Identification of metabolic regulators of glycerolipid synthesis and storage

甘油脂合成和储存代谢调节剂的鉴定

• 批准号: 10220022
• 负责人: Kivanc Birsoy
• 金额: $ 49.94万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220022
• 关键词: Address; Binding; Biochemical; Biological Assay; Cardiovascular Diseases; Cells; Cellular Membrane; Chemicals; Chronic Disease; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cues; Data; Disease; Drug Design; Drug Targeting; Endoplasmic Reticulum; Enzymes; Equilibrium; Family; Fatty Acids; Fatty Liver; Genetic; Genetic Screening; Genetic Transcription; Goals; Health; Hepatocyte; Homologous Protein; Impairment; Insulin Resistance; Invertebrates; Lipids; Mammalian Cell; Mammals; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Metabolic; Metabolic Diseases; Metabolic Pathway; Modification; Mus; Nutrient; PPP3CA gene; PPP3CB gene; Palmitates; Pathway interactions; Pharmacology; Phospholipids; Physiology; Production; Proteins; Publishing; Regulation; Role; Saturated Fatty Acids; Signal Transduction; Structure; Testing; Triglycerides; Work; base; cell type; design; enzyme activity; experimental study; fly; genetic regulatory protein; improved; in vivo; innovation; insight; lipid metabolism; mouse model; non-alcoholic fatty liver disease; novel; sensor; structural biology; therapeutic evaluation; therapeutic target; transcription factor

Mammalian cells require fatty acids to continuously synthesize cellular membranes and generate energy. At the cellular level, these fatty acids are either taken up or synthesized de novo from other nutrients and incorporated into glycerolipids as major constituents of membrane phospholipids and triacylglycerols. Balancing glycerolipid synthesis with fatty acid availability must involve strict regulatory mechanisms. While there has been substantial progress to identify transcription factors involved in lipid metabolism, there are still several gaps in our understanding of allosteric mechanisms that regulate glycerolipid synthesis and storage. Addressing this through a systematic analysis of regulatory or enzymatic components of lipid synthesis and storage can provide significant insights in the field of metabolic disorders. Our long-term goal is to elucidate these regulatory components and to understand their roles in normal and disease physiology. We previously devised a CRISPR-based genetic screening strategy utilizing a toxic saturated fatty acid, palmitate, and systematically defined key metabolic enzymes and regulators of the glycerolipid synthesis pathway. We discovered calcineurin B homologous protein 1 (CHP1) as an essential regulatory protein of glycerolipid synthesis and storage. Through a myristoyl modification, CHP1 binds to and activates an endoplasmic reticulum GPAT (GPAT4), the first committed enzyme for the de novo synthesis of triacylglycerols and membrane lipids. Our preliminary data, which form the premise of our application, point to an unexpected mode of glycerolipid synthesis and storage regulation by CHP1. Given the conserved and critical role of CHP1 in glycerolipid synthesis, a chemical designed to impair the CHP1-GPAT4 complex could be used to treat metabolic disorders associated with dysfunctional lipid accumulation. However, the lack of the precise regulatory mechanisms and structural information of the CHP1-GPAT complex precludes sufficient mechanistic understanding to guide drug design. In this proposal, building on our previous data, I aim to test the hypothesis that CHP1 regulates ER GPAT function and may be used as a therapeutic target for metabolic disorders with dysfunctional lipid accumulation. To address this, we will first identify the precise mechanism by which CHP1 activates GPAT4 through structural and biochemical studies (Aim1). We will then determine whether upstream metabolic cues regulate the CHP1-GPAT4 complex in mammalian cells (Aim 2). Finally, we will test the therapeutic potential of targeting CHP1 in murine models of hepatic steatosis (Aim 3). Our proposal is highly innovative because we aim to identify new regulatory mechanisms for lipid storage and synthesis that could potentially be drug targets for disorders associated with dysfunctional lipid accumulation. Finally, this endeavor represents the first attempt to apply structural biology and genetics to the GPAT family of enzymes and will bring much needed insight to this elusive membrane protein and to pharmacological targeting of metabolic diseases.

哺乳动物细胞需要脂肪酸来不断合成细胞膜并产生能量。在细胞水平上，这些脂肪酸要么被吸收，要么从其他营养物质中重新合成，并作为膜磷脂和三酰基甘油的主要成分并入甘油中。平衡甘油脂合成与脂肪酸可用性必须涉及严格的调节机制。虽然在确定参与脂质代谢的转录因子方面已经取得了实质性进展，但我们对调节甘油脂合成和储存的变构机制的理解仍然存在一些空白。通过对脂质合成和储存的调节或酶成分的系统分析来解决这个问题，可以为代谢紊乱领域提供重要的见解。我们的长期目标是阐明这些调控成分，并了解它们在正常和疾病生理学中的作用。我们之前设计了一种基于crispr的遗传筛选策略，利用有毒饱和脂肪酸棕榈酸酯，并系统地定义了甘油脂合成途径的关键代谢酶和调节因子。我们发现calcalineurin B同源蛋白1 （CHP1）是甘油脂合成和储存的重要调控蛋白。通过肉豆蔻酰基修饰，CHP1结合并激活内质网GPAT (GPAT4)，这是三酰基甘油和膜脂从头合成的第一个承诺的酶。我们的初步数据构成了我们申请的前提，指出了一种意想不到的由CHP1调节的甘油脂合成和储存模式。鉴于CHP1在甘油脂合成中的保守和关键作用，一种旨在破坏CHP1- gpat4复合物的化学物质可用于治疗与功能失调的脂质积累相关的代谢紊乱。然而，缺乏精确的调控机制和CHP1-GPAT复合物的结构信息阻碍了对机制的充分理解来指导药物设计。在本提案中，基于我们之前的数据，我的目标是验证CHP1调节ER GPAT功能的假设，并可能作为代谢紊乱与功能失调的脂质积累的治疗靶点。为了解决这个问题，我们将首先通过结构和生化研究（Aim1）确定CHP1激活GPAT4的确切机制。然后，我们将确定上游代谢线索是否调节哺乳动物细胞中的CHP1-GPAT4复合物（目的2）。最后，我们将在小鼠肝脂肪变性模型中测试靶向CHP1的治疗潜力（目的3）。我们的建议是高度创新的，因为我们的目标是确定脂质储存和合成的新调控机制，这可能是与功能失调的脂质积累相关的疾病的潜在药物靶点。最后，这一努力代表了将结构生物学和遗传学应用于GPAT酶家族的首次尝试，并将为这种难以捉摸的膜蛋白和代谢性疾病的药理学靶向提供急需的见解。