Molecular Mechanism and In Vivo Study of ER Associated Degradation

描述(申请人提供)：蛋白质质量控制需要去除错误折叠的蛋白质。如果错误折叠的蛋白质持续存在，这种错配的积累会导致未折叠蛋白质反应(UPR)的激活，最终对细胞和生物体造成病理后果。在内质网中不能折叠的蛋白质，无论是管状的还是膜状的，通常都会被逆转或‘错位’到胞浆中进行降解。这种错位在很大程度上是泛素依赖的，错误折叠的胞浆蛋白的降解也是如此。该项目的总体目标是研究内质网相关降解(ERAD)的机制，以及这一过程的中断如何导致或是各种蛋白质错折叠疾病发展的重要因素。为了实现这一目标，将使用新型捕捉和释放探针与光交联剂相结合来确定参与错位的新成分：ERAD泛素化结合酶Ubc6e的体内泛素受体和Ubc6e的体内相互作用伙伴。目前，将内质网应激控制的研究扩展到生物体水平是困难的。Ploegh实验室已经开发出一些工具，现在可以让我们以一种无毒的方式阻止ER错位过程，这一方法将被开发并扩展到两个新的活体小鼠模型。新的位错组分的识别将是我们对ERAD机制基本知识的重要扩展。由于许多疾病都被归因于错误折叠蛋白质的积累，因此拟议的实验与许多疾病有关。

DESCRIPTION (provided by applicant): Protein quality control entails the removal of misfolded proteins. Accumulation of such misfits leads to activation of the unfolded protein response (UPR) with eventual pathological consequences to the cell and organism, should misfolded proteins persist. Proteins, either luminal or membrane-disposed, that fail to fold in the ER are often retro-translocated or 'dislocated' into the cytosol for degradation. This dislocation is to a large extent ubiquitin- dependent, as is the degradation of misfolded cytosolic proteins. The overall goal of the project is to study the mechanism of ER associated degradation (ERAD) and how disruption of this process leads to, or is an important factor in, the development of various protein misfolding diseases. To achieve this goal, novel catch-and-release probes will be applied in combination with photo-crosslinkers to identify new components involved in dislocation: in vivo ubiquitin acceptors of the ERAD ubiquitinating conjugating enzyme Ubc6e and in vivo interaction partners of Ubc6e. It is currently difficult to extend the study of ER stress control to the organismal level. The Ploegh lab has developed tools that now allow us to block the ER dislocation process in a non-toxic manner, an approach that will be exploited and extended to two new in vivo mouse models. Identification of novel dislocation components will be an important extension of our basic knowledge of ERAD machinery. Because many diseases have been attributed to the accumulation of misfolded proteins, the proposed experiments are relevant to numerous diseases.