Global identification of endogenous ERAD substrates

项目概要/摘要 我们研究的长期目标是通过以下方式实现对该过程的全局和机械理解： 哪些蛋白质穿过内质网 (ER) 被识别并靶向降解。急诊室 负责大约三分之一的细胞蛋白质组的折叠、修饰和运输， 包括细胞表面的整合膜蛋白（例如通道和受体）和 分泌的可溶性蛋白质（例如生长因子和细胞外蛋白酶）。蛋白质的质量 从 ER 部署的数据由现在称为 ER 相关系统的严密监控和控制 降解（ERAD）。 ERAD 采用广泛的组件网络来介导错误折叠的蛋白质 检测、错位进入细胞质、泛素化和蛋白酶体降解。除了它的作用之外 清除错误折叠的蛋白质（即质量控制），ERAD 还调节蛋白质的丰度（即质量控制）。 量控制）来调节重要的细胞生物学过程。例如，ERAD 介导甾醇 胆固醇合成所需酶、HMG CoA 还原酶和角鲨烯的依赖性降解 单加氧酶。由于缺乏研究 ERAD 的全球方法，我们对 ERAD 池的理解 由不同 ERAD 途径降解的内源底物仍然有限。为了克服这个 障碍，我们开发了一种 VCP 抑制剂底物捕获方法 (VISTA) 来识别内源 ERAD 基材。在我们提出的研究中，我们将应用这种独特的策略来：1）定义途径特异性 ERAD 底物和 2) 鉴定代谢调节的 ERAD 底物。我们的研究将描述一个新的特征 对生物医学研究界具有广泛实用性的方法，并将增进我们对 ERAD 在细胞蛋白质稳态中的作用。

PROJECT SUMMARY / ABSTRACT The long-term goal of our research is to achieve a global and mechanistic understanding of the process by which proteins transiting the endoplasmic reticulum (ER) are recognized and targeted for degradation. The ER is responsible for the folding, modification, and trafficking of approximately one-third of the cellular proteome, including integral membrane proteins that are presented on the cell surface (e.g. channels and receptors) and soluble proteins that are secreted (e.g. growth factors and extracellular proteases). The quality of the proteins that are deployed from the ER is tightly monitored and controlled by a system now known as ER-associated degradation (ERAD). ERAD employs an extensive network of components that mediate misfolded protein detection, dislocation into the cytoplasm, ubiquitination, and proteasomal degradation. In addition to its role in the clearance of misfolded proteins (i.e. quality control), ERAD also regulates the abundance of proteins (i.e. quantity control) to modulate important cell biology processes. For example, ERAD mediates the sterol- dependent degradation of enzymes necessary for cholesterol synthesis, HMG CoA reductase and squalene monooxygenase. Due to the lack of global methods to study ERAD, our understanding of the pools of endogenous substrates that are degraded by different ERAD pathways remains limited. To overcome this obstacle, we developed a VCP-inhibitor substrate trapping approach (VISTA) to identify endogenous ERAD substrates. In our proposed research we will apply this unique strategy to: 1) define pathway-specific ERAD substrates and 2) identify metabolically regulated ERAD substrates. Our studies will characterize a new method of broad utility to the biomedical research community and will advance our understanding of the role of ERAD in cellular protein homeostasis.