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

蛋白质参与几乎所有的细胞事件，其正确的功能是由大量的蛋白质质量控制系统保证的。它们监测单个蛋白质的折叠、组装、定位和水平，并促进有缺陷和多余分子的清除。质量控制的缺陷常常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标志。膜和分泌蛋白的生物发生发生在内质网（ER）中，内质网相关蛋白降解（ERAD）的监测下。这种质量控制过程由不同的分支组成，对不同的基材类别具有特异性。虽然ERAD分支在简单的真核生物中有很好的特征，如出芽酵母，但在哺乳动物中只有少数分支被详细研究过。我们努力扩大对哺乳动物ERAD的认识，最近发现了一个新的ERAD分支，由RNF185/Membralin泛素连接酶复合物定义。我们发现该复合物由内质网膜蛋白RNF185、Membralin和TMUB1/2组成，并促进膜蛋白亚群的降解。这些蛋白质如何组装成复合物并处理它们的底物尚不清楚。同样，RNF185/Membralin复合物的全部底物以及它们的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的了解不多，但缺乏Membralin的小鼠在出生后不久就会因与星形胶质细胞功能障碍相关的严重中枢神经系统缺陷而死亡。重要的是，在肌萎缩性侧索硬化症（ALS）患者分离的脊髓中也观察到类似的缺陷，这将星形胶质细胞中的Membralin功能与神经变性联系起来。Membralin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。本研究将遗传、生化和蛋白质组学方法结合在培养细胞和hips衍生的星形胶质细胞中，以系统地解决这些开放性问题。我们的工作将提供对RNF185/Membralin ERAD分支的全面了解，并阐明其在相关细胞系统中的生理作用。这一发现可能为诸如ALS等早发性神经退行性疾病的机制中断提供信息，并最终为治疗干预提供新的途径。

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.