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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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Schnyder corneal dystrophy (SCD)-associated N102S mutant of UBIAD1 blocks sterol-accelerated ERAD of full-length HMG CoA reductase.SV-589 cells were set up for experiments on day 0 at 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 1, cells were transfected with 3 µg/dish of pCMV-HMG-Red-T7 in the absence or presence of the indicated concentration of wild type (A) or N102S (B) versions of pCMV-Myc-UBIAD1 as described in ‘Materials and methods’. 4 hr after transfection, cells received a direct addition of medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate (final concentrations). Following incubation for 16 hr at 37°C, cells were treated with identical medium in the absence or presence of 1 µg/ml 25-HC plus 20 µM geranylgeraniol (GGOH) as indicated. After 4 hr at 37°C, cells were harvested and subjected to subcellular fractionation. Aliquots of resulting membrane fractions were then subjected to SDS-PAGE and immunoblot analysis was carried out with anti-T7 IgG (against reductase), IgG-9E10 (against UBIAD1 and Insgi-1), and anti-calnexin IgG. Asterisks denote a non-specific cross-reactive band. Proteins corresponding to reductase in (A and B) were quantified using ImageJ software. The intensities of these signals in the absence of 25-HC plus geranylgeraniol were arbitrarily set as 1.DOI:http://dx.doi.org/10.7554/eLife.05560.015
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fig7s1: Schnyder corneal dystrophy (SCD)-associated N102S mutant of UBIAD1 blocks sterol-accelerated ERAD of full-length HMG CoA reductase.SV-589 cells were set up for experiments on day 0 at 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 1, cells were transfected with 3 µg/dish of pCMV-HMG-Red-T7 in the absence or presence of the indicated concentration of wild type (A) or N102S (B) versions of pCMV-Myc-UBIAD1 as described in ‘Materials and methods’. 4 hr after transfection, cells received a direct addition of medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate (final concentrations). Following incubation for 16 hr at 37°C, cells were treated with identical medium in the absence or presence of 1 µg/ml 25-HC plus 20 µM geranylgeraniol (GGOH) as indicated. After 4 hr at 37°C, cells were harvested and subjected to subcellular fractionation. Aliquots of resulting membrane fractions were then subjected to SDS-PAGE and immunoblot analysis was carried out with anti-T7 IgG (against reductase), IgG-9E10 (against UBIAD1 and Insgi-1), and anti-calnexin IgG. Asterisks denote a non-specific cross-reactive band. Proteins corresponding to reductase in (A and B) were quantified using ImageJ software. The intensities of these signals in the absence of 25-HC plus geranylgeraniol were arbitrarily set as 1.DOI:http://dx.doi.org/10.7554/eLife.05560.015

Mentions: To date, 24 mutations in the UBIAD1 gene have been identified in SCD families (Nickerson et al., 2013; Nowinska et al., 2014); these mutations result in the alteration of 20 amino acid residues in UBAID1 (Figure 3A). Asparagine-102 (N102), which localizes to the prenyltransferase domain of UBIAD1 (Cheng and Li, 2014; Huang et al., 2014), is the most frequently altered residue (∼30%) in SCD families. Considering that SCD is an autosomal dominant disorder, we reasoned that overexpression of UBIAD1 harboring the SCD-associated N102S mutation would block reductase ERAD. Figure 7A shows that when overexpressed in SV-589 cells, the T7-tagged membrane domain of reductase became degraded in the presence of 25-HC and geranylgeraniol (top panel, lanes 1 and 2). Co-expression of a plasmid encoding wild type UBIAD1 stabilized reductase in a dose-dependent fashion and 25-HC plus geranylgeraniol continued to accelerate its ERAD (lanes 3–10). Notably, the highest level of wild type UBIAD1 co-expressed with reductase blunted the enzyme's accelerated degradation (lanes 11 and 12). Co-expression of UBIAD1 (N102S) also stabilized the membrane domain of reductase (Figure 7B, top panel, compare lane 1 with lanes 3, 5, 7, 9, and 11), suggesting the protein continued to bind to the enzyme. However, UBIAD1 (N102S) blunted sterol-accelerated ERAD of reductase, even at low levels of expression (Figure 7B, top panel, compare lane 2 with lanes 4, 8, 10, and 12). Similar results were obtained with T7-tagged, full-length reductase (Figure 7—figure supplement 1). The protein was subjected to sterol-accelerated degradation when transfected alone or together with up to 100 ng of plasmid encoding wild type UBIAD1 (Figure 7—figure supplement 1A, top panel, lanes 1–8); co-transfection of higher levels (300 and 1000 ng) of the UBIAD1-encoding plasmid inhibited reductase degradation (lanes 9–12). Consistent with results obtained with the reductase membrane domain, inhibition of full-length reductase degradation was observed upon co-transfection with a significantly lower amount (30 ng) of plasmid encoding UBIAD1 (N102S) (Figure 7—figure supplement 1B, top panel, compare lanes 1–4 with lanes 5–12). Glycine-177 (G177, see Figure 3A) is the second-most frequently altered UBIAD1 residue in SCD families (Nickerson et al., 2013). The results of Figure 7C show that UBAID1 harboring the SCD-associated G177R mutation blocked reductase ERAD to a degree similar to that observed with UBIAD1 (N102S) (top panel, compare lanes 1 and 2 with lanes 3–6).10.7554/eLife.05560.014Figure 7.The Schnyder corneal dystrophy (SCD)-associated N102S and G177R mutants of UBIAD1 block sterol-accelerated ERAD 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)

Schnyder corneal dystrophy (SCD)-associated N102S mutant of UBIAD1 blocks sterol-accelerated ERAD of full-length HMG CoA reductase.SV-589 cells were set up for experiments on day 0 at 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 1, cells were transfected with 3 µg/dish of pCMV-HMG-Red-T7 in the absence or presence of the indicated concentration of wild type (A) or N102S (B) versions of pCMV-Myc-UBIAD1 as described in ‘Materials and methods’. 4 hr after transfection, cells received a direct addition of medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate (final concentrations). Following incubation for 16 hr at 37°C, cells were treated with identical medium in the absence or presence of 1 µg/ml 25-HC plus 20 µM geranylgeraniol (GGOH) as indicated. After 4 hr at 37°C, cells were harvested and subjected to subcellular fractionation. Aliquots of resulting membrane fractions were then subjected to SDS-PAGE and immunoblot analysis was carried out with anti-T7 IgG (against reductase), IgG-9E10 (against UBIAD1 and Insgi-1), and anti-calnexin IgG. Asterisks denote a non-specific cross-reactive band. Proteins corresponding to reductase in (A and B) were quantified using ImageJ software. The intensities of these signals in the absence of 25-HC plus geranylgeraniol were arbitrarily set as 1.DOI:http://dx.doi.org/10.7554/eLife.05560.015
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Related In: Results  -  Collection

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fig7s1: Schnyder corneal dystrophy (SCD)-associated N102S mutant of UBIAD1 blocks sterol-accelerated ERAD of full-length HMG CoA reductase.SV-589 cells were set up for experiments on day 0 at 4 × 105 cells per 60-mm dish in medium A containing 10% FCS. On day 1, cells were transfected with 3 µg/dish of pCMV-HMG-Red-T7 in the absence or presence of the indicated concentration of wild type (A) or N102S (B) versions of pCMV-Myc-UBIAD1 as described in ‘Materials and methods’. 4 hr after transfection, cells received a direct addition of medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate (final concentrations). Following incubation for 16 hr at 37°C, cells were treated with identical medium in the absence or presence of 1 µg/ml 25-HC plus 20 µM geranylgeraniol (GGOH) as indicated. After 4 hr at 37°C, cells were harvested and subjected to subcellular fractionation. Aliquots of resulting membrane fractions were then subjected to SDS-PAGE and immunoblot analysis was carried out with anti-T7 IgG (against reductase), IgG-9E10 (against UBIAD1 and Insgi-1), and anti-calnexin IgG. Asterisks denote a non-specific cross-reactive band. Proteins corresponding to reductase in (A and B) were quantified using ImageJ software. The intensities of these signals in the absence of 25-HC plus geranylgeraniol were arbitrarily set as 1.DOI:http://dx.doi.org/10.7554/eLife.05560.015
Mentions: To date, 24 mutations in the UBIAD1 gene have been identified in SCD families (Nickerson et al., 2013; Nowinska et al., 2014); these mutations result in the alteration of 20 amino acid residues in UBAID1 (Figure 3A). Asparagine-102 (N102), which localizes to the prenyltransferase domain of UBIAD1 (Cheng and Li, 2014; Huang et al., 2014), is the most frequently altered residue (∼30%) in SCD families. Considering that SCD is an autosomal dominant disorder, we reasoned that overexpression of UBIAD1 harboring the SCD-associated N102S mutation would block reductase ERAD. Figure 7A shows that when overexpressed in SV-589 cells, the T7-tagged membrane domain of reductase became degraded in the presence of 25-HC and geranylgeraniol (top panel, lanes 1 and 2). Co-expression of a plasmid encoding wild type UBIAD1 stabilized reductase in a dose-dependent fashion and 25-HC plus geranylgeraniol continued to accelerate its ERAD (lanes 3–10). Notably, the highest level of wild type UBIAD1 co-expressed with reductase blunted the enzyme's accelerated degradation (lanes 11 and 12). Co-expression of UBIAD1 (N102S) also stabilized the membrane domain of reductase (Figure 7B, top panel, compare lane 1 with lanes 3, 5, 7, 9, and 11), suggesting the protein continued to bind to the enzyme. However, UBIAD1 (N102S) blunted sterol-accelerated ERAD of reductase, even at low levels of expression (Figure 7B, top panel, compare lane 2 with lanes 4, 8, 10, and 12). Similar results were obtained with T7-tagged, full-length reductase (Figure 7—figure supplement 1). The protein was subjected to sterol-accelerated degradation when transfected alone or together with up to 100 ng of plasmid encoding wild type UBIAD1 (Figure 7—figure supplement 1A, top panel, lanes 1–8); co-transfection of higher levels (300 and 1000 ng) of the UBIAD1-encoding plasmid inhibited reductase degradation (lanes 9–12). Consistent with results obtained with the reductase membrane domain, inhibition of full-length reductase degradation was observed upon co-transfection with a significantly lower amount (30 ng) of plasmid encoding UBIAD1 (N102S) (Figure 7—figure supplement 1B, top panel, compare lanes 1–4 with lanes 5–12). Glycine-177 (G177, see Figure 3A) is the second-most frequently altered UBIAD1 residue in SCD families (Nickerson et al., 2013). The results of Figure 7C show that UBAID1 harboring the SCD-associated G177R mutation blocked reductase ERAD to a degree similar to that observed with UBIAD1 (N102S) (top panel, compare lanes 1 and 2 with lanes 3–6).10.7554/eLife.05560.014Figure 7.The Schnyder corneal dystrophy (SCD)-associated N102S and G177R mutants of UBIAD1 block sterol-accelerated ERAD 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