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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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Model depicting hypothetical mechanisms of RIDα/ORP1L-mediated transport of cholesterol from endosome to ER in NPC1-deficient cells. Cholesterol may be trafficked from endosomes to the ER by vesicular or nonvesicular mechanisms, including membrane contact sites, where close apposition of donor and acceptor membranes supports transport down a concentration gradient (A), vesicular transport, where cholesterol is packaged in transport vesicles to be delivered to the ER (B), or by transport proteins that bind cholesterol in a hydrophobic pocket protected from the cytosol and transport it to the ER (C). RIDα mediates the transport of cholesterol to the ER independent of NPC1 but dependent on NPC2 and ORP1L. Once delivered, cholesterol is acted upon by ACAT for incorporation into LDs and does not affect SREBP-regulated gene expression. CE, cholesterol ester; Ch, cholesterol.
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Figure 9: Model depicting hypothetical mechanisms of RIDα/ORP1L-mediated transport of cholesterol from endosome to ER in NPC1-deficient cells. Cholesterol may be trafficked from endosomes to the ER by vesicular or nonvesicular mechanisms, including membrane contact sites, where close apposition of donor and acceptor membranes supports transport down a concentration gradient (A), vesicular transport, where cholesterol is packaged in transport vesicles to be delivered to the ER (B), or by transport proteins that bind cholesterol in a hydrophobic pocket protected from the cytosol and transport it to the ER (C). RIDα mediates the transport of cholesterol to the ER independent of NPC1 but dependent on NPC2 and ORP1L. Once delivered, cholesterol is acted upon by ACAT for incorporation into LDs and does not affect SREBP-regulated gene expression. CE, cholesterol ester; Ch, cholesterol.

Mentions: ORP1L binds cholesterol, and this interaction has a profound effect on its conformation downstream of GTP-Rab7 (Rocha et al., 2009). High LE cholesterol levels occlude the ORP1L FFAT motif involved in VAP-A binding, whereas this motif is exposed when LE cholesterol levels are low. In this way, cholesterol levels and ORP1L in association with Rab7 regulate the positioning of LEs. Because RIDα interacts with ORP1L in its cholesterol binding ORD (Shah et al., 2007), we hypothesize that RIDα may modulate the effect of cholesterol on ORP1L conformation. Thus, ORP1L may be able to promote VAP-A interactions in the presence of high cholesterol while bound to RIDα. We previously showed that RIDα does not localize to LAMP1-positive LEs but promotes formation of a hybrid intermediate endosome compartment positive for ORP1L via an unconventional autophagy-based pathway (Cianciola and Carlin, 2009). We hypothesize that RIDα binding to ORP1L allows for exposure of the FFAT motif responsible for binding to VAP-A on the ER surface independent of cholesterol levels. The ORP1L/VAP-A interaction would create endosome–ER contact sites that allow for cholesterol to be transported to the ER by way of passive diffusion down a concentration gradient between closely apposed membranes (Figure 9A). Although we favor this mechanism, the possibility exists that ORP1L may facilitate ER cholesterol trafficking through vesicular transport (Figure 9B) or by acting as a cholesterol transport protein (Figure 9C). However, the notion that ORP1L acts as a cholesterol transport protein is not supported in the literature, and our data indicate that cholesterol binding by ORP1L is unessential for RIDα NPC1 rescue. The yeast oxysterol-binding protein Kes1/Osh4p binds dehydroergosterol and phosphatidylinositol 4-phosphate in a hydrophobic pocket for transport of these lipids between membranes along opposite routes in vitro (de Saint-Jean et al., 2011). However, reports have also demonstrated that Osh proteins do not play a role in the transport of sterol from PM to ER and LDs in living cells (Georgiev et al., 2011) and that a Kes1/Osh4p sterol-binding mutant is a gain-of-function mutation that inhibits cell growth (Alfaro et al., 2011; Mousley et al., 2012). We propose that RIDα reengineers ORP1L to act as a bridge linking cholesterol-loaded LEs with VAP-A–positive ER by transforming its cholesterol-sensing capacity in NPC1-deficient cells. Alternatively, ORP1L may have additional functions aside from mediating endosome–ER membrane contact sites with VAP-A that have yet to be identified. Further experiments are underway to investigate the role of ORP1L in ER cholesterol transport during RIDα NPC1 rescue.


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)

Model depicting hypothetical mechanisms of RIDα/ORP1L-mediated transport of cholesterol from endosome to ER in NPC1-deficient cells. Cholesterol may be trafficked from endosomes to the ER by vesicular or nonvesicular mechanisms, including membrane contact sites, where close apposition of donor and acceptor membranes supports transport down a concentration gradient (A), vesicular transport, where cholesterol is packaged in transport vesicles to be delivered to the ER (B), or by transport proteins that bind cholesterol in a hydrophobic pocket protected from the cytosol and transport it to the ER (C). RIDα mediates the transport of cholesterol to the ER independent of NPC1 but dependent on NPC2 and ORP1L. Once delivered, cholesterol is acted upon by ACAT for incorporation into LDs and does not affect SREBP-regulated gene expression. CE, cholesterol ester; Ch, cholesterol.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814149&req=5

Figure 9: Model depicting hypothetical mechanisms of RIDα/ORP1L-mediated transport of cholesterol from endosome to ER in NPC1-deficient cells. Cholesterol may be trafficked from endosomes to the ER by vesicular or nonvesicular mechanisms, including membrane contact sites, where close apposition of donor and acceptor membranes supports transport down a concentration gradient (A), vesicular transport, where cholesterol is packaged in transport vesicles to be delivered to the ER (B), or by transport proteins that bind cholesterol in a hydrophobic pocket protected from the cytosol and transport it to the ER (C). RIDα mediates the transport of cholesterol to the ER independent of NPC1 but dependent on NPC2 and ORP1L. Once delivered, cholesterol is acted upon by ACAT for incorporation into LDs and does not affect SREBP-regulated gene expression. CE, cholesterol ester; Ch, cholesterol.
Mentions: ORP1L binds cholesterol, and this interaction has a profound effect on its conformation downstream of GTP-Rab7 (Rocha et al., 2009). High LE cholesterol levels occlude the ORP1L FFAT motif involved in VAP-A binding, whereas this motif is exposed when LE cholesterol levels are low. In this way, cholesterol levels and ORP1L in association with Rab7 regulate the positioning of LEs. Because RIDα interacts with ORP1L in its cholesterol binding ORD (Shah et al., 2007), we hypothesize that RIDα may modulate the effect of cholesterol on ORP1L conformation. Thus, ORP1L may be able to promote VAP-A interactions in the presence of high cholesterol while bound to RIDα. We previously showed that RIDα does not localize to LAMP1-positive LEs but promotes formation of a hybrid intermediate endosome compartment positive for ORP1L via an unconventional autophagy-based pathway (Cianciola and Carlin, 2009). We hypothesize that RIDα binding to ORP1L allows for exposure of the FFAT motif responsible for binding to VAP-A on the ER surface independent of cholesterol levels. The ORP1L/VAP-A interaction would create endosome–ER contact sites that allow for cholesterol to be transported to the ER by way of passive diffusion down a concentration gradient between closely apposed membranes (Figure 9A). Although we favor this mechanism, the possibility exists that ORP1L may facilitate ER cholesterol trafficking through vesicular transport (Figure 9B) or by acting as a cholesterol transport protein (Figure 9C). However, the notion that ORP1L acts as a cholesterol transport protein is not supported in the literature, and our data indicate that cholesterol binding by ORP1L is unessential for RIDα NPC1 rescue. The yeast oxysterol-binding protein Kes1/Osh4p binds dehydroergosterol and phosphatidylinositol 4-phosphate in a hydrophobic pocket for transport of these lipids between membranes along opposite routes in vitro (de Saint-Jean et al., 2011). However, reports have also demonstrated that Osh proteins do not play a role in the transport of sterol from PM to ER and LDs in living cells (Georgiev et al., 2011) and that a Kes1/Osh4p sterol-binding mutant is a gain-of-function mutation that inhibits cell growth (Alfaro et al., 2011; Mousley et al., 2012). We propose that RIDα reengineers ORP1L to act as a bridge linking cholesterol-loaded LEs with VAP-A–positive ER by transforming its cholesterol-sensing capacity in NPC1-deficient cells. Alternatively, ORP1L may have additional functions aside from mediating endosome–ER membrane contact sites with VAP-A that have yet to be identified. Further experiments are underway to investigate the role of ORP1L in ER cholesterol transport during RIDα NPC1 rescue.

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