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。由于低密度脂蛋白相关的血清胆固醇升高与人类心血管疾病的风险大大增加有关，因此了解胆固醇稳态的分子基础具有重要意义。SREBP-2通路控制细胞的胆固醇合成和摄取。内质网（ER）中调节SREBP-2激活的胆固醇感知机制的核心成分已经被详细表征。相比之下，对其调控的某些其他方面知之甚少，包括内质网中这些成分是否受蛋白质/脂质纳米结构域的调控。Erlin-1和erlin-2是密切相关的内质网蛋白，与富含胆固醇的抗洗涤剂膜分离。Erlins先前被认为与er相关的IP3受体降解有关。基于最近将erlins与SREBP-2通路联系起来的生化和功能结果，本项目将研究erlins在内质网中组织富含胆固醇的膜筏状纳米结构域的假设，这些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。这些突变体将被分析其分裂成耐洗涤剂的筏状部分的能力，并补充内源性erlins沉默诱导的SREBP-2的胆固醇敏感调节的丧失。进一步的工作将分析重组野生型erlin-2和缺乏筏形结构的erlin-2突变体直接结合胆固醇和神经酰胺的能力，并将通过交联和质谱分析研究SREBP-2机制中与erlins最近邻的成分。总之，这些研究有望描述erlins在组织内质网筏和调节SREBP-2通路中的作用的主要特征。因此，这项工作应该揭示一个以前未被认识的机制，涉及内质网纳米结构域，可能对胆固醇生物合成的调节至关重要。

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.