NgBR as a regulator of endothelial function

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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.