NgBR as a regulator of endothelial function

NgBR 作为内皮功能的调节剂

基本信息

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
项目类别：
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

项目摘要

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.

项目概要我们的实验室发现了 Nogo-B 受体 (NgBR) 和一种与 RTN-4B 相互作用的结合蛋白（称为 Nogo-B)、Niemann Pick C2 蛋白和顺式异戊二烯基转移酶 (hCIT)。 NgBR与NPC2的相互作用通过稳定 Niemann Pick C2 蛋白 (NPC2) 来调节细胞内胆固醇代谢 NgBR 在胆固醇稳态的基本方面发挥重要作用的可能性。最近，我们发现 NgBR 与 hCIT 的异聚相互作用是顺式异戊二烯基转移酶活性所必需的（顺-PTase）和酵母、小鼠和人中的多醇合成。 Dolichol 是聚糖的专性载体（糖）用于蛋白质糖基化反应。此外，我们还发现了功能丧失突变通过外显子组测序发现 NgBR (R290H) 的保守 C 末端，导致先天性疾病糖基化。从受影响的患者中分离出的成纤维细胞表现出游离胆固醇和与缺乏 NgBR 的小鼠的成纤维细胞相同，多醇成分的减少。人类 NgBR-R290H 突变和酵母中的直系同源物强调了顺式 PTase 的这种进化上保守的残基的重要性活动和功能。因此，这些数据为 NgBR 的重要作用提供了坚实的遗传基础。胆固醇代谢、多醇合成和蛋白质糖基化。有趣的是，NgBR 监管胆固醇稳态和糖基化的发生独立于 Nogo-B，因为 Nogo-B 的丢失不会影响影响游离胆固醇水平或 cisPTase 活性。因此，考虑到这些新数据，我们推测 NgBR 与 NPC2 和 hCIT 相互作用调节胆固醇等内质网 (ER) 功能并提出通过单独的域和实验进行多醇合成，以充分表征这些体外和体内途径。我们将： (1) 表征 NgBR/hCIT 蛋白复合物和致病原因突变体； (2) 剖析 NgBR 如何调节胆固醇代谢和蛋白质糖基化， (3) 利用转基因小鼠破译NgBR在体内的作用。总的来说，这些目标将建立在我们的发现并推动该领域向前发展。