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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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SCD-associated UBIAD1 (N102S) resists geranylgeraniol-mediated displacement from HMG CoA reductase in two independent experiments (A and B).UBIAD1−/pCDNA3.1, UBIAD1−/pMyc-UBIAD1 (WT), and UBIAD1−/pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 3, cells were depleted of sterols as described in the legend to Figure 4. After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presence of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1.DOI:http://dx.doi.org/10.7554/eLife.05560.017
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fig8s1: SCD-associated UBIAD1 (N102S) resists geranylgeraniol-mediated displacement from HMG CoA reductase in two independent experiments (A and B).UBIAD1−/pCDNA3.1, UBIAD1−/pMyc-UBIAD1 (WT), and UBIAD1−/pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 3, cells were depleted of sterols as described in the legend to Figure 4. After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presence of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1.DOI:http://dx.doi.org/10.7554/eLife.05560.017

Mentions: We next compared the geranylgeraniol-mediated displacement of wild type and N102S UBIAD1 from reductase using co-immunoprecipitation. UBIAD1− cells stably transfected with empty mock vector, pCMV-Myc-UBIAD1, or pCMV-Myc-UBIAD1 (N102S) were briefly incubated with 25-HC in the absence or presence of geranylgeraniol. The cells were then harvested, lysed, and subjected to anti-reductase immunoprecipitation. The results show that wild type UBIAD1 co-precipitated with reductase in the 25-HC-treated cells (Figure 8A, second panel, lane e). This co-precipitation was inhibited by geranylgeraniol (lanes g and h). UBIAD1 (N102S) similarly co-precipitated with reductase (lane i); however, the mutant UBAID1 resisted geranylgeraniol-mediated displacement and remained associated with reductase (lanes j–l). UBIAD1 (N102S) exhibited a similar resistance to geranylgeraniol-mediated displacement from reductase in two independent experiments shown in Figure 8—figure supplement 1.10.7554/eLife.05560.016Figure 8.SCD-associated UBIAD1 mutant resists geranylgeraniol-mediated displacement from HMG CoA reductase and remains sequestered in ER membranes.


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)

SCD-associated UBIAD1 (N102S) resists geranylgeraniol-mediated displacement from HMG CoA reductase in two independent experiments (A and B).UBIAD1−/pCDNA3.1, UBIAD1−/pMyc-UBIAD1 (WT), and UBIAD1−/pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 3, cells were depleted of sterols as described in the legend to Figure 4. After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presence of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1.DOI:http://dx.doi.org/10.7554/eLife.05560.017
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4374513&req=5

fig8s1: SCD-associated UBIAD1 (N102S) resists geranylgeraniol-mediated displacement from HMG CoA reductase in two independent experiments (A and B).UBIAD1−/pCDNA3.1, UBIAD1−/pMyc-UBIAD1 (WT), and UBIAD1−/pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 3, cells were depleted of sterols as described in the legend to Figure 4. After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presence of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1.DOI:http://dx.doi.org/10.7554/eLife.05560.017
Mentions: We next compared the geranylgeraniol-mediated displacement of wild type and N102S UBIAD1 from reductase using co-immunoprecipitation. UBIAD1− cells stably transfected with empty mock vector, pCMV-Myc-UBIAD1, or pCMV-Myc-UBIAD1 (N102S) were briefly incubated with 25-HC in the absence or presence of geranylgeraniol. The cells were then harvested, lysed, and subjected to anti-reductase immunoprecipitation. The results show that wild type UBIAD1 co-precipitated with reductase in the 25-HC-treated cells (Figure 8A, second panel, lane e). This co-precipitation was inhibited by geranylgeraniol (lanes g and h). UBIAD1 (N102S) similarly co-precipitated with reductase (lane i); however, the mutant UBAID1 resisted geranylgeraniol-mediated displacement and remained associated with reductase (lanes j–l). UBIAD1 (N102S) exhibited a similar resistance to geranylgeraniol-mediated displacement from reductase in two independent experiments shown in Figure 8—figure supplement 1.10.7554/eLife.05560.016Figure 8.SCD-associated UBIAD1 mutant resists geranylgeraniol-mediated displacement from HMG CoA reductase and remains sequestered in ER membranes.

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