Regulation of Cellular Cholesterol Homeostasis

描述(由申请人提供)： 细胞脂平衡是维持双层流动性、膜不通透性和细胞器特性所必需的。系统脂平衡失调是冠状动脉疾病和肥胖相关的II型糖尿病的病理核心。这项研究的长期目标是将细胞调节事件的知识转化为整个生物体的知识，并促进我们对这些日益广泛的疾病的理解。作为实现这一目标的第一步，我们将使用胆固醇作为模型脂质来了解细胞如何测量这些主要不可溶分子的水平，然后调节它们的产生。 哺乳动物细胞中的胆固醇动态平衡受到反馈机制的调节，该反馈机制监测细胞膜中的胆固醇水平，并改变胆固醇供应所需基因的转录。这些基因的转录是由一种名为SREBP的ER膜结合转录因子控制的，该转录因子在甾醇耗竭的细胞中通过蛋白分解激活并从膜上释放。另外两个ER膜蛋白，SCAP和INSIG，通过控制SREBP的ER退出和获得高尔基体定位的蛋白酶，作为SREBP活性的正负调节因子。到目前为止，SCAP和INSIG如何感知类固醇和调节SREBP的ER退出尚不清楚。 为了加速发现新的蛋白质和化学调节因子，我们将在哺乳动物细胞研究的同时，分析SREBP、SCAP和INSIG同源基因在遗传易处理的酵母模型中的功能。我们的序列数据库搜索显示，裂解酵母S.pombe，而不是萌芽酵母S.cerevisiae，分别含有SREBP、SCAP和INSIG的未鉴定同源物：slp1+、scp1+和ins1+。本项目的主要目的是利用S.pombe分离SREBP和SCAP的未知调控因子。我们推测，Scp1通过与哺乳动物细胞相似的机制感知类固醇并调节SLp1的裂解。在本项目中，将结合遗传学、分子和生物化学方法来实现以下特定目标：1)确认S.pombe基因是哺乳动物SREBP、SCAP和INSIG的功能同源基因；2)定义SIP1调控的转录程序并鉴定SIP1激活的内源化学调节因子；3)分离SIP1激活所需的新基因；4)采用亲和纯化的方法分离Scp1和Ins1相互作用的蛋白质。人们期望S.pombe中的新型调控因子将在哺乳动物中具有可识别的同源基因，从而为我们对人类胆固醇稳态的研究提供新的见解，并为预防和治疗心脏病提供潜在的治疗靶点。

DESCRIPTION (provided by applicant): Cellular lipid homeostasis is required to maintain bilayer fluidity, membrane impermeability, and organelle identity. Disturbances in systemic lipid homeostasis lie at the core of the pathologies for both coronary artery disease and obesity related, type II diabetes. The long-term goal of this research is to translate knowledge of cellular regulatory events to that of the whole organism and to advance our understanding of these increasingly widespread diseases. As a first step toward this goal, we will use cholesterol as a model lipid to understand how cells measure levels of these largely insoluble molecules and then modulate their production. Cholesterol homeostasis in mammalian cells is regulated by a feedback mechanism that monitors the level of cholesterol in membranes and alters transcription of genes required for cholesterol supply. Transcription of these genes is controlled by an ER membrane-bound transcription factor called SREBP that is activated and released from the membrane by proteolysis in sterol-depleted cells. Two additional ER membrane proteins, SCAP and INSIG, act as positive and negative regulators of SREBP activity by controlling ER exit of SREBP and access to Golgi-localized proteases. To date, it is unknown how SCAP and INSIG sense sterols and regulate ER exit of SREBP. To accelerate discovery of novel protein and chemical regulators, we will analyze the function of SREBP, SCAP, and INSIG orthologues in a genetically tractable yeast model in parallel to our studies in mammalian cells. Our sequence database searches reveal that the fission yeast S. pombe, but not the budding yeast S. cerevisiae, contains uncharacterized homologues of SREBP, SCAP, and INSIG: slp1+, scpl+ , and ins1+ , respectively. The primary goal of this proiect is to use S. pombe to isolate unknown regulators of SREBP and SCAP. We hypothesize that Scp1 senses sterols and regulates cleavage of Slp1 through a mechanism similar to that in mammalian cells. In this project, a combination of genetic, molecular, and biochemical approaches will be used to accomplish the following specific aims: 1) To confirm that the S. pombe genes are functional orthologues of mammalian SREBP, SCAP, and INSIG; 2) To define the transcriptional program that Sip1 controls and identify the endogenous chemical regulator of Sip1 activation; 3) To isolate novel genes required for the activation of Sip1; 4) To isolate Scp1 and Ins1 interacting proteins biochemically using affinity purification. The expectation is that novel regulators in S. pombe will have identifiable orthologues in mammals, thus providing new insight into our studies of cholesterol homeostasis in humans and potential therapeutic targets for prevention and treatment of heart disease.