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The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

Schumacher MM, Elsabrouty R, Seemann J, Jo Y, DeBose-Boyd RA - Elife (2015)

Bottom Line: Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism.CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation.The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.

ABSTRACT
Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

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RNA interference-mediated knockdown of UBAD1 alleviates requirement for geranylgeraniol in sterol-accelerated reductase degradation.SV-589 cells were set up for experiments on day 0, transfected with the indicated siRNA duplexes on day 3, and depleted of sterols as described in the legend to Figure 3. Notably, the siRNA duplex targeting UBIAD1 (5′-UCUUGGAGCCGCAGGAUGUUU-3′, Dharmacon/ThermoScientfic) was distinct from that used in Figures 3, 6. The sterol-depleted cells were then treated with medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate in the absence or presence of 1 µg/ml 25-HC and 20 µM geranylgeraniol (GGOH). Following incubation for 4 hr at 37°C, cells were harvested for subcellular fractionation. Aliquots of resulting membrane fractions (20 µg protein/lane) were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase) and IgG-H8 (against UBIAD1).DOI:http://dx.doi.org/10.7554/eLife.05560.013
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fig6s1: RNA interference-mediated knockdown of UBAD1 alleviates requirement for geranylgeraniol in sterol-accelerated reductase degradation.SV-589 cells were set up for experiments on day 0, transfected with the indicated siRNA duplexes on day 3, and depleted of sterols as described in the legend to Figure 3. Notably, the siRNA duplex targeting UBIAD1 (5′-UCUUGGAGCCGCAGGAUGUUU-3′, Dharmacon/ThermoScientfic) was distinct from that used in Figures 3, 6. The sterol-depleted cells were then treated with medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate in the absence or presence of 1 µg/ml 25-HC and 20 µM geranylgeraniol (GGOH). Following incubation for 4 hr at 37°C, cells were harvested for subcellular fractionation. Aliquots of resulting membrane fractions (20 µg protein/lane) were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase) and IgG-H8 (against UBIAD1).DOI:http://dx.doi.org/10.7554/eLife.05560.013

Mentions: The role of UBIAD1 in sterol-accelerated reductase ERAD was next examined using RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout (Cong et al., 2013; Mali et al., 2013). For RNAi experiments, SV-589 cells were transfected with siRNA duplexes targeting the GFP or UBIAD1 mRNAs and subsequently treated in the absence or presence of 25-HC and geranylgeraniol prior to subcellular fractionation. Figure 6A shows that 25-HC stimulated reductase degradation from membranes of control siRNA-transfected cells (top panel, lane 2); this degradation was enhanced by geranylgeraniol (lanes 3 and 4). In UBIAD1 knockdown cells, 25-HC stimulated complete degradation of reductase, even in the absence of geranylgeraniol (lanes 5–8). Nearly identical results were obtained using an siRNA duplex targeting a different region of the UBIAD1 mRNA (Figure 6—figure supplement 1). Geranylgeraniol also augmented the ERAD of reductase that was stimulated by Apomine (Figure 6B, top panel, lanes 1–4); however, the 1,1-bisphosphonate ester caused reductase to become completely degraded in UBIAD1 knockdown cells (lanes 5–8). Results consistent with those observed using RNAi were obtained in cells subjected to CRISPR/Cas9-mediated knockout of UBIAD1. In wild type SV-589 cells, Apomine stimulated reductase ERAD through a mechanism augmented by geranylgeraniol (Figure 6C, top panel, lanes 1–4), whereas the compound caused complete degradation of reductase in cells deficient in UBIAD1 (designated UBIAD1−) (lanes 5–8). Figure 6D shows that in a time-dependent fashion, geranylgeraniol augmented Apomine-induced degradation of reductase in SV-589 cells (compare lanes 1–6 with lanes 7–12). However, Apomine alone caused complete degradation of reductase in UBIAD1− cells (Figure 6D, lanes 13–18) and augmentation by geranylgeraniol was markedly diminished (lanes 19–24). Importantly, transfection of UBIAD1− cells with pCMV-Myc-UBIAD1 restored the requirement of geranylgeraniol for maximal degradation of reductase (Figure 6E, top panel, compare lanes 1–3 with lanes 4–6).10.7554/eLife.05560.012Figure 6.RNA interference-mediated knockdown or CRISPR/Cas9-mediated knockout of UBIAD1 alleviates geranylgeraniol requirement in sterol-accelerated degradation of HMG CoA reductase.


The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

Schumacher MM, Elsabrouty R, Seemann J, Jo Y, DeBose-Boyd RA - Elife (2015)

RNA interference-mediated knockdown of UBAD1 alleviates requirement for geranylgeraniol in sterol-accelerated reductase degradation.SV-589 cells were set up for experiments on day 0, transfected with the indicated siRNA duplexes on day 3, and depleted of sterols as described in the legend to Figure 3. Notably, the siRNA duplex targeting UBIAD1 (5′-UCUUGGAGCCGCAGGAUGUUU-3′, Dharmacon/ThermoScientfic) was distinct from that used in Figures 3, 6. The sterol-depleted cells were then treated with medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate in the absence or presence of 1 µg/ml 25-HC and 20 µM geranylgeraniol (GGOH). Following incubation for 4 hr at 37°C, cells were harvested for subcellular fractionation. Aliquots of resulting membrane fractions (20 µg protein/lane) were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase) and IgG-H8 (against UBIAD1).DOI:http://dx.doi.org/10.7554/eLife.05560.013
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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fig6s1: RNA interference-mediated knockdown of UBAD1 alleviates requirement for geranylgeraniol in sterol-accelerated reductase degradation.SV-589 cells were set up for experiments on day 0, transfected with the indicated siRNA duplexes on day 3, and depleted of sterols as described in the legend to Figure 3. Notably, the siRNA duplex targeting UBIAD1 (5′-UCUUGGAGCCGCAGGAUGUUU-3′, Dharmacon/ThermoScientfic) was distinct from that used in Figures 3, 6. The sterol-depleted cells were then treated with medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate in the absence or presence of 1 µg/ml 25-HC and 20 µM geranylgeraniol (GGOH). Following incubation for 4 hr at 37°C, cells were harvested for subcellular fractionation. Aliquots of resulting membrane fractions (20 µg protein/lane) were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase) and IgG-H8 (against UBIAD1).DOI:http://dx.doi.org/10.7554/eLife.05560.013
Mentions: The role of UBIAD1 in sterol-accelerated reductase ERAD was next examined using RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout (Cong et al., 2013; Mali et al., 2013). For RNAi experiments, SV-589 cells were transfected with siRNA duplexes targeting the GFP or UBIAD1 mRNAs and subsequently treated in the absence or presence of 25-HC and geranylgeraniol prior to subcellular fractionation. Figure 6A shows that 25-HC stimulated reductase degradation from membranes of control siRNA-transfected cells (top panel, lane 2); this degradation was enhanced by geranylgeraniol (lanes 3 and 4). In UBIAD1 knockdown cells, 25-HC stimulated complete degradation of reductase, even in the absence of geranylgeraniol (lanes 5–8). Nearly identical results were obtained using an siRNA duplex targeting a different region of the UBIAD1 mRNA (Figure 6—figure supplement 1). Geranylgeraniol also augmented the ERAD of reductase that was stimulated by Apomine (Figure 6B, top panel, lanes 1–4); however, the 1,1-bisphosphonate ester caused reductase to become completely degraded in UBIAD1 knockdown cells (lanes 5–8). Results consistent with those observed using RNAi were obtained in cells subjected to CRISPR/Cas9-mediated knockout of UBIAD1. In wild type SV-589 cells, Apomine stimulated reductase ERAD through a mechanism augmented by geranylgeraniol (Figure 6C, top panel, lanes 1–4), whereas the compound caused complete degradation of reductase in cells deficient in UBIAD1 (designated UBIAD1−) (lanes 5–8). Figure 6D shows that in a time-dependent fashion, geranylgeraniol augmented Apomine-induced degradation of reductase in SV-589 cells (compare lanes 1–6 with lanes 7–12). However, Apomine alone caused complete degradation of reductase in UBIAD1− cells (Figure 6D, lanes 13–18) and augmentation by geranylgeraniol was markedly diminished (lanes 19–24). Importantly, transfection of UBIAD1− cells with pCMV-Myc-UBIAD1 restored the requirement of geranylgeraniol for maximal degradation of reductase (Figure 6E, top panel, compare lanes 1–3 with lanes 4–6).10.7554/eLife.05560.012Figure 6.RNA interference-mediated knockdown or CRISPR/Cas9-mediated knockout of UBIAD1 alleviates geranylgeraniol requirement in sterol-accelerated degradation of HMG CoA reductase.

Bottom Line: Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism.CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation.The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States.

ABSTRACT
Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

Show MeSH
Related in: MedlinePlus