocytes also depend on its partners RNF185 and TMUB1/2 is currently unknown. This proposal combines genetic, biochemical and proteomics approaches in cultured cells and hIPS-derived astrocytes to systematically address these open questions. Our work will provide comprehensive insight into the RNF185/Membralin ERAD branch and shed light on its physiological role in a relevant cellular system. The findings may inform on the mechanisms disrupted during early onset neurodegenerative disorders such as ALS and ultimately hint at novel avenues for therapeutic intervention.