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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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Identification of proteins associated with HMG-Red(TM1-8)-BirA*.(A) HEK-293S/pHMG-Red(TM1-8)-BirA* cells were set up on day 0, depleted of sterols on day 3, harvested on day 4 for lysis and affinity purification using streptavidin-coupled beads as described in ‘Materials and methods’. Precipitated proteins were subjected to SDS-PAGE and the gel was subjected to staining with colloidal blue. Three segments of the gel (indicated by boxes) were excised and the identities of the proteins were determined by tandem mass spectrometry. The spectral count for the most abundant proteins identified in each segment is indicated in parentheses. (B) SV-589 cells were set up on day 0, depleted of sterols on day 3, and subjected to treatment in the absence or presence of 25-HC (1 µg/ml) for 45 min at 37°C on day 4 as described in the legend to Figure 3. Cells were then harvested, lysed in PBS containing 1% digitonin, and the resulting lysates were subjected to immunoprecipitation with polyclonal anti-reductase antibodies as described in ‘Materials and methods’. Aliquots of precipitated material (IP Pellet) and lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with antibodies against the indicated protein.DOI:http://dx.doi.org/10.7554/eLife.05560.006
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fig3s1: Identification of proteins associated with HMG-Red(TM1-8)-BirA*.(A) HEK-293S/pHMG-Red(TM1-8)-BirA* cells were set up on day 0, depleted of sterols on day 3, harvested on day 4 for lysis and affinity purification using streptavidin-coupled beads as described in ‘Materials and methods’. Precipitated proteins were subjected to SDS-PAGE and the gel was subjected to staining with colloidal blue. Three segments of the gel (indicated by boxes) were excised and the identities of the proteins were determined by tandem mass spectrometry. The spectral count for the most abundant proteins identified in each segment is indicated in parentheses. (B) SV-589 cells were set up on day 0, depleted of sterols on day 3, and subjected to treatment in the absence or presence of 25-HC (1 µg/ml) for 45 min at 37°C on day 4 as described in the legend to Figure 3. Cells were then harvested, lysed in PBS containing 1% digitonin, and the resulting lysates were subjected to immunoprecipitation with polyclonal anti-reductase antibodies as described in ‘Materials and methods’. Aliquots of precipitated material (IP Pellet) and lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with antibodies against the indicated protein.DOI:http://dx.doi.org/10.7554/eLife.05560.006

Mentions: Figure 3—figure supplement 1B shows an experiment in which we used co-immunoprecipitation to measure the interaction of endogenous reductase with several proteins that were identified as associated with the reductase-BirA* chimera (Figure 3—figure supplement 1A). SV-589 cells, a line of transformed human fibroblasts (Yamamoto et al., 1984), were depleted of sterols and subsequently treated in the absence or presence of 25-HC. Following treatments, the cells were harvested for lysis in detergent-containing buffer; the resulting lysates were then immunoprecipitated with polyclonal antibodies against reductase. Immunoblot analysis revealed that treatment of cells with 25-HC triggered co-immunoprecipitation of endogenous reductase with Insig-1 (Figure 3—figure supplement 1B, lanes 3 and 4); the control ER membrane protein calnexin failed to interact with reductase, regardless of the absence or presence of 25-HC (lanes 5 and 6). ERGIC-53 similarly failed to appear in the anti-reductase immunoprecipitates (lanes 9 and 10). In contrast, lamina-associated peptide-2 (lanes 7 and 8), peroxiredoxin-4 (lanes 11 and 12), PGRMC2 (lanes 13 and 14), and annexin-A1 (lanes 15 and 16) co-precipitated with reductase in both the absence and presence of 25-HC; the significance and specificity of these interactions are unclear. In the absence of 25-HC, a small amount of UBIAD1 (UbiA prenyltransferase domain-containing protein-1) appeared in the pellet fraction of the reductase immunoprecipitation (lane 17); this appearance was markedly enhanced when the cells were subjected to treatment with 25-HC (lane 18).


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)

Identification of proteins associated with HMG-Red(TM1-8)-BirA*.(A) HEK-293S/pHMG-Red(TM1-8)-BirA* cells were set up on day 0, depleted of sterols on day 3, harvested on day 4 for lysis and affinity purification using streptavidin-coupled beads as described in ‘Materials and methods’. Precipitated proteins were subjected to SDS-PAGE and the gel was subjected to staining with colloidal blue. Three segments of the gel (indicated by boxes) were excised and the identities of the proteins were determined by tandem mass spectrometry. The spectral count for the most abundant proteins identified in each segment is indicated in parentheses. (B) SV-589 cells were set up on day 0, depleted of sterols on day 3, and subjected to treatment in the absence or presence of 25-HC (1 µg/ml) for 45 min at 37°C on day 4 as described in the legend to Figure 3. Cells were then harvested, lysed in PBS containing 1% digitonin, and the resulting lysates were subjected to immunoprecipitation with polyclonal anti-reductase antibodies as described in ‘Materials and methods’. Aliquots of precipitated material (IP Pellet) and lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with antibodies against the indicated protein.DOI:http://dx.doi.org/10.7554/eLife.05560.006
© Copyright Policy
Related In: Results  -  Collection

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

fig3s1: Identification of proteins associated with HMG-Red(TM1-8)-BirA*.(A) HEK-293S/pHMG-Red(TM1-8)-BirA* cells were set up on day 0, depleted of sterols on day 3, harvested on day 4 for lysis and affinity purification using streptavidin-coupled beads as described in ‘Materials and methods’. Precipitated proteins were subjected to SDS-PAGE and the gel was subjected to staining with colloidal blue. Three segments of the gel (indicated by boxes) were excised and the identities of the proteins were determined by tandem mass spectrometry. The spectral count for the most abundant proteins identified in each segment is indicated in parentheses. (B) SV-589 cells were set up on day 0, depleted of sterols on day 3, and subjected to treatment in the absence or presence of 25-HC (1 µg/ml) for 45 min at 37°C on day 4 as described in the legend to Figure 3. Cells were then harvested, lysed in PBS containing 1% digitonin, and the resulting lysates were subjected to immunoprecipitation with polyclonal anti-reductase antibodies as described in ‘Materials and methods’. Aliquots of precipitated material (IP Pellet) and lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with antibodies against the indicated protein.DOI:http://dx.doi.org/10.7554/eLife.05560.006
Mentions: Figure 3—figure supplement 1B shows an experiment in which we used co-immunoprecipitation to measure the interaction of endogenous reductase with several proteins that were identified as associated with the reductase-BirA* chimera (Figure 3—figure supplement 1A). SV-589 cells, a line of transformed human fibroblasts (Yamamoto et al., 1984), were depleted of sterols and subsequently treated in the absence or presence of 25-HC. Following treatments, the cells were harvested for lysis in detergent-containing buffer; the resulting lysates were then immunoprecipitated with polyclonal antibodies against reductase. Immunoblot analysis revealed that treatment of cells with 25-HC triggered co-immunoprecipitation of endogenous reductase with Insig-1 (Figure 3—figure supplement 1B, lanes 3 and 4); the control ER membrane protein calnexin failed to interact with reductase, regardless of the absence or presence of 25-HC (lanes 5 and 6). ERGIC-53 similarly failed to appear in the anti-reductase immunoprecipitates (lanes 9 and 10). In contrast, lamina-associated peptide-2 (lanes 7 and 8), peroxiredoxin-4 (lanes 11 and 12), PGRMC2 (lanes 13 and 14), and annexin-A1 (lanes 15 and 16) co-precipitated with reductase in both the absence and presence of 25-HC; the significance and specificity of these interactions are unclear. In the absence of 25-HC, a small amount of UBIAD1 (UbiA prenyltransferase domain-containing protein-1) appeared in the pellet fraction of the reductase immunoprecipitation (lane 17); this appearance was markedly enhanced when the cells were subjected to treatment with 25-HC (lane 18).

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