Regulation of Cellular Cholesterol Homeostasis

描述（由申请人提供）：脂质稳态对细胞功能至关重要，脂质稳态的破坏会导致疾病。血清胆固醇升高是心脏病和动脉粥样硬化的主要危险因素，动脉粥样硬化是美国成年人的主要杀手。脂肪酸和甘油三酯在肝脏中的积累会导致脂肪肝，通常会发展为非酒精性脂肪性肝炎，肝硬化和癌症。II型糖尿病是发展脂肪肝的主要风险因素，因为过量的血清葡萄糖被肝脏转化为脂肪酸。令人担忧的是，预计到2050年，糖尿病将影响四分之一的美国人口。了解细胞脂质稳态是如何调节的，将为这些日益常见的疾病确定治疗机会。 膜结合的碱性螺旋-环-螺旋亮氨酸拉链转录因子称为固醇调节元件结合蛋白（SREBP），是细胞脂质稳态的中心调节因子，控制胆固醇、脂肪酸和甘油三酯的合成和摄取。使用裂殖酵母，我们发现SREBP途径在真菌中是保守的，除了控制脂质稳态外，还控制对低氧的适应。裂变酵母SREBP通过一种新的途径被蛋白水解激活，该途径需要高尔基体Dsc E3连接酶和AAA-ATP酶Cdc 48。我们在新型隐球菌和烟曲霉中的研究表明，这种氧响应途径在真菌门中是保守的，并且SREBP途径对于这些重要的机会性人类真菌病原体的毒力是必不可少的。因此，我们的研究影响了脂质稳态，蛋白质和降解，以及真菌发病机制的研究。 在这项提案中，我们将继续我们的SREBP途径和Dsc E3连接酶的研究，以了解细胞如何调节脂质稳态，以应对环境中的氧气变化。我们假设Dsc E3连接酶泛素化高尔基体中的SREBP，使其靶向Rbd 2-Cdc 48进行切割和膜释放。为了验证这一假设，我们提出了以下具体目标：目的1。确定SREBP与DSC E3连接酶结合的要求。AIM 2.测试劈裂是否需要SREBP遍在。AIM 3.以测试RBD 2是否是SREBP蛋白酶。目标4.目的：探讨CDC 48在SREBP裂解中的作用。 该项目的长期目标是使用裂殖酵母作为发现工具，以确定哺乳动物细胞中SREBP调节的新机制和SREBP通路抑制的新靶点，以治疗真菌疾病。到目前为止，我们的工作已经强调了环境氧作为脂质合成的关键调节剂。期望我们提出的研究将描述SREBP如何被E3连接酶识别用于泛素化和被菱形蛋白酶识别用于裂解的新机制，从而推进我们对SREBP通路和脂质稳态疾病的理解。

DESCRIPTION (provided by applicant): Lipid homeostasis is essential for cell function and disruptions to lipid homeostasis lead to disease. Elevated serum cholesterol is a primary risk factor for heart disease and atherosclerosis, a leading killer of adults in the United States. Fatt acid and triglyceride accumulation in the liver causes fatty liver that frequently progresses to non-alcoholic steatohepatitis, liver cirrhosis and cancer. Type II diabetes mellitus is a major ris factor for developing fatty liver as excess serum glucose is converted into fatty acid by the liver Alarmingly, diabetes is projected to affect one-quarter of the U.S. population by 2050. Knowing how cellular lipid homeostasis is regulated will identify therapeutic opportunities for these increasingly common diseases. Membrane-bound, basic helix-loop-helix leucine zipper transcription factors called sterol regulatory element-binding proteins (SREBPs) are the central regulators of cellular lipid homeostasis, controlling synthesis and uptake of cholesterol, fatty acids, and triglycerides. Using fission yeast, we discovered that the SREBP pathway is conserved in fungi, controlling adaptation to low oxygen in addition to lipid homeostasis. Fission yeast SREBP is proteolytically activated through a novel pathway that requires the Golgi Dsc E3 ligase and the AAA-ATPase Cdc48. Our studies in Cryptococcus neoformans and in Aspergillus fumigatus by others demonstrated that this oxygen-responsive pathway is conserved across fungal phyla, and that the SREBP pathway is essential for virulence in these important opportunistic human fungal pathogens. Thus, our studies impact research of lipid homeostasis, protein and degradation, and fungal pathogenesis. In this proposal, we will continue our studies of the SREBP pathway and the Dsc E3 ligase to understand how cells regulate lipid homeostasis in response to changes in environmental oxygen. We hypothesize that the Dsc E3 ligase ubiquitinates SREBP in the Golgi to target it to Rbd2-Cdc48 for cleavage and membrane release. To test this hypothesis, we propose the following specific aims: AIM 1. TO IDENTIFY REQUIREMENTS FOR SREBP BINDING TO DSC E3 LIGASE. AIM 2. TO TEST WHETHER CLEAVAGE REQUIRES SREBP UBIQUITINATION. AIM 3. TO TEST WHETHER RBD2 IS A SREBP PROTEASE. AIM 4. TO DETERMINE THE FUNCTION OF CDC48 IN SREBP CLEAVAGE. The long-term goal of this project is to use fission yeast as a discovery tool to identify new mechanisms for regulation of SREBPs in mammalian cells and new targets for SREBP pathway inhibition toward treatments of fungal disease. To date, our work has highlighted environmental oxygen as a key regulator of lipid synthesis. The expectation is that our proposed studies will describe new mechanisms for how SREBPs are recognized by E3 ligases for ubiquitination and by rhomboid proteases for cleavage advancing our understanding of the SREBP pathway and diseases of lipid homeostasis.