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.

蛋白质动态平衡确保了蛋白质的适当水平来完成它们的任务。有针对性的蛋白质降解 成为调节膜胆固醇和鞘磷脂的关键机制。它们中的蛋白质是如何 内质网中存在的途径被认为是退化的，但人们对此知之甚少。错误折叠的ER蛋白被识别 由保守的ERAD机器。然而，多蛋白质复合体的成员并不总是错误折叠，除了它们的 有同源的合作伙伴，需要有针对性的退化途径。多种甾醇和脂肪生物合成蛋白的降解 是由它们的蛋白质-蛋白质相互作用调节的，还是由它们在负反馈环中的代谢产物诱导的。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。SREBP2最初是一种完整的内质网膜蛋白。在……里面 在低胆固醇的条件下，SREBP2由胆固醇传感器SCAP运输到高尔基体。在那里，SREBP2被切割 释放其可溶的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 胆固醇的合成和吸收。在我的博士后工作中，我发现了C-末端调控结构域中的一个新的退化 SREBP2。该基序对于SREBP2前体在没有SCAP的情况下的降解和对 由SREBP2裂解在高尔基体中产生的C-末端SREBP2产物的降解。此C端子SREBP2 产品必须被清除，才能允许SCAP回收并与其他SREBP2前体相互作用。SREBP2的降解 是由内质网驻留的E3连接酶TRC8介导的。我开发了表达和纯化SCAP-SREBP2复合体的系统 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方法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是由TRC8识别的，以及SREBP2和SCAP之间的相互作用如何拮抗这一点。 在R00期间，我将通过确定定向降级的机制来建立我的独立职业生涯 完成对神经鞘脂膜水平的调节。内质网驻留的丝氨酸棕榈酰转移酶(SPT)复合体 进行鞘磷脂合成中的限速步骤。在哺乳动物中，SPT的酶活性受 三个高度保守的蛋白质(ORMDL1-3)，它们与SPT形成直接的复合体。虽然几乎没有 生化或生物物理对SPT功能的洞察，最近的研究表明ORMDL的活性是通过 对过量鞘磷脂代谢物的降解。此外，这种退化可以由非规范的 埃拉德小路。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 它们与E3连接酶相互作用的生物物理研究。我将进一步阐明ORMDL如何通过以下方式调节SPT活动 用冷冻-EM方法高分辨率测定ORMDL-SPT的复杂结构。这些研究将揭示 内质网蛋白质如何被识别为降解以及这些机制如何维持脂质的基本科学原理 动态平衡。