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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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Related in: MedlinePlus

Overexpression of ORP1L after siRNA knockdown restores the ability of RIDα to rescue the cholesterol storage phenotype in NPC1-deficient cells. (A) shNPC1-RIDα cells were transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L, and equal aliquots of total cellular protein were immunoblotted with antibodies to ORP1L, GFP, or actin for loading control. (B–E) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (B), ORP1L siRNA targeting the 3′ UTR (C), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (D) or Δ560–563 GFP-ORP1L (E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (F–I) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (F), ORP1L siRNA targeting the 3′ UTR (G), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (H) or Δ560–563 GFP-ORP1L (I) and stained with antibody to FLAG-RIDα and with LipidTOX deep red. (J) Quantification of esterified cholesterol in shNPC1-RIDα cells transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM (*p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
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Figure 7: Overexpression of ORP1L after siRNA knockdown restores the ability of RIDα to rescue the cholesterol storage phenotype in NPC1-deficient cells. (A) shNPC1-RIDα cells were transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L, and equal aliquots of total cellular protein were immunoblotted with antibodies to ORP1L, GFP, or actin for loading control. (B–E) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (B), ORP1L siRNA targeting the 3′ UTR (C), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (D) or Δ560–563 GFP-ORP1L (E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (F–I) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (F), ORP1L siRNA targeting the 3′ UTR (G), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (H) or Δ560–563 GFP-ORP1L (I) and stained with antibody to FLAG-RIDα and with LipidTOX deep red. (J) Quantification of esterified cholesterol in shNPC1-RIDα cells transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM (*p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Mentions: To further our understanding of the involvement of ORP1L in the ability of RIDα to rescue the NPC1 cholesterol storage phenotype, we developed a knockdown/rescue system in which ORP1L expression was silenced with siRNA directed against the 3′ untranslated region (UTR) and then rescued with green fluorescent protein (GFP)–ORP1L overexpression, which was resistant to knockdown. shNPC1-RIDα cells were transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type GFP-ORP1L or a sterol binding mutant (Δ560–563 GFP-ORP1L; Vihervaara et al., 2011). Equal aliquots of total cellular protein were then immunoblotted for ORP1L and GFP, with actin for loading control (Figure 7A). We routinely achieved ∼90% knockdown of ORP1L, whereas GFP-ORP1L expression was unaffected. shNPC1-RIDα cells treated similarly to those in Figure 7A were loaded with LDL and then examined by confocal microscopy after cells were stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. shNPC1-RIDα cells treated with siCntl showed the expected rescue of the LSO phenotype (Figure 7B), and knockdown of ORP1L again caused LSOs to persist (Figure 7C). However, shNPC1-RIDα cells transfected with siORP1L targeting the 3′ UTR and overexpressing wild-type GFP-ORP1L displayed a lack of enlarged LAMP1/filipin-positive LEs indicative of LSOs upon LDL loading (Figure 7D). Of interest, rescue of ORP1L expression with the sterol binding mutant Δ560–563 GFP-ORP1L in shNPC1-RIDα cells caused a reduction in the size of LAMP1/filipin-positive LEs consistent with RIDα NPC1 rescue (Figure 7E), as well as with the hypothesis that RIDα binding the ORP1L ORD transforms its ability to sense cholesterol (Shah et al., 2007). Next we used the ORP1L knockdown/rescue technique to study LD formation in shNPC1-RIDα and loaded with LDL by LipidTOX deep red staining of LDs. LipidTOX deep red neutral lipid stain was used because the BODIPY 493/503 emission overlapped with that of GFP, and LipidTOX deep red colocalizes exclusively with BODIPY 493/503 (Klapper et al., 2011). LD formation was induced in shNPC1-RIDα cells treated with control siRNA (Figure 7F), and LD formation was again inhibited when cells were treated with ORP1L siRNA targeting the 3′ UTR (Figure 7G). LD formation was induced in shNPC1-RIDα cells cotransfected with siORP1L targeting the 3′ UTR and wild-type GFP-ORP1L (Figure 7H), as well as in Δ560–563 GFP-ORP1L–transfected cells (Figure 7I). We then used the Amplex Red cholesterol assay to quantify cholesterol esters in shNPC1-RIDα cells with knockdown/rescue of ORP1L expression. Knockdown of ORP1L targeting the 3′ UTR caused a statistically significant decrease in the formation of esterified cholesterol compared with control siRNA treatment, and rescue of ORP1L expression with wild-type or Δ560–563 GFP-ORP1L restored the ability of RIDα to induce an increase in esterified cholesterol (Figure 7J). Collectively these data reveal that RIDα NPC1 rescue depends on ORP1L but is independent of its ability to bind cholesterol and suggest for the first time a possible role for ORP1L in transport of cholesterol to the ER and subsequent LD formation in the absence of functional NPC1.


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)

Overexpression of ORP1L after siRNA knockdown restores the ability of RIDα to rescue the cholesterol storage phenotype in NPC1-deficient cells. (A) shNPC1-RIDα cells were transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L, and equal aliquots of total cellular protein were immunoblotted with antibodies to ORP1L, GFP, or actin for loading control. (B–E) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (B), ORP1L siRNA targeting the 3′ UTR (C), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (D) or Δ560–563 GFP-ORP1L (E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (F–I) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (F), ORP1L siRNA targeting the 3′ UTR (G), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (H) or Δ560–563 GFP-ORP1L (I) and stained with antibody to FLAG-RIDα and with LipidTOX deep red. (J) Quantification of esterified cholesterol in shNPC1-RIDα cells transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM (*p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
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Related In: Results  -  Collection

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Figure 7: Overexpression of ORP1L after siRNA knockdown restores the ability of RIDα to rescue the cholesterol storage phenotype in NPC1-deficient cells. (A) shNPC1-RIDα cells were transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L, and equal aliquots of total cellular protein were immunoblotted with antibodies to ORP1L, GFP, or actin for loading control. (B–E) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (B), ORP1L siRNA targeting the 3′ UTR (C), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (D) or Δ560–563 GFP-ORP1L (E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (F–I) Confocal images of shNPC1-RIDα cells transfected with Cntl siRNA (F), ORP1L siRNA targeting the 3′ UTR (G), or cotransfected with ORP1L siRNA targeting the 3′ UTR and wild-type (H) or Δ560–563 GFP-ORP1L (I) and stained with antibody to FLAG-RIDα and with LipidTOX deep red. (J) Quantification of esterified cholesterol in shNPC1-RIDα cells transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type or Δ560–563 GFP-ORP1L using the Amplex Red Cholesterol Assay kit as described in Materials and Methods. Values were normalized to total cellular protein and are displayed as mean ± SEM (*p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
Mentions: To further our understanding of the involvement of ORP1L in the ability of RIDα to rescue the NPC1 cholesterol storage phenotype, we developed a knockdown/rescue system in which ORP1L expression was silenced with siRNA directed against the 3′ untranslated region (UTR) and then rescued with green fluorescent protein (GFP)–ORP1L overexpression, which was resistant to knockdown. shNPC1-RIDα cells were transfected with Cntl siRNA or ORP1L siRNA targeting the 3′ UTR or cotransfected with ORP1L siRNA targeting the 3′ UTR along with wild-type GFP-ORP1L or a sterol binding mutant (Δ560–563 GFP-ORP1L; Vihervaara et al., 2011). Equal aliquots of total cellular protein were then immunoblotted for ORP1L and GFP, with actin for loading control (Figure 7A). We routinely achieved ∼90% knockdown of ORP1L, whereas GFP-ORP1L expression was unaffected. shNPC1-RIDα cells treated similarly to those in Figure 7A were loaded with LDL and then examined by confocal microscopy after cells were stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. shNPC1-RIDα cells treated with siCntl showed the expected rescue of the LSO phenotype (Figure 7B), and knockdown of ORP1L again caused LSOs to persist (Figure 7C). However, shNPC1-RIDα cells transfected with siORP1L targeting the 3′ UTR and overexpressing wild-type GFP-ORP1L displayed a lack of enlarged LAMP1/filipin-positive LEs indicative of LSOs upon LDL loading (Figure 7D). Of interest, rescue of ORP1L expression with the sterol binding mutant Δ560–563 GFP-ORP1L in shNPC1-RIDα cells caused a reduction in the size of LAMP1/filipin-positive LEs consistent with RIDα NPC1 rescue (Figure 7E), as well as with the hypothesis that RIDα binding the ORP1L ORD transforms its ability to sense cholesterol (Shah et al., 2007). Next we used the ORP1L knockdown/rescue technique to study LD formation in shNPC1-RIDα and loaded with LDL by LipidTOX deep red staining of LDs. LipidTOX deep red neutral lipid stain was used because the BODIPY 493/503 emission overlapped with that of GFP, and LipidTOX deep red colocalizes exclusively with BODIPY 493/503 (Klapper et al., 2011). LD formation was induced in shNPC1-RIDα cells treated with control siRNA (Figure 7F), and LD formation was again inhibited when cells were treated with ORP1L siRNA targeting the 3′ UTR (Figure 7G). LD formation was induced in shNPC1-RIDα cells cotransfected with siORP1L targeting the 3′ UTR and wild-type GFP-ORP1L (Figure 7H), as well as in Δ560–563 GFP-ORP1L–transfected cells (Figure 7I). We then used the Amplex Red cholesterol assay to quantify cholesterol esters in shNPC1-RIDα cells with knockdown/rescue of ORP1L expression. Knockdown of ORP1L targeting the 3′ UTR caused a statistically significant decrease in the formation of esterified cholesterol compared with control siRNA treatment, and rescue of ORP1L expression with wild-type or Δ560–563 GFP-ORP1L restored the ability of RIDα to induce an increase in esterified cholesterol (Figure 7J). Collectively these data reveal that RIDα NPC1 rescue depends on ORP1L but is independent of its ability to bind cholesterol and suggest for the first time a possible role for ORP1L in transport of cholesterol to the ER and subsequent LD formation in the absence of functional NPC1.

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