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: 10682426
• 负责人: Kivanc Birsoy
• 金额: $ 48.11万
• 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/10682426
• 关键词: Address; Binding; Biochemical; Biological Assay; Cardiovascular Diseases; Cells; Cellular Membrane; Chemicals; Chronic Disease; Clustered Regularly Interspaced Short Palindromic Repeats; Compensation; Complex; Cues; Data; Disease; Drug Design; Drug Targeting; Endoplasmic Reticulum; Enzymes; 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; Microsomes; Modification; Mus; Nutrient; PPP3CA gene; PPP3CB gene; Palmitates; Pathway interactions; Phospholipids; Physiology; Production; Proteins; Publishing; Regulation; Role; Saturated Fatty Acids; Signal Transduction; Structure; Testing; Triglycerides; Work; 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; pharmacologic; posttranscriptional; 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持续合成细胞膜并产生能量。在细胞水平上，这些脂肪酸从其他营养素中摄取或从头合成，并作为膜磷脂和三酰基甘油的主要成分掺入甘油酯中。平衡甘油脂质合成与脂肪酸的可用性必须涉及严格的调节机制。虽然已经有实质性的进展，以确定参与脂质代谢的转录因子，仍然有一些差距，在我们的理解变构机制，调节甘油脂的合成和储存。通过对脂质合成和储存的调节或酶组分的系统分析来解决这一问题，可以在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，利用有毒的饱和脂肪酸棕榈酸酯和系统定义的关键代谢酶和甘油脂质合成途径的调节剂。我们发现钙调神经磷酸酶B同源蛋白1（CHP 1）作为一个重要的调节蛋白的甘油脂的合成和储存。通过肉豆蔻酰修饰，CHP 1结合并激活内质网GPAT（GPAT 4），这是三酰甘油和膜脂质从头合成的第一个关键酶。我们的初步数据，这形成了我们的应用程序的前提，指向一个意想不到的模式的甘油脂质合成和储存调节CHP 1。鉴于CHP 1在甘油脂质合成中的保守和关键作用，设计用于损害CHP 1-GPAT 4复合物的化学品可用于治疗与功能失调性脂质积累相关的代谢紊乱。然而，缺乏精确的调控机制和结构信息的CHP 1-GPAT复合物排除了足够的机制的理解，以指导药物设计。在这个建议中，基于我们以前的数据，我的目的是测试假设，CHP 1调节ER GPAT功能，并可用作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确定CHP 1激活GPAT 4的确切机制（Aim 1）。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CHP 1-GPAT 4复合物（目的2）。最后，我们将在肝脂肪变性的鼠模型中测试靶向CHP 1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新的脂质储存和合成的调节机制，可能是与功能失调的脂质积累相关的疾病的药物靶点。最后，这一奋进代表了首次尝试将结构生物学和遗传学应用于GPAT家族的酶，并将为这种难以捉摸的膜蛋白和代谢疾病的药理学靶向带来急需的洞察力。