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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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Geranylgeraniol, but not 25-HC, FOH, or cholesterol, stimulates translocation of endogenous UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.(A) SV-589 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining as described in the legend to Figure 5. Coverslips were mounted using Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Images were obtained with a Zeiss Axio Observer Epifluorescence microscope using a 63× oil Plan-Apochromat objective and Ziess Axiocam color digital camera in black and white mode. For the purpose of presentation, brightness levels were adjusted across the entire image using ImageJ software (National Institution of Health, USA). (B) SV-589 cells were set up on day 0 at 3 × 104 cells/well of a twelve-well plate with glass coverslips. On day 1, the cells were transfected using FuGENE6 with 200 ng DsRed-Golgi (Clontech Laboratories, Inc., Mountain View, CA); the total amount of DNA/well was adjusted to 500 ng by the addition of empty pcDNA3.1 vector. 4 hr after transfection, cells received a direct addition of medium A containing 10% FCS or 10% NC-LPPS supplemented with 10 µM compactin and 50 µM mevalonate (final concentrations) as indicated. Following incubation for 16 hr at 37°C, cells were treated for 4 hr in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol. Cells were subsequently fixed and subjected to immunostaining and imaging as described in (A). Shown are cropped images representing a 64 × 64 micron-portion of the original images 219 × 174 microns in size.DOI:http://dx.doi.org/10.7554/eLife.05560.010
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fig5s2: Geranylgeraniol, but not 25-HC, FOH, or cholesterol, stimulates translocation of endogenous UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.(A) SV-589 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining as described in the legend to Figure 5. Coverslips were mounted using Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Images were obtained with a Zeiss Axio Observer Epifluorescence microscope using a 63× oil Plan-Apochromat objective and Ziess Axiocam color digital camera in black and white mode. For the purpose of presentation, brightness levels were adjusted across the entire image using ImageJ software (National Institution of Health, USA). (B) SV-589 cells were set up on day 0 at 3 × 104 cells/well of a twelve-well plate with glass coverslips. On day 1, the cells were transfected using FuGENE6 with 200 ng DsRed-Golgi (Clontech Laboratories, Inc., Mountain View, CA); the total amount of DNA/well was adjusted to 500 ng by the addition of empty pcDNA3.1 vector. 4 hr after transfection, cells received a direct addition of medium A containing 10% FCS or 10% NC-LPPS supplemented with 10 µM compactin and 50 µM mevalonate (final concentrations) as indicated. Following incubation for 16 hr at 37°C, cells were treated for 4 hr in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol. Cells were subsequently fixed and subjected to immunostaining and imaging as described in (A). Shown are cropped images representing a 64 × 64 micron-portion of the original images 219 × 174 microns in size.DOI:http://dx.doi.org/10.7554/eLife.05560.010

Mentions: SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 2 µg/dish of pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent as described in ‘Materials and methods’. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium as added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescense using IgG-9E10 against the Myc epitope. For experiments, SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol, 30 µM farnesol, or 1 µg/ml 25-HC for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining and analysis as described in the legend to Figure 5—figure supplement 2.


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)

Geranylgeraniol, but not 25-HC, FOH, or cholesterol, stimulates translocation of endogenous UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.(A) SV-589 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining as described in the legend to Figure 5. Coverslips were mounted using Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Images were obtained with a Zeiss Axio Observer Epifluorescence microscope using a 63× oil Plan-Apochromat objective and Ziess Axiocam color digital camera in black and white mode. For the purpose of presentation, brightness levels were adjusted across the entire image using ImageJ software (National Institution of Health, USA). (B) SV-589 cells were set up on day 0 at 3 × 104 cells/well of a twelve-well plate with glass coverslips. On day 1, the cells were transfected using FuGENE6 with 200 ng DsRed-Golgi (Clontech Laboratories, Inc., Mountain View, CA); the total amount of DNA/well was adjusted to 500 ng by the addition of empty pcDNA3.1 vector. 4 hr after transfection, cells received a direct addition of medium A containing 10% FCS or 10% NC-LPPS supplemented with 10 µM compactin and 50 µM mevalonate (final concentrations) as indicated. Following incubation for 16 hr at 37°C, cells were treated for 4 hr in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol. Cells were subsequently fixed and subjected to immunostaining and imaging as described in (A). Shown are cropped images representing a 64 × 64 micron-portion of the original images 219 × 174 microns in size.DOI:http://dx.doi.org/10.7554/eLife.05560.010
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fig5s2: Geranylgeraniol, but not 25-HC, FOH, or cholesterol, stimulates translocation of endogenous UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids.(A) SV-589 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining as described in the legend to Figure 5. Coverslips were mounted using Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Images were obtained with a Zeiss Axio Observer Epifluorescence microscope using a 63× oil Plan-Apochromat objective and Ziess Axiocam color digital camera in black and white mode. For the purpose of presentation, brightness levels were adjusted across the entire image using ImageJ software (National Institution of Health, USA). (B) SV-589 cells were set up on day 0 at 3 × 104 cells/well of a twelve-well plate with glass coverslips. On day 1, the cells were transfected using FuGENE6 with 200 ng DsRed-Golgi (Clontech Laboratories, Inc., Mountain View, CA); the total amount of DNA/well was adjusted to 500 ng by the addition of empty pcDNA3.1 vector. 4 hr after transfection, cells received a direct addition of medium A containing 10% FCS or 10% NC-LPPS supplemented with 10 µM compactin and 50 µM mevalonate (final concentrations) as indicated. Following incubation for 16 hr at 37°C, cells were treated for 4 hr in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol. Cells were subsequently fixed and subjected to immunostaining and imaging as described in (A). Shown are cropped images representing a 64 × 64 micron-portion of the original images 219 × 174 microns in size.DOI:http://dx.doi.org/10.7554/eLife.05560.010
Mentions: SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 105 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 2 µg/dish of pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent as described in ‘Materials and methods’. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium as added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescense using IgG-9E10 against the Myc epitope. For experiments, SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol, 30 µM farnesol, or 1 µg/ml 25-HC for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining and analysis as described in the legend to Figure 5—figure supplement 2.

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