Biophysical study of the recognition of ER proteins for degradation and lipid homeostasis

蛋白质稳态确保蛋白质的适当水平来完成其任务。靶向蛋白质降解是 作为调节膜胆固醇和鞘脂的关键机制出现。这些细胞中的蛋白质 ER中存在的降解途径知之甚少。错误折叠的ER蛋白被识别 由保守的ERAD机器。然而，多蛋白质复合物的成员并不总是错误折叠，除了它们的蛋白质外。 同源伙伴，需要有针对性的降解途径。许多甾醇和脂类生物合成蛋白的降解 由它们的蛋白质-蛋白质相互作用调节或由它们的代谢产物在负反馈回路中诱导。这 该提案将阐明甾醇和鞘脂代谢中选择性降解的分子和生物物理基础。 胆固醇水平由Scap-SREBP系统调节。SREBP 2开始作为一个完整的ER膜蛋白。在 在低胆固醇条件下，SREBP 2通过胆固醇传感器Scap转运到高尔基体。在那里，SREBP 2被切割， 释放其可溶性N-末端转录因子结构域，该结构域运输到细胞核并上调基因， 胆固醇的合成和摄取。在我的博士后工作中，我在C-末端调控域中鉴定了一种新的降解决定子， SREBP 2该基序对于SREBP 2前体在Scap不存在下的降解和SREBP 2前体的降解是必需的。 通过SREBP 2的切割在高尔基体中产生的C-末端SREBP 2产物的降解。该C末端SREBP 2 产物必须被清除以允许Scap再循环并与另外的SREBP 2前体相互作用。SREBP 2的降解 是由TRC 8介导的，TRC 8是一种ER驻留的E3连接酶。我开发了表达和纯化Scap-SREBP 2复合物的系统， 结构研究。在K99期间，我将使用尖端的冷冻电镜方法确定SREBP 2-Scap的结构 并将使用基于细胞和生物物理学的方法来表征SREBP 2和TRC 8之间的相互作用。这些研究 将揭示SREBP 2被TRC 8识别，以及SREBP 2和Scap之间的相互作用如何拮抗TRC 8。 在R 00期间，我将通过确定定向降解的机制来建立我的独立职业生涯。 完成鞘脂膜水平的调节。ER-驻留丝氨酸棕榈酰转移酶（SPT）复合物 在鞘脂合成中进行限速步骤。在哺乳动物中，SPT的酶活性受到以下因素的负调控： 三个高度保守的蛋白质（ORMDL 1 -3），其与SPT形成直接复合物。虽然很少有 最近的研究表明，ORMDL活性通过以下途径调节： 响应于过量的鞘脂代谢物的降解。此外，这种退化可以由非正则表达式进行。 ERAD途径。我将使用功能分析来确定ORMDLs中的E3连接酶识别基序，并进行一项研究。 生物物理研究其与其E3连接酶的相互作用。我将进一步阐明ORMDLs如何通过以下方式调节SPT活性： 使用cryo-EM方法以高分辨率确定ORMDL-SPT复合物结构。这些研究将揭示 ER蛋白如何被识别降解以及这些机制如何维持脂质的基本科学原理 体内平衡

Protein homeostasis ensures the proper levels of proteins to accomplish their tasks. Targeted protein degradation is emerging as a critical mechanism for the regulation of membrane cholesterol and sphingolipids. How the proteins in these pathways, which exist in the ER, are recognized for degradation is poorly understood. Misfolded ER proteins are recognized by conserved ERAD machinery. However, members of multi-protein complexes are not always misfolded apart from their cognate partners and require targeted degradation pathways. The degradation of many sterol and lipid biosynthesis proteins is regulated by their protein-protein interactions or is induced by their metabolic products in negative feedback loops. This proposal will elucidate the molecular and biophysical basis for selective degradation in sterol and sphingolipid metabolism. Cholesterol levels are regulated by the Scap-SREBP system. SREBP2 begins as an integral ER membrane protein. In conditions of low cholesterol, SREBP2 is transported by the cholesterol-sensor Scap to the Golgi. There, SREBP2 is cleaved to release its soluble N-terminal transcription factor domain, which traffics to the nucleus and upregulates genes for cholesterol synthesis and uptake. In my postdoctoral work, I identified a novel degron in the C-terminal regulatory domain of SREBP2. This motif is necessary for the degradation of the SREBP2 precursor in the absence of Scap and for the degradation of the C-terminal SREBP2 product created in the Golgi by the cleavage of SREBP2. This C-terminal SREBP2 product must be cleared to allow Scap recycle and interact with additional SREBP2 precursors. The degradation of SREBP2 is mediated by TRC8, an ER-resident E3 ligase. I developed systems to express and purify Scap-SREBP2 complexes for structural studies. In the K99 period, I will determine the structure of SREBP2-Scap using cutting edge cryo-EM methods and will use cell-based and biophysical methods to characterize the interaction between SREBP2 and TRC8. These studies will reveal SREBP2 is recognized by TRC8 and how this is antagonized by the interaction between SREBP2 and Scap. In the R00 period, I will establish my independent career by determining the mechanisms by which targeted degradation accomplishes the regulation of membrane levels sphingolipids. The ER-resident serine palmitoyltransferase (SPT) complex conducts the rate-limiting step in sphingolipid synthesis. In mammals, SPT’s enzymatic activity is negatively regulated by three highly conserved proteins (ORMDL1-3), which form a direct complex with the SPT. While there is very little biochemical or biophysical insight into how SPT functions, recent studies show that ORMDL activity is regulated through degradation in response to excess sphingolipid metabolites. Moreover, this degradation may be carried out by non-canonical ERAD pathways. I will use functional assays to determine the E3 ligase recognition motifs in the ORMDLs and conduct a biophysical study of their interaction with their E3 ligases. I will further elucidate how ORMDLs regulate SPT activity by determining the ORMDL-SPT complex structure at high resolution using cryo-EM methods. These studies will uncover the basic science principles of how ER proteins are recognized for degradation and how these mechanisms maintain lipid homeostasis.