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

ER 蛋白降解和脂质稳态识别的生物物理学研究

• 批准号: 10189043
• 负责人: Daniel Luke Kober
• 金额: $ 10万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10189043
• 关键词: Anabolism; Architecture; Basic Science; Binding; Biochemical; Biological Assay; Biological Models; Biophysical Process; Biophysics; C-terminal; Cell Nucleus; Cells; Cholesterol; Cleaved cell; Complex; Cryoelectron Microscopy; Data; Degradation Pathway; Detection; Dissection; Endoplasmic Reticulum Degradation Pathway; Ensure; Epitopes; Feedback; Genes; Golgi Apparatus; Homeostasis; Immune; In Vitro; Light; Lipids; Mammals; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Metabolic; Metabolism; Methods; Molecular; Molecular Conformation; Multiprotein Complexes; Mutate; N-terminal; Pathway interactions; Phenotype; Protein Biosynthesis; Protein Family; Protein Region; Proteins; RNF139 gene; Regulation; Resolution; Scientist; Signal Transduction; Sphingolipids; Sphingosine; Sterol Biosynthesis Pathway; Sterols; Stimulus; Structure; System; Testing; Transcriptional Regulation; Work; X-Ray Crystallography; base; biophysical analysis; biophysical techniques; career; experimental study; inhibitor/antagonist; insight; lipid biosynthesis; member; novel; prevent; protein complex; protein degradation; protein expression; protein metabolism; protein protein interaction; proteostasis; response; sensor; serine palmitoyltransferase; stoichiometry; transcription factor; ubiquitin-protein ligase; uptake

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.

蛋白质稳态可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调节的关键机制。这些蛋白质如何 急诊室中存在的途径被认为是降解的认可。识别错误折叠的ER蛋白 由保守的伊拉德机械。但是，多蛋白络合物的成员除了他们的 同源伙伴，需要有针对性的降解途径。许多立体声和脂质生物合成蛋白的降解 受其蛋白质 - 蛋白质相互作用的调节，或者由负反馈回路中的代谢产物诱导。这 建议将阐明固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。 SREBP2开始于整体ER膜蛋白。在 胆固醇低，SREBP2的条件是通过胆固醇传感器的SCAP转运到高尔基体的。在那里，SREBP2已清洁 释放其固体N末端转录因子结构域，该结构域流动到核并上调基因 胆固醇的合成和摄取。在我的博士后工作中，我在C末端调节域中确定了一个新颖的DEGRON SREBP2。在没有SCAP和对于SREBP2前体降解的必要条件是必要的 SREBP2的裂解在高尔基体中产生的C末端SREBP2产物的降解。此C端SREBP2 必须清除产品以允许SCAP回收并与其他SREBP2前体相互作用。 SREBP2的降解 由TRC8介导的E3连接酶TRC8介导。我开发了用于表达和净化Scap-srebp2复合物的系统 结构研究。在K99期间，我将使用尖端冷冻EM方法确定SREBP2-SCAP的结构 并将使用基于细胞的和生物物理方法来表征SREBP2和TRC8之间的相互作用。这些研究 将揭示SREBP2由TRC8识别，以及如何通过SREBP2和SCAP之间的相互作用拮抗。 在R00时期，我将通过确定目标退化的机制来建立自己的独立职业 完成膜水平鞘脂的调节。 ER居住的丝氨酸棕榈酰转移酶（SPT）复合物 在鞘脂合成中执行限速步骤。在哺乳动物中，SPT的酶活性受负调节 三种高度保守的蛋白质（ORMDL1-3），它​​们与SPT形成直接的复合物。虽然很少 最近的研究表明，生化或生物物理学的洞察力表明，ORMDL活性通过 响应超过鞘脂代谢物的降解。此外，这种降解可以通过非典型的 Erad途径。我将使用功能分析来确定ORMDL中的E3连接酶识别基序并进行A 对其与E3连接酶相互作用的生物物理研究。我将进一步阐明ORMDL如何通过 使用冷冻方法在高分辨率下确定ORMDL-SPT复合物结构。这些研究将发现 基础科学原理是如何识别降解以及这些机制如何保持脂质的基础科学原则 稳态。