Global identification of endogenous ERAD substrates

项目摘要/摘要 我们研究的长期目标是通过以下方式实现对这一过程的全面和机械性的理解 哪些穿过内质网(ER)的蛋白质被识别并被降解。急诊室 负责大约三分之一细胞蛋白质组的折叠、修饰和运输， 包括存在于细胞表面的完整的膜蛋白(如通道和受体)和 分泌的可溶性蛋白质(如生长因子和胞外蛋白酶)。蛋白质的质量 从ER部署的资源由现在称为ER关联的系统严密监视和控制 退化(ERAD)。Erad使用广泛的成分网络来调节错误折叠的蛋白质 检测、错位进入细胞质、泛素化和蛋白酶体降解。除了它在以下方面的作用 除了清除错误折叠的蛋白质(即质量控制)外，ERAD还调节蛋白质的丰度(即， 数量控制)来调节重要的细胞生物学过程。例如，ERAD介导了类固醇- 胆固醇合成所必需的酶、HMG辅酶A还原酶和角鲨烯的依赖降解 单加氧酶。由于缺乏研究ERAD的全球方法，我们对ERAD池的理解 被不同ERAD途径降解的内源底物仍然有限。要克服这一点 我们开发了一种VCP抑制剂底物捕捉法(Vista)来鉴定内源ERAD 底物。在我们提出的研究中，我们将应用这一独特的策略来：1)定义特定于通路的ERAD 底物和2)鉴定代谢调节的ERAD底物。我们的研究将描述一种新的 方法对生物医学研究界具有广泛的实用价值，并将促进我们对 细胞蛋白质动态平衡中的erad。

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.