NgBR as a regulator of endothelial function

NgBR 作为内皮功能的调节剂

• 批准号: 9151876
• 负责人: William C Sessa
• 金额: $ 50.22万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9151876
• 关键词: Affect; Alleles; Amino Acids; Apoptotic; Binding; Binding Proteins; Biochemical Genetics; Biochemistry; Biology; Blood Vessels; Blood flow; Cholesterol; Cholesterol Homeostasis; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Disorders; Cysteine; Data; Dimethylallyltranstransferase; Disease; Dolichol; Early Diagnosis; Endoplasmic Reticulum; Endothelial Cells; Epilepsy; Equilibrium; Exhibits; Fibroblasts; Genetic; Human; Impaired cognition; Impaired wound healing; In Vitro; Injury; Knock-in Mouse; Knock-out; Mammalian Cell; Mediating; Membrane; Mind; Morphology; Multienzyme Complexes; Mus; Mutate; Mutation; Neurologic; Orthologous Gene; Pathway interactions; Patients; Physiological; Polysaccharides; Protein Family; Protein Glycosylation; Proteins; Public Health; Reaction; Recovery; Regulation; Research; Role; Side; Smooth Muscle Myocytes; Symptoms; Technology; Vascular Endothelial Growth Factors; Vascular Smooth Muscle; Yeasts; base; exome sequencing; expression cloning; genetic approach; glycosylation; in vivo; insight; loss of function mutation; macrophage; man; mutant; novel therapeutics; protein complex; protein transport; receptor; receptor function; research study; sugar

Project Summary Our lab discovered Nogo-B receptor (NgBR) and a binding protein that interacts with RTN-4B (called Nogo-B), Niemann Pick C2 protein and cis-isoprenyltransferase (hCIT). The interaction of NgBR with NPC2 regulates intracellular cholesterol metabolism by stabilizing the Niemann Pick C2 protein (NPC2) raising the possibility that NgBR exerts a fundamental role in basic aspects of cholesterol homeostasis. Recently, we have discovered the heteromeric interaction of NgBR with hCIT is required for cis-prenyltransferase activity (cis-PTase) and dolichol synthesis in yeast, mice and man. Dolichol is an obligate carrier of glycans (sugars) for protein glycosylation reactions. In addition, we identified a loss of function mutation in the conserved C terminus of NgBR (R290H) via exome sequencing that causes a congenital disorder of glycosylation. Fibroblasts isolated from affected patients exhibit enhanced accumulation of free cholesterol and reduced dolichol profiles identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast underscores the importance of this evolutionarily conserved residue for cis-PTase activity and function. Thus, these data provide a firm genetic basis for the essential role of NgBR in cholesterol metabolism, dolichol synthesis and protein glycosylation. Interestingly, NgBR regulation of cholesterol homeostasis and glycosylation occur independent of Nogo-B, since the loss of Nogo-B does not influence free cholesterol levels or cisPTase activity. Thus, with these new data in mind, we surmise that NgBR interaction with NPC2 and hCIT regulates endoplasmic reticulum (ER) functions such as cholesterol and dolichol synthesis via separate domains and experiments are proposed to fully characterize these pathways in vitro and in vivo. We will: (1) Characterize the NgBR/hCIT protein complex and disease causing mutants; (2) Dissect how NgBR regulates both cholesterol metabolism and protein glycosylation and, (3) Decipher the role of NgBR in vivo using genetically modified mice. Collectively, these aims will build upon our discoveries and move the field forward.

项目摘要 我们的实验室发现了Nogo-B受体（NgBR）和一种与RTN-4 B相互作用的结合蛋白（称为 Nogo-B）、尼曼匹克C2蛋白和顺式异戊二烯基转移酶（hCIT）。NgBR与NPC 2的相互作用 通过稳定尼曼匹克C2蛋白（NPC 2）来调节细胞内胆固醇代谢， NgBR可能在胆固醇稳态的基本方面发挥重要作用。最近我们 已经发现NgBR与hCIT的异聚体相互作用是顺式异戊二烯基转移酶活性所必需的 （顺式-PTase）和多萜醇的合成。多萜醇是聚糖的专性载体 （糖）用于蛋白质糖基化反应。此外，我们还发现了该基因中的功能缺失突变。 通过外显子组测序发现NgBR的保守C末端（R290 H）， 糖基化 从受影响的患者分离的成纤维细胞表现出增强的游离胆固醇积累， 与来自缺乏NgBR的小鼠的成纤维细胞相同地降低了多萜醇谱。人NgBR-R290 H突变 酵母中的同源物强调了这个进化上保守的顺式PTase残基的重要性 活动和功能。因此，这些数据为NgBR在以下方面的重要作用提供了坚实的遗传基础： 胆固醇代谢、多萜醇合成和蛋白质糖基化。有趣的是， 胆固醇稳态和糖基化的发生与Nogo-B无关，因为Nogo-B的丢失并不 影响游离胆固醇水平或顺式PTase活性。因此，考虑到这些新数据，我们推测， NgBR与NPC 2和hCIT的相互作用调节内质网（ER）功能，如胆固醇 和dolichol合成通过单独的领域和实验提出了充分表征这些 在体外和体内的途径。我们将：（1）研究NgBR/hCIT蛋白复合物的结构和致病性 突变体;（2）剖析NgBR如何调节胆固醇代谢和蛋白质糖基化， (3)使用转基因小鼠破译NgBR在体内的作用。总的来说，这些目标将基于 我们的发现，并推动该领域的发展。