Erlins in regulation of cholesterol-based endoplasmic reticulum functions

说明(申请人提供)：胆固醇在人类和许多其他生物体中发挥着关键作用，它是细胞膜的结构元素，也是类固醇激素的前体。由于与低密度脂蛋白相关的血清胆固醇升高与人类心血管疾病风险的显著增加有关，因此了解胆固醇稳态的分子基础是非常重要的。SREBP-2途径控制细胞胆固醇的合成和吸收。内质网(ER)中调节SREBP-2活性的胆固醇传感机制的核心组件已经被详细描述。相比之下，人们对其调控的某些其他方面知之甚少，包括这些成分是否受内质网中的蛋白质/脂纳米结构域的调控。Erlin-1和Erlin-2是密切相关的ER蛋白，与富含胆固醇的抗洗涤剂膜分离。Erlins以前被认为与内质网相关的IP3受体的降解有关。基于最近将Erlins与SREBP-2途径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，这些纳米结构域对调节胆固醇的生物合成非常重要。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配到耐洗涤剂的RAFT组分的能力，并补充由于内源性erlins沉默而导致的SREBP-2对固醇敏感调节的丧失。此外，还将分析RAFT形成缺陷的重组野生型Erlin-2和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与erlins最接近的SREBP-2机器的组件。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途径中的作用的基础。因此，这项工作应该有助于揭示一种以前未被认识到的涉及ER纳米结构域的机制，该机制可能对调节胆固醇的生物合成至关重要。

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.