Nogo-B Receptor Stabilizes Niemann-Pick Type C2 Protein and Regulates Intracellular Cholesterol Trafficking

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study, we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells, wherein these pathways are severely compromised, causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further, NPCD Epstein-Barr virus-transformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone, respectively, replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results, suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally, normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein, and overexpression of NPC1 increased HIV-1 release. Taken together, our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.

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Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking

Journal of Clinical Investigation ◽

10.1172/jci32561 ◽

2008 ◽

Cited By ~ 11

Author(s):

Jessie R. Zhang ◽

Trey Coleman ◽

S. Joshua Langmade ◽

David E. Scherrer ◽

Lindsay Lane ◽

...

Keyword(s):

Cholesterol Trafficking ◽

Intracellular Cholesterol ◽

Niemann Pick

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Interaction of NPC2 protein with Lysobisphosphatidic Acid is required for normal endolysosomal cholesterol trafficking

10.1101/559849 ◽

2019 ◽

Author(s):

Leslie A. McCauliff ◽

Annette Langan ◽

Ran Li ◽

Olga Ilnytska ◽

Debosreeta Bose ◽

...

Keyword(s):

Functional Relationship ◽

Cholesterol Transport ◽

Unesterified Cholesterol ◽

Cholesterol Trafficking ◽

Cholesterol Accumulation ◽

Genes Encoding ◽

Lysobisphosphatidic Acid ◽

Intracellular Cholesterol ◽

Niemann Pick ◽

Stimulation Of

AbstractUnesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates by the LE/LY phospholipid lysobisphosphatidic acid (LBPA) and in these studies sought to determine their functional relationship in normal LE/LY cholesterol egress. Here we demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Using its precursor phosphatidylglycerol (PG), we show that PG-induced LBPA enrichment results in clearance of accumulated cholesterol from NPC1-deficient cells but is ineffective in cells lacking functional NPC2. Together these studies reveal a heretofore unknown aspect of intracellular cholesterol trafficking, in which NPC2 and LBPA function together in an obligate step of sterol egress from the LE/LY compartment, which appears to be independent of NPC1.

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Intracellular cholesterol trafficking is dependent upon NPC2 interaction with lysobisphosphatidic acid

eLife ◽

10.7554/elife.50832 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 11

Author(s):

Leslie A McCauliff ◽

Annette Langan ◽

Ran Li ◽

Olga Ilnytska ◽

Debosreeta Bose ◽

...

Keyword(s):

Cholesterol Transport ◽

Unesterified Cholesterol ◽

Cholesterol Trafficking ◽

Cholesterol Accumulation ◽

Functional Interaction ◽

Genes Encoding ◽

Lysobisphosphatidic Acid ◽

Intracellular Cholesterol ◽

Niemann Pick ◽

Stimulation Of

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein.

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Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae)

Biochemical Journal ◽

10.1042/bj3580079 ◽

2001 ◽

Vol 358 (1) ◽

pp. 79-86 ◽

Cited By ~ 18

Author(s):

Dmitri SVIRIDOV ◽

Noel FIDGE ◽

Gabrielle BEAUMIER-GALLON ◽

Christopher FIELDING

Keyword(s):

Cell Surface ◽

Cholesterol Efflux ◽

Partial Purification ◽

Cholesterol Transport ◽

Dependent Manner ◽

Cholesterol Trafficking ◽

Apolipoprotein A ◽

Intracellular Cholesterol ◽

Plasma Apolipoprotein ◽

Stimulation Of

We have studied the effect of lipid-free human plasma apolipoprotein A-I (apoA-I) on the transport of newly synthesized cholesterol to cell-surface cholesterol-rich domains, which in human skin fibroblasts are mainly represented by caveolae. Changes in transport of newly synthesized cholesterol were assessed after labelling cells with [14C]acetate at 15°C and warming cells to permit the transfer of cholesterol, followed by the selective oxidation of cholesterol in cholesterol-rich domains (caveolae) in the plasma membrane before their partial purification. ApoA-I, but not BSA added in an equimolar concentration, enhanced the transport of cholesterol to the caveolae up to 5-fold in a dose- and time-dependent manner. The effect of apoA-I on cholesterol transport exceeded its effect on cholesterol efflux, resulting in an accumulation of intracellular cholesterol in caveolae. Methyl-β-cyclodextrin, added at a concentration promoting cholesterol efflux to the same extent as apoA-I, also stimulated cholesterol trafficking, but was 3-fold less effective than apoA-I. Progesterone inhibited the transport of newly synthesized cholesterol to the caveolae. Treatment of cells with apoA-I stimulated the expression of caveolin, increasing the amount of caveolin protein and mRNA by approx. 2-fold. We conclude that apoA-I induces the transport of intracellular cholesterol to cell-surface caveolae, possibly in part through the stimulation of caveolin expression.

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Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis

Circulation Research ◽

10.1161/circresaha.121.318881 ◽

2021 ◽

Author(s):

Sara Awan ◽

Magalie Lambert ◽

Ali Imtiaz ◽

Fabien Alpy ◽

Catherine Tomasetto ◽

...

Keyword(s):

Dietary Cholesterol ◽

Cell Types ◽

Cholesterol Content ◽

Cholesterol Homeostasis ◽

Cholesterol Trafficking ◽

Atherosclerotic Lesions ◽

Crucial Component ◽

Multiple Cell ◽

Niemann Pick

Background: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and two lysosomal proteins, the Niemann-Pick C1 (NPC1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. Methods: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells (VSMCs). To establish whether Wnt5a also protects against cholesterol accumulation in human VSMCs, we used a CRISPR/Cas9 guided nuclease approach to generate human VSMCs knockout for Wnt5a. Results: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum (ER). Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the ER, and promoted atherosclerosis. Conclusions: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

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