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Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.

Cianciola NL, Greene DJ, Morton RE, Carlin CR - Mol. Biol. Cell (2013)

Bottom Line: Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7.The molecular identity of putative alternative pathways, however, is poorly characterized.We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195 Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.

ABSTRACT
Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

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RIDα induces formation of LDs during Ad infection of NPC1-deficient cells. (A– C) Confocal images of shControl (A), shNPC1 (B), or shNPC2 cells (C) mock infected or infected with RIDα-overexpressing in724 (middle) or RIDα- dl753 (right) Ad2. Cells were stained with BODIPY 493/503 and DAPI. Bars, 10 μm.
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Figure 8: RIDα induces formation of LDs during Ad infection of NPC1-deficient cells. (A– C) Confocal images of shControl (A), shNPC1 (B), or shNPC2 cells (C) mock infected or infected with RIDα-overexpressing in724 (middle) or RIDα- dl753 (right) Ad2. Cells were stained with BODIPY 493/503 and DAPI. Bars, 10 μm.

Mentions: Our studies reveal a role for RIDα in the formation of LDs independent of NPC1. To address physiological relevance, we next sought to determine whether RIDα plays a similar role in LD formation during infection with group C Ad serotype 2. We first verified that NPC1 and NPC2 are not required for Ad infection. This was determined by showing that shControl, shNPC1, and shNPC2 cells infected with an Ad that overexpresses RIDα (in724) all produced similar levels of early region 1A (E1A), the first virally encoded protein synthesized in acute infections (Supplemental Figure S3; Nevins et al., 1979). Infected cells were then stained for LDs with BODIPY 493/503. Mock-infected shControl cells displayed appropriate formation of LDs when loaded with LDL (Figure 8A), and infection of shControl cells with RIDα-overexpressing in724 Ad had no effect on LD formation (Figure 8A). Mock-infected shNPC1 cells displayed a lack of LDs when loaded with LDL (Figure 8B); however, shNPC1 cells infected with the RIDα-overexpressing in724 virus displayed a marked increase in LD formation with LDL load (Figure 8B). Mock-infected shNPC2 cells also displayed a lack of LDs upon LDL loading (Figure 8C). However, similar to ectopic expression of tagged RIDα in NPC2-mutant fibroblasts, infection of shNPC2 cells with RIDα-overexpressing in724 Ad did not induce formation of LDs with LDL load (Figure 8C). Next we infected cells with a RIDα- Ad (dl753) to show that RIDα is necessary for LD formation. Similar to infection with in724, shControl cells infected with RIDα- dl753 Ad displayed appropriate formation of LDs upon LDL loading (Figure 8A). As expected, infection of shNPC1 cells with the RIDα- dl753 Ad did not induce formation of LDs (Figure 8B), demonstrating that RIDα is the viral protein mediating alterations to the LD phenotype. Finally, infection of shNPC2 cells with the RIDα- dl753 Ad had no effect on LD formation (Figure 8C). Together these data demonstrate that RIDα induces LD formation by an NPC1-independent mechanism during an acute Ad infection as well when expressed ectopically as a tagged transgene.


Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.

Cianciola NL, Greene DJ, Morton RE, Carlin CR - Mol. Biol. Cell (2013)

RIDα induces formation of LDs during Ad infection of NPC1-deficient cells. (A– C) Confocal images of shControl (A), shNPC1 (B), or shNPC2 cells (C) mock infected or infected with RIDα-overexpressing in724 (middle) or RIDα- dl753 (right) Ad2. Cells were stained with BODIPY 493/503 and DAPI. Bars, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: RIDα induces formation of LDs during Ad infection of NPC1-deficient cells. (A– C) Confocal images of shControl (A), shNPC1 (B), or shNPC2 cells (C) mock infected or infected with RIDα-overexpressing in724 (middle) or RIDα- dl753 (right) Ad2. Cells were stained with BODIPY 493/503 and DAPI. Bars, 10 μm.
Mentions: Our studies reveal a role for RIDα in the formation of LDs independent of NPC1. To address physiological relevance, we next sought to determine whether RIDα plays a similar role in LD formation during infection with group C Ad serotype 2. We first verified that NPC1 and NPC2 are not required for Ad infection. This was determined by showing that shControl, shNPC1, and shNPC2 cells infected with an Ad that overexpresses RIDα (in724) all produced similar levels of early region 1A (E1A), the first virally encoded protein synthesized in acute infections (Supplemental Figure S3; Nevins et al., 1979). Infected cells were then stained for LDs with BODIPY 493/503. Mock-infected shControl cells displayed appropriate formation of LDs when loaded with LDL (Figure 8A), and infection of shControl cells with RIDα-overexpressing in724 Ad had no effect on LD formation (Figure 8A). Mock-infected shNPC1 cells displayed a lack of LDs when loaded with LDL (Figure 8B); however, shNPC1 cells infected with the RIDα-overexpressing in724 virus displayed a marked increase in LD formation with LDL load (Figure 8B). Mock-infected shNPC2 cells also displayed a lack of LDs upon LDL loading (Figure 8C). However, similar to ectopic expression of tagged RIDα in NPC2-mutant fibroblasts, infection of shNPC2 cells with RIDα-overexpressing in724 Ad did not induce formation of LDs with LDL load (Figure 8C). Next we infected cells with a RIDα- Ad (dl753) to show that RIDα is necessary for LD formation. Similar to infection with in724, shControl cells infected with RIDα- dl753 Ad displayed appropriate formation of LDs upon LDL loading (Figure 8A). As expected, infection of shNPC1 cells with the RIDα- dl753 Ad did not induce formation of LDs (Figure 8B), demonstrating that RIDα is the viral protein mediating alterations to the LD phenotype. Finally, infection of shNPC2 cells with the RIDα- dl753 Ad had no effect on LD formation (Figure 8C). Together these data demonstrate that RIDα induces LD formation by an NPC1-independent mechanism during an acute Ad infection as well when expressed ectopically as a tagged transgene.

Bottom Line: Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7.The molecular identity of putative alternative pathways, however, is poorly characterized.We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195 Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.

ABSTRACT
Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

Show MeSH
Related in: MedlinePlus