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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 does not rescue ER retention of defective NPC1 I1061T mutant protein in patient fibroblasts. (A–C) Confocal images of normal (A), mock-transfected homozygous NPC1 I1061T mutant (B), or homozygous NPC1 I1061T mutant fibroblasts transfected with FLAG-RIDα (C) and stained with antibodies to NPC1 and FLAG-RIDα and costained with antibodies to calreticulin (left) or LAMP1 (right). (D, E) Confocal images of homozygous NPC1 I1061T mutant fibroblasts mock transfected (D, left) or transfected with FLAG-RIDα (D, right; E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin (D) or with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI (E). Mock-transfected cells in E are shown in the same field and designated with an asterisk Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
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Figure 5: RIDα does not rescue ER retention of defective NPC1 I1061T mutant protein in patient fibroblasts. (A–C) Confocal images of normal (A), mock-transfected homozygous NPC1 I1061T mutant (B), or homozygous NPC1 I1061T mutant fibroblasts transfected with FLAG-RIDα (C) and stained with antibodies to NPC1 and FLAG-RIDα and costained with antibodies to calreticulin (left) or LAMP1 (right). (D, E) Confocal images of homozygous NPC1 I1061T mutant fibroblasts mock transfected (D, left) or transfected with FLAG-RIDα (D, right; E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin (D) or with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI (E). Mock-transfected cells in E are shown in the same field and designated with an asterisk Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Mentions: The NPC1 I1061T mutant can function in cholesterol egress but is retained in the ER and degraded (Gelsthorpe et al., 2008). Histone deacetylases (HDACs) are a diverse family of enzymes that are important mediators of chromatin remodeling and gene expression and also target nonhistone proteins for deacetylation (Glozak et al., 2005). Treatment of NPC1-mutant fibroblasts with HDAC inhibitor was shown to rescue the cholesterol storage phenotype (Munkacsi et al., 2011; Pipalia et al., 2011) by an unknown mechanism that involves increased expression of the NPC1 I1061T mutant protein and delivery to LE/Ly, where it can mediate cholesterol egress. Therefore one possible mechanism for RIDα NPC1 rescue is that RIDα promotes ER escape of NPC1 similar to HDAC inhibitor treatment. The NPC1 mutant fibroblasts used up to this point in this study are a compound heterozygote for the I1061T and P237S mutations in NPC1. To specifically test a role for RIDα in correcting the trafficking of the NPC1 I1061T mutant, we used homozygous NPC1 I1061T mutant fibroblasts and examined the colocalization of NPC1 with the ER marker calreticulin and the LE marker LAMP1 in cells transfected with FLAG-RIDα and loaded with LDL. Normal human fibroblasts exhibited colocalization of NPC1 with LAMP1 but not calreticulin (Figure 5A), demonstrating that wild-type NPC1 is efficiently trafficked out of the ER to its functional location on LAMP1-positive endosomes. In contrast, the NPC1 I1061T mutant protein exhibited significant overlap with calreticulin-positive ER and did not colocalize with LAMP1-positive LEs in mock-transfected homozygous NPC1 I1061T mutant fibroblasts (Figure 5B), confirming ER retention of the I1061T NPC1 mutant protein. However, RIDα did not correct the ER retention of the NPC1 I1061T mutant, as it remained colocalized with calreticulin but not LAMP1 (Figure 5C). FLAG-RIDα expression rescued the cholesterol storage phenotype in homozygous NPC1 I1061T mutant fibroblasts (Figure 5D) and induced formation of LDs upon LDL loading (Figure 5E). These data indicate that RIDα does not restore NPC1 function by promoting exit of the I1061T mutant protein from the ER.


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 does not rescue ER retention of defective NPC1 I1061T mutant protein in patient fibroblasts. (A–C) Confocal images of normal (A), mock-transfected homozygous NPC1 I1061T mutant (B), or homozygous NPC1 I1061T mutant fibroblasts transfected with FLAG-RIDα (C) and stained with antibodies to NPC1 and FLAG-RIDα and costained with antibodies to calreticulin (left) or LAMP1 (right). (D, E) Confocal images of homozygous NPC1 I1061T mutant fibroblasts mock transfected (D, left) or transfected with FLAG-RIDα (D, right; E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin (D) or with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI (E). Mock-transfected cells in E are shown in the same field and designated with an asterisk 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 5: RIDα does not rescue ER retention of defective NPC1 I1061T mutant protein in patient fibroblasts. (A–C) Confocal images of normal (A), mock-transfected homozygous NPC1 I1061T mutant (B), or homozygous NPC1 I1061T mutant fibroblasts transfected with FLAG-RIDα (C) and stained with antibodies to NPC1 and FLAG-RIDα and costained with antibodies to calreticulin (left) or LAMP1 (right). (D, E) Confocal images of homozygous NPC1 I1061T mutant fibroblasts mock transfected (D, left) or transfected with FLAG-RIDα (D, right; E) and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin (D) or with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI (E). Mock-transfected cells in E are shown in the same field and designated with an asterisk Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
Mentions: The NPC1 I1061T mutant can function in cholesterol egress but is retained in the ER and degraded (Gelsthorpe et al., 2008). Histone deacetylases (HDACs) are a diverse family of enzymes that are important mediators of chromatin remodeling and gene expression and also target nonhistone proteins for deacetylation (Glozak et al., 2005). Treatment of NPC1-mutant fibroblasts with HDAC inhibitor was shown to rescue the cholesterol storage phenotype (Munkacsi et al., 2011; Pipalia et al., 2011) by an unknown mechanism that involves increased expression of the NPC1 I1061T mutant protein and delivery to LE/Ly, where it can mediate cholesterol egress. Therefore one possible mechanism for RIDα NPC1 rescue is that RIDα promotes ER escape of NPC1 similar to HDAC inhibitor treatment. The NPC1 mutant fibroblasts used up to this point in this study are a compound heterozygote for the I1061T and P237S mutations in NPC1. To specifically test a role for RIDα in correcting the trafficking of the NPC1 I1061T mutant, we used homozygous NPC1 I1061T mutant fibroblasts and examined the colocalization of NPC1 with the ER marker calreticulin and the LE marker LAMP1 in cells transfected with FLAG-RIDα and loaded with LDL. Normal human fibroblasts exhibited colocalization of NPC1 with LAMP1 but not calreticulin (Figure 5A), demonstrating that wild-type NPC1 is efficiently trafficked out of the ER to its functional location on LAMP1-positive endosomes. In contrast, the NPC1 I1061T mutant protein exhibited significant overlap with calreticulin-positive ER and did not colocalize with LAMP1-positive LEs in mock-transfected homozygous NPC1 I1061T mutant fibroblasts (Figure 5B), confirming ER retention of the I1061T NPC1 mutant protein. However, RIDα did not correct the ER retention of the NPC1 I1061T mutant, as it remained colocalized with calreticulin but not LAMP1 (Figure 5C). FLAG-RIDα expression rescued the cholesterol storage phenotype in homozygous NPC1 I1061T mutant fibroblasts (Figure 5D) and induced formation of LDs upon LDL loading (Figure 5E). These data indicate that RIDα does not restore NPC1 function by promoting exit of the I1061T mutant protein from the ER.

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