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.

蛋白质几乎参与所有细胞事件，并且通过大量蛋白质质量控​​制系统确保其正确的功能。这些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。质量控制的缺陷通常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标志。膜和分泌蛋白的生物发生发生在ER相关蛋白降解（ERAD）的监视下的内质网（ER）中。该质量控制过程由各种分支组成，具有针对不同底物类的特异性。虽然Erad分支在简单的真核生物（如萌芽的酵母）中进行了充分的特征，但在哺乳动物中，仅研究了一些分支。我们为扩大对哺乳动物Erad的知识的努力，最近导致了RNF185/膜泛素泛素连接酶综合体定义的新的ERAD分支。我们表明该复合物由ER膜蛋白RNF185，膜和TMUB1/2组成，并促进了膜蛋白的一部分降解。这些蛋白质如何组装成复杂并处理其底物是未知的。同样，RNF185/Membralin复合物的底物的完整曲目以及它们的降解如何影响细胞生理学仍然是要解决的重要问题。虽然对RNF185或TMUB1/2的知之甚少，但由于严重的中枢神经系统缺陷，与星形胶质细胞功能障碍有关的严重中枢神经系统缺陷，缺乏膜林死亡的小鼠死亡。重要的是，在从肌萎缩性侧索硬化症（ALS）患者中分离出的脊髓中观察到了相似的缺陷，将星形胶质细胞中的膜蛋白功能与神经退行性变成联系。当前尚不清楚星形胶质细胞中膜蛋白的这些基本功能是否还取决于其伴侣RNF185和TMUB1/2。该建议结合了培养细胞和臀部衍生的星形胶质细胞中的遗传，生化和蛋白质组学方法，以系统地解决这些开放问题。我们的工作将为RNF185/膜蛋白ERAD分支提供全面的见解，并阐明其在相关的细胞系统中的生理作用。这些发现可能会导致在早期发作神经退行性疾病（例如ALS）中破坏的机制，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.

• 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