Elucidating the contributions of ubiquitin ligase complexes to endoplasmic reticulum associated degradation (ERAD)

蛋白质是细胞的分子机器，执行广泛的基本功能。一系列广泛的遗传性人类疾病可以追溯到不能正确折叠的单一蛋白质。遗传密码的改变导致蛋白质无法达到功能所需的正确三维结构。其他疾病与细胞内不适合蛋白质正确折叠的条件有关。对于那些在细胞表面分泌或表达的蛋白质，蛋白质折叠发生在称为内质网（ER）的细胞内区室中。在ER中，每种蛋白质都通过质量控制机制进行评估，以决定它是否正确折叠。当蛋白质错误折叠时，一个复杂的补救方案将这些形式从ER中移除并破坏它们。总的来说，这种机制被称为ER相关降解（ERAD）。由于细胞表面和分泌的蛋白质占所有人类蛋白质的约30%，因此区分和降解如此广泛的潜在错误折叠蛋白质是一个重大挑战。ERAD使用能够识别、靶向、递送和降解具有各种构型的非天然蛋白质的相互关联的因子的网络。该方案中的一个中心组成部分是嵌入ER膜中的泛素连接酶家族。一个例子是Hrd 1，它通过在ER膜两侧支架不同的辅助因子来形成ERAD活性的联系。该MRC项目旨在研究含Hrd 1复合物的组成和组织，并确定整个ER-居民泛素连接酶家族对ERAD的贡献。 拟议研究的好处将是促进我们对错误折叠蛋白质如何在细胞中降解的理解，以及这种基本机制如何减轻细胞应激，这是许多疾病的标志。我们目前对ERAD的看法是基于对单个组分和底物的有限分析。拟议的研究旨在通过以下方式推进这一图景：1）详细表征进行ERAD的功能性Hrd 1复合物，2）使用经过验证的分离策略和质谱法来识别参与ERAD的其他ER驻留泛素连接酶复合物，3）确定细胞如何使用不同ERAD组分的表达来应对压力。随着与蛋白质错误折叠相关的疾病数量的增加，预计本文提出的全面分析将促进我们对这些疾病可能发生的理解。此外，这项研究可能有助于直接努力设计新的和替代的治疗策略，用于治疗与蛋白质错误折叠和运输相关的疾病，如囊性纤维化和α 1-抗胰蛋白酶缺乏症。

Proteins are the molecular machines of cells, performing a wide range of essential functions. A broad range of inherited human diseases can be traced back to single proteins that do not fold correctly. A change in the genetic code results in a protein that is unable to reach the correct 3-dimensional structure necessary to function. Other disorders are associated with conditions within cells that are unsuitable for proteins in general to fold correctly. For those proteins that are secreted or expressed on the surface of cells, protein folding occurs in the intracellular compartment known as the endoplasmic reticulum (ER). Within the ER, each protein is evaluated by quality control mechanisms that decide whether it's folded correctly. When proteins misfold, a complex remediation scheme is engaged which removes those forms from the ER and destroys them. Collectively, this mechanism is known as ER-associated degradation (ERAD). As cell surface and secreted proteins represent approximately 30% of all the human proteins made, distinguishing and degrading such a wide range of potentially misfolded proteins presents a significant challenge. ERAD uses a network of interconnected factors able to identify, target, deliver and degrade non-native proteins with various configurations. A central component in this scheme is the family of ubiquitin ligases embedded in the ER membrane. One example is Hrd1, which forms nexuses of ERAD activity by scaffolding different accessory factors on both sides of the ER membrane. This MRC project aims to characterise both the composition and organisation of Hrd1-containing complexes and to ascertain the contributions that the entire family of ER-resident ubiquitin ligases make to ERAD. The benefits of the proposed research will be to advance our understanding of how misfolded proteins are degraded in cells, and how this fundamental mechanism alleviates cellular stress that is the hallmark of many diseases. Our current view of ERAD has been based on a limited analysis of individual components and substrates. The proposed research aims to advance this picture by 1) characterising in detail, the functional Hrd1 complexes the carry out ERAD, 2) using a proven isolation strategy and mass spectrometry to characterise other ER-resident ubiquitin ligase complexes that participate in ERAD and 3) determining how cells use expression of the different ERAD components to respond to stress. As the number of diseases linked to protein misfolding grows, it is anticipated that the comprehensive analysis proposed here will advance our understanding of how these conditions may arise. Moreover, this study may help direct efforts to devise novel and alternative therapeutic strategies for the treatment of disorders of associated with protein misfolding and trafficking, such as cystic fibrosis and alpha1-antitrypsin deficiency.