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

Rnf185/Membralin 泛素连接酶复合物的表征及其内源性底物的鉴定

• 批准号: BB/W001519/1
• 负责人: Pedro Carvalho
• 金额: $ 59.39万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW001519%2F1
• 关键词: Characterization; Rnf185; Membralin; ubiquitin; ligase

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.

蛋白质参与几乎所有的细胞活动，它们的正确功能由大量的蛋白质质量控制系统来确保。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。质量控制的缺陷通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。膜蛋白和分泌蛋白的生物合成在内质网（ER）中在ER相关蛋白降解（ERAD）的监视下进行。该质量控制过程由对不同底物类别具有特异性的各个分支组成。虽然ERAD分支在简单的真核生物如芽殖酵母中得到了很好的表征，但在哺乳动物中只有少数分支被详细研究。我们努力扩大对哺乳动物ERAD的了解，最近导致了一个新的ERAD分支的鉴定，该分支由RNF 185/Membralin泛素连接酶复合物定义。我们发现，这种复合物由ER膜蛋白RNF 185，Membralin和TMUB 1/2组成，并促进膜蛋白的一个子集的降解。这些蛋白质如何组装成复合物并加工其底物尚不清楚。类似地，RNF 185/Membralin复合物的全部底物库以及它们的降解如何影响细胞生理学仍然是需要解决的重要问题。虽然对RNF 185或TMUB 1/2知之甚少，但缺乏Membralin的小鼠在出生后不久就死于与星形胶质细胞功能障碍相关的严重中枢神经系统缺陷。重要的是，在从肌萎缩侧索硬化症（ALS）患者分离的脊髓中观察到类似的缺陷，将星形胶质细胞中的膜蛋白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RNF 185和TMUB 1/2目前尚不清楚。该提案结合了培养细胞和HIPS-derived星形胶质细胞的遗传学、生物化学和蛋白质组学方法，以系统地解决这些悬而未决的问题。我们的工作将提供全面的深入了解RNF 185/Membralin ERAD分支，并阐明其在相关细胞系统中的生理作用。这些发现可能会为ALS等早发性神经退行性疾病中被破坏的机制提供信息，并最终暗示了治疗干预的新途径。

项目成果

Order through destruction: how ER-associated protein degradation contributes to organelle homeostasis.

通过破坏的顺序：与ER相关的蛋白质降解如何有助于细胞器稳态。

• DOI: 10.15252/embj.2021109845
• 发表时间: 2022-03-15
• 期刊: The EMBO journal
• 作者: Christianson JC;Carvalho P
• 通讯作者: Carvalho P