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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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Specificity of sterol-dependent UBIAD1-HMG CoA reductase association.SV-589 cells were set up on day 0 at 1 × 105 cells per 100-mm dish in medium A containing 10% FCS. On day 3, cells were transfected with siRNAs targeting mRNAs encoding GFP, Insig-1 and Insig-2, UBIAD1, or reductase as indicated and described in ‘Materials and methods’. Cells transfected with siRNA duplexes against reductase received 200 mM mevalonate to provide essential nonsterol isoprenoids. On day 4, cells were depleted of sterols through incubation for 16 hr at 37°C in medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate. The cells then received identical medium in the absence or presence of 1 µg/ml 25-HC. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against either reductase (A and B) or UBIAD1 (C). The resulting precipitated material and lysates were subjected to SDS-PAGE and 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.05560.007
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fig4: Specificity of sterol-dependent UBIAD1-HMG CoA reductase association.SV-589 cells were set up on day 0 at 1 × 105 cells per 100-mm dish in medium A containing 10% FCS. On day 3, cells were transfected with siRNAs targeting mRNAs encoding GFP, Insig-1 and Insig-2, UBIAD1, or reductase as indicated and described in ‘Materials and methods’. Cells transfected with siRNA duplexes against reductase received 200 mM mevalonate to provide essential nonsterol isoprenoids. On day 4, cells were depleted of sterols through incubation for 16 hr at 37°C in medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate. The cells then received identical medium in the absence or presence of 1 µg/ml 25-HC. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against either reductase (A and B) or UBIAD1 (C). The resulting precipitated material and lysates were subjected to SDS-PAGE and 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.05560.007

Mentions: We next used RNA interference (RNAi) to examine a role for Insigs in the 25-HC-induced binding of reductase to UBIAD1. SV-589 cells were transfected with duplexes of small interfering RNAs (siRNAs) against mRNAs encoding green fluorescent protein (GFP), which is not expressed in the cells, or Insig-1 and Insig-2. Following transfection, the cells were depleted of sterols, incubated in the absence or presence of 25-HC, and lysed for subsequent immunoprecipitation with polyclonal anti-reductase. Immunoblotting of precipitated proteins revealed that 25-HC stimulated co-precipitation of both UBIAD1 and Insig-1 with reductase in SV-589 cells transfected with control GFP siRNA (Figure 4A, second and third panels, lane 2). The sterol-induced co-precipitation of UBIAD1 with reductase was markedly reduced in cells that received siRNAs against Insig-1 and Insig-2 (second panel, lanes 3 and 4). The experiment of Figure 4B shows that knockdown of UBIAD1 reduced, but did not eliminate the sterol-induced binding of reductase to Insig-1 (third panel, compare lanes 2 and 4). Finally, we examined the requirement of reductase for the association of UBIAD1 with Insig-1. Immunoblot analysis of anti-UBIAD1 immunoprecipitates revealed that as expected, reductase and Insig-1 co-precipitated with UBIAD1 when control siRNA-transfected cells were treated with 25-HC (Figure 4C, top and bottom panels, lane 2). However, RNAi-mediated knockdown of reductase abolished co-precipitation of Insig-1 with UBIAD1 (bottom panel, compare lane 4 with lane 2).10.7554/eLife.05560.007Figure 4.Specificity of sterol-dependent UBIAD1-HMG CoA reductase association.


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)

Specificity of sterol-dependent UBIAD1-HMG CoA reductase association.SV-589 cells were set up on day 0 at 1 × 105 cells per 100-mm dish in medium A containing 10% FCS. On day 3, cells were transfected with siRNAs targeting mRNAs encoding GFP, Insig-1 and Insig-2, UBIAD1, or reductase as indicated and described in ‘Materials and methods’. Cells transfected with siRNA duplexes against reductase received 200 mM mevalonate to provide essential nonsterol isoprenoids. On day 4, cells were depleted of sterols through incubation for 16 hr at 37°C in medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate. The cells then received identical medium in the absence or presence of 1 µg/ml 25-HC. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against either reductase (A and B) or UBIAD1 (C). The resulting precipitated material and lysates were subjected to SDS-PAGE and 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.05560.007
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Specificity of sterol-dependent UBIAD1-HMG CoA reductase association.SV-589 cells were set up on day 0 at 1 × 105 cells per 100-mm dish in medium A containing 10% FCS. On day 3, cells were transfected with siRNAs targeting mRNAs encoding GFP, Insig-1 and Insig-2, UBIAD1, or reductase as indicated and described in ‘Materials and methods’. Cells transfected with siRNA duplexes against reductase received 200 mM mevalonate to provide essential nonsterol isoprenoids. On day 4, cells were depleted of sterols through incubation for 16 hr at 37°C in medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate. The cells then received identical medium in the absence or presence of 1 µg/ml 25-HC. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against either reductase (A and B) or UBIAD1 (C). The resulting precipitated material and lysates were subjected to SDS-PAGE and 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.05560.007
Mentions: We next used RNA interference (RNAi) to examine a role for Insigs in the 25-HC-induced binding of reductase to UBIAD1. SV-589 cells were transfected with duplexes of small interfering RNAs (siRNAs) against mRNAs encoding green fluorescent protein (GFP), which is not expressed in the cells, or Insig-1 and Insig-2. Following transfection, the cells were depleted of sterols, incubated in the absence or presence of 25-HC, and lysed for subsequent immunoprecipitation with polyclonal anti-reductase. Immunoblotting of precipitated proteins revealed that 25-HC stimulated co-precipitation of both UBIAD1 and Insig-1 with reductase in SV-589 cells transfected with control GFP siRNA (Figure 4A, second and third panels, lane 2). The sterol-induced co-precipitation of UBIAD1 with reductase was markedly reduced in cells that received siRNAs against Insig-1 and Insig-2 (second panel, lanes 3 and 4). The experiment of Figure 4B shows that knockdown of UBIAD1 reduced, but did not eliminate the sterol-induced binding of reductase to Insig-1 (third panel, compare lanes 2 and 4). Finally, we examined the requirement of reductase for the association of UBIAD1 with Insig-1. Immunoblot analysis of anti-UBIAD1 immunoprecipitates revealed that as expected, reductase and Insig-1 co-precipitated with UBIAD1 when control siRNA-transfected cells were treated with 25-HC (Figure 4C, top and bottom panels, lane 2). However, RNAi-mediated knockdown of reductase abolished co-precipitation of Insig-1 with UBIAD1 (bottom panel, compare lane 4 with lane 2).10.7554/eLife.05560.007Figure 4.Specificity of sterol-dependent UBIAD1-HMG CoA reductase association.

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