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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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The proteasome inhibitor MG-132 and geranylgeraniol inhibit sterol-induced binding of UBIAD1 to HMG CoA reductase.SV-589 cells were set up for experiments on day 0 and depleted of sterols on day 3 as described in the legend to Figure 3. Following sterol-depletion, cells received medium A containing 10% NC-LPPS, 10 µM compactin, 50 µM mevalonate in the absence or presence of 10 µM MG-132 for 1 hr at 37°C, followed by treatment in the absence or presence of 1 µg/ml 25-HC (A) or the indicated concentration of 25-HC in the absence or presence of 15 µM geranylgeraniol (B). Following incubation for 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against reductase. Aliquots of the resulting immunoprecipitates and lysates were subjected to immunoblot analysis with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), IgG-17H1 (against Insig-1), and anti-calnexin IgG.DOI:http://dx.doi.org/10.7554/eLife.05560.009
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fig5s1: The proteasome inhibitor MG-132 and geranylgeraniol inhibit sterol-induced binding of UBIAD1 to HMG CoA reductase.SV-589 cells were set up for experiments on day 0 and depleted of sterols on day 3 as described in the legend to Figure 3. Following sterol-depletion, cells received medium A containing 10% NC-LPPS, 10 µM compactin, 50 µM mevalonate in the absence or presence of 10 µM MG-132 for 1 hr at 37°C, followed by treatment in the absence or presence of 1 µg/ml 25-HC (A) or the indicated concentration of 25-HC in the absence or presence of 15 µM geranylgeraniol (B). Following incubation for 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against reductase. Aliquots of the resulting immunoprecipitates and lysates were subjected to immunoblot analysis with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), IgG-17H1 (against Insig-1), and anti-calnexin IgG.DOI:http://dx.doi.org/10.7554/eLife.05560.009

Mentions: Results of Figure 3B show that UBIAD1 continued to appear in anti-reductase immunoprecipitates after prolonged treatment with 25-HC, even though total reductase was reduced by accelerated degradation. We reasoned that the proteasome inhibitor MG-132 would block this degradation, leading to increased co-immunoprecipitation of UBIAD1 with stabilized reductase. Immunoblot analysis of anti-reductase immunoprecipitates from lysates of cells treated in the absence of MG-132 shows that 25-HC stimulated reductase degradation (Figure 5—figure supplement 1A, top panel, compare lanes 1 and 2), which was blocked by MG-132 (lanes 3 and 4). Insig-1 co-precipitated with reductase in the presence of 25-HC (second panel, lane 2) and this co-precipitation was enhanced by MG-132 (lane 4). UBIAD1 also co-precipitated with reductase in 25-HC-treated cells (third panel, lane 2); however, the interaction was reduced, rather than enhanced, in the presence of MG-132 (lane 4).


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)

The proteasome inhibitor MG-132 and geranylgeraniol inhibit sterol-induced binding of UBIAD1 to HMG CoA reductase.SV-589 cells were set up for experiments on day 0 and depleted of sterols on day 3 as described in the legend to Figure 3. Following sterol-depletion, cells received medium A containing 10% NC-LPPS, 10 µM compactin, 50 µM mevalonate in the absence or presence of 10 µM MG-132 for 1 hr at 37°C, followed by treatment in the absence or presence of 1 µg/ml 25-HC (A) or the indicated concentration of 25-HC in the absence or presence of 15 µM geranylgeraniol (B). Following incubation for 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against reductase. Aliquots of the resulting immunoprecipitates and lysates were subjected to immunoblot analysis with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), IgG-17H1 (against Insig-1), and anti-calnexin IgG.DOI:http://dx.doi.org/10.7554/eLife.05560.009
© Copyright Policy
Related In: Results  -  Collection

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

fig5s1: The proteasome inhibitor MG-132 and geranylgeraniol inhibit sterol-induced binding of UBIAD1 to HMG CoA reductase.SV-589 cells were set up for experiments on day 0 and depleted of sterols on day 3 as described in the legend to Figure 3. Following sterol-depletion, cells received medium A containing 10% NC-LPPS, 10 µM compactin, 50 µM mevalonate in the absence or presence of 10 µM MG-132 for 1 hr at 37°C, followed by treatment in the absence or presence of 1 µg/ml 25-HC (A) or the indicated concentration of 25-HC in the absence or presence of 15 µM geranylgeraniol (B). Following incubation for 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against reductase. Aliquots of the resulting immunoprecipitates and lysates were subjected to immunoblot analysis with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), IgG-17H1 (against Insig-1), and anti-calnexin IgG.DOI:http://dx.doi.org/10.7554/eLife.05560.009
Mentions: Results of Figure 3B show that UBIAD1 continued to appear in anti-reductase immunoprecipitates after prolonged treatment with 25-HC, even though total reductase was reduced by accelerated degradation. We reasoned that the proteasome inhibitor MG-132 would block this degradation, leading to increased co-immunoprecipitation of UBIAD1 with stabilized reductase. Immunoblot analysis of anti-reductase immunoprecipitates from lysates of cells treated in the absence of MG-132 shows that 25-HC stimulated reductase degradation (Figure 5—figure supplement 1A, top panel, compare lanes 1 and 2), which was blocked by MG-132 (lanes 3 and 4). Insig-1 co-precipitated with reductase in the presence of 25-HC (second panel, lane 2) and this co-precipitation was enhanced by MG-132 (lane 4). UBIAD1 also co-precipitated with reductase in 25-HC-treated cells (third panel, lane 2); however, the interaction was reduced, rather than enhanced, in the presence of MG-132 (lane 4).

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