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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

基本信息

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
项目类别：
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

项目摘要

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.

鞘脂是一种种类繁多但与代谢相关的脂类，具有独特的功能和物理特性