Biochemistry and genetics of the ORMDL regulators of sphingolipid biosynthesis

鞘脂生物合成 ORMDL 调节因子的生物化学和遗传学

• 批准号: 9195971
• 负责人: BRIAN W. WATTENBERG
• 金额: $ 38.13万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9195971
• 关键词: Address; Affect; Agonist; Alleles; Anabolism; Architecture; Asthma; Beryllium; Binding; Binding Sites; Biochemistry; Cardiovascular Diseases; Cell Line; Cell membrane; Cell-Free System; Cells; Ceramides; Childhood Asthma; Complex; Conserved Sequence; Detergents; Disease; Endoplasmic Reticulum; Enzymes; Epithelial; Genes; Genetic; Genotype; Goals; Homologous Gene; Indium; Individual; Inflammatory; Knowledge; Lipids; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Proteins; Metabolic; Metabolism; Molecular; Mutation; Pathway interactions; Phenotype; Protein Isoforms; Regulation; Research; Risk; Single Nucleotide Polymorphism; Smooth Muscle Myocytes; Specificity; Sphingolipids; Stimulus; System; Technology; Testing; Yeasts; asthmatic; cell type; eosinophil; genome editing; genome wide association study; hamstring; intercellular communication; novel; physical property; population based; reconstitution; response; risk variant; serine palmitoyltransferase; tool

Sphingolipids are a diverse but metabolically related group of lipids with unique functional and physical properties that are critical for cell signaling and membrane architecture. Misregulation of particular sphingolipids has been implicated in such diverse diseases as cancer, inflammatory disease, and cardiovascular disease. In particular, as outlined below, genome-wide association studies have strongly implicated the subject of this proposal, the sphingolipid metabolic regulator ORMDL, in the risk for childhood asthma. The overall level of cellular sphingolipid is determined by the initiating, and rate limiting enzyme in the biosynthetic pathway, serine palmitoyltransferase (SPT). Not surprisingly, SPT activity is homeostatically controlled-i.e. SPT activity is strongly inhibited by elevated levels of cellular sphingolipid. We have demonstrated that this homeostatic control is mediated by a set of small membrane proteins found in the endoplasmic reticulum, the ORMDLs. The goal of the studies proposed here is to establish the mechanism by which the ORMDLs mediate SPT inhibition in response to elevated sphingolipid levels and to begin to uncover how those mechanisms impact on risk for asthma. These studies will answer basic questions concerning how ORMDLs sense cellular sphingolipid levels and how changes in sphingolipid levels are transmitted through ORMDL to affect SPT activity. We will test the hypothesis that ceramide directly binds ORMDL to trigger SPT regulation. We will determine the structural elements in ORMDL that are essential for mediating sphingolipid regulation of SPT, will use a recently constructed cell-free system to quantitate the levels of sphingolipid sensed by the ORMDL system, and will test the hypothesis that elevation of sphingolipid induces changes in ORMDL topology. Finally we will construct a reconstituted system with purified ORMDL and SPT to test the hypothesis that the ORMDLs and SPT are the only required components of this regulatory system. In addition we propose to address an essential question concerning the genetics of ORMDL expression that has emerged from genome-wide association studies in asthma. Single nucleotide polymorphisms (SNPs) adjacent to the gene of one of the ORMDL isoforms, ORMDL3, are strongly and consistently associated with the risk for childhood asthma and with elevated expression of ORMDL3. Although the population-based studies are intriguing and suggestive, a detailed examination of this phenomenon has been hamstrung by the inability to directly test the effect of these SNPs on ORMDL3 expression. It has not been possible to directly measure the degree to which these SNPs affect ORMDL3 expression nor in what cell types and conditions this elevated expression occurs. Here we propose to use emerging Crispr/Cas9 genome editing technology to produce paired cell lines, in asthma-relevant cell types, containing either a risk or non-risk genotype at the most prominent risk-associated SNP. These will be used to examine the extent, cell type, and stimuli specificity of elevation of ORMDL3 expression induced by the risk allele and how this affects sphingolipid metabolism.

鞘脂是具有独特功能和物理的多样化但代谢相关的脂质组 对于细胞信号传导和膜结构至关重要的属性。对特定的不利 鞘脂与癌症，炎症性疾病和 心血管疾病。特别是，如下所述，全基因组关联研究强烈 暗示了该提案的主题，即鞘脂代谢调节剂ORMDL的风险 哮喘。细胞鞘脂的总体水平取决于启动，并限制酶的酶 生物合成途径，丝氨酸棕榈酰转移酶（SPT）。毫不奇怪，SPT活动是体内平衡的 控制 - i。 SPT活性受到细胞鞘脂升高的升高。我们有 证明这种稳态控制是由一组小膜蛋白介导的 内质网，ORMDL。这里提出的研究的目的是通过 ORMDL响应于升高的鞘脂水平介导SPT抑制作用，并开始发现 这些机制如何影响哮喘的风险。这些研究将回答有关如何 ORMDL感知细胞鞘脂水平以及鞘脂水平的变化如何通过 ORMDL影响SPT活动。我们将测试神经酰胺直接结合ORMDL以触发SPT的假设 规定。我们将确定ORMDL中的结构元素，这些元素对于介导鞘脂至关重要 SPT的调节将使用最近构造的无细胞系统来定量鞘脂的水平 通过ORMDL系统感应，将检验以下假设：鞘脂升高会引起变化 ORMDL拓扑。最后，我们将使用纯化的ORMDL和SPT构建一个重构系统，以测试 假设ORMDL和SPT是该调节系统唯一所需的组成部分。此外 我们建议解决有关ORMDL表达的遗传学的基本问题 来自哮喘全基因组关联研究。与邻近的单核苷酸多态性（SNP） ORMDL同工型ORMDL3之一的基因与患有的风险密切相关 儿童哮喘和ORMDL3表达升高。尽管基于人群的研究是 令人着迷和暗示性的是，对这种现象的详细检查一直无法通过无法 直接测试这些SNP对ORMDL3表达的影响。不可能直接测量 这些SNP影响ORMDL3表达的程度，或在哪种细胞类型和条件下升高 表达发生。在这里，我们建议使用新兴CRISPR/CAS9基因组编辑技术来生产 配对的细胞系，与哮喘相关的细胞类型中，最多包含风险或非风险基因型 突出的风险相关SNP。这些将用于检查范围，细胞类型和刺激特异性 风险等位基因引起的ORMDL3表达的升高以及这如何影响鞘脂代谢。