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

基本信息

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
项目类别：
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

项目摘要

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，这些脂肪酸要么被其他营养物质吸收，要么从头合成，并并入甘油脂中，作为膜磷脂和三酰甘油的主要成分。平衡甘油脂合成与脂肪酸可用性必须涉及严格的监管机制。尽管在鉴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差距。通过对脂质合成和储存的调节或酶促成分进行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节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 的基因筛选策略，利用有毒的饱和脂肪酸、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剂。我们发现钙调磷酸酶 B 同源蛋白 1 (CHP1) 是甘油脂合成和储存的重要调节蛋白。通过肉豆蔻酰修饰，CHP1 结合并激活内质网 GPAT (GPAT4)，这是从头合成三酰甘油和膜脂的第一个定型酶。我们的初步数据构成了我们应用的前提，指出了 CHP1 的甘油脂合成和储存调节的意想不到的模式。鉴于 CHP1 在甘油脂合成中的保守且关键的作用，一种旨在损害 CHP1-GPAT4 复合物的化学物质可用于治疗与脂质积累功能失调相关的代谢紊乱。然而，由于缺乏 CHP1-GPAT 复合物的精确调控机制和结构信息，无法对机制有足够的了解来指导药物设计。在本提案中，基于我们之前的数据，我的目的是检验 CHP1 调节 ER GPAT 功能的假设，并可用作脂质蓄积功能失调的代谢性疾病的治疗靶点。为了解决这个问题，我们首先通过结构和生化研究确定 CHP1 激活 GPAT4 的精确机制 (Aim1)。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（目标 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（目标 3）。我们的建议具有高度创新性，因为我们的目标是确定脂质储存和合成的新调节机制，这些机制可能成为与脂质积累功能失调相关的疾病的药物靶标。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疾病的药理学靶向带来急需的见解。