Erlins in regulation of cholesterol-based endoplasmic reticulum functions

描述（由申请人提供）：胆固醇在人类和许多其他生物体中起着关键作用，作为膜的结构元素，并提供类固醇激素的前体。由于LDL相关血清胆固醇升高与人类心血管疾病风险的大幅增加有关，因此了解胆固醇稳态的分子基础至关重要。SREBP-2途径控制细胞的胆固醇合成和摄取。内质网（ER）中调节SREBP-2激活的胆固醇传感机制的核心组分已被详细表征。相比之下，很少有人知道他们的某些其他方面的调控，包括这些组件是否由蛋白质/脂质纳米结构域在ER的调节。Erlin-1和erlin-2是与富含胆固醇的耐洗涤剂膜断裂密切相关的ER蛋白。Erlin先前已与ER相关的IP 3受体降解有关。基于最近将erlin与SREBP-2通路联系起来的生物化学和功能结果，该项目将研究erlin在ER中组织富含胆固醇的膜筏样纳米结构域的假设，这些纳米结构域对胆固醇生物合成的调节很重要。这项工作将产生erlin-2突变体，预计这些突变体在组织这些纳米结构域方面存在缺陷。将分析这些突变体分配成洗涤剂抗性筏级分的能力，以及补充由内源性erlin沉默诱导的SREBP-2的甾醇敏感性调节的丧失的能力。额外的工作将分析重组野生型erlin-2和erlin-2突变体在筏形成缺陷的能力，直接结合胆固醇和神经酰胺，并将调查的SREBP-2机器的组成部分，是最近的邻居的erlin交联和质谱。总之，这些研究有望描绘erlin的主要特征，这些特征是它们在组织ER筏和调节SREBP-2通路中的作用的基础。因此，这项工作应该揭示一个以前未被认识的机制，涉及ER纳米结构域，这可能是至关重要的调节胆固醇生物合成。

DESCRIPTION (provided by applicant): Cholesterol plays a key role in humans and many other organisms, by serving as a structural element of membranes and by providing a precursor for steroid hormones. Since elevated LDL-associated serum cholesterol is linked to greatly increased risk of cardiovascular disease in humans, understanding the molecular basis for cholesterol homeostasis is of major importance. The SREBP-2 pathway controls cholesterol synthesis and uptake by cells. Core components of the cholesterol sensing machinery in the endoplasmic reticulum (ER) that regulate the activation of SREBP-2 have been characterized in detail. In contrast, little is known about certain other aspects of their regulation, including whether these components are regulated by protein/lipid nanodomains in the ER. Erlin-1 and erlin-2 are closely related ER proteins that fractionate with cholesterol-rich, detergent-resistant membranes. Erlins previously have been linked to ER-associated degradation of the IP3 receptor. Based on recent biochemical and functional results linking erlins to the SREBP-2 pathway, this project will investigate the hypothesis that erlins organize cholesterol-rich, membrane raft-like nanodomains in the ER that are important for regulation of cholesterol biosynthesis. The work will generate erlin-2 mutants that are predicted to be deficient in organizing these nanodomains. These mutants will be analyzed for their ability to partition into a detergent-resistant raft fraction, and to complement the loss of sterol-sensitive regulation of SREBP-2 that is induced by the silencing of endogenous erlins. Additional work will analyze the ability of recombinant wild-type erlin-2 and erlin-2 mutants deficient in raft formation to directly bind cholesterol and ceramide, and will investigate the components of the SREBP-2 machinery that are nearest neighbors of erlins by crosslinking and mass spectrometry. Together, these studies are expected to delineate major features of erlins that underlie their role in organizing ER rafts and in regulating the SREBP-2 pathway. Thus, the work should shed light on a previously unrecognized mechanism involving ER nanodomains that may be crucial for regulation of cholesterol biosynthesis.