Abstract

UBIAD1 (UbiA prenyltransferase domain-containing protein-1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K2 We previously reported that sterols stimulate binding of UBIAD1 to endoplasmic reticulum (ER)-localized 3-hydroxy-3-methylglutaryl (HMG) CoA reductase. UBIAD1 binding inhibits sterol-accelerated, ER-associated degradation (ERAD) of reductase, one of several mechanisms for feedback control of this rate-limiting enzyme in the branched pathway that produces cholesterol and nonsterol isoprenoids such as GGpp. Accumulation of GGpp in ER membranes triggers release of UBIAD1 from reductase, permitting its maximal ERAD and ER-to-Golgi transport of UBIAD1. Mutant UBIAD1 variants associated with Schnyder corneal dystrophy (SCD), a human disorder characterized by corneal accumulation of cholesterol, resist GGpp-induced release from reductase and remain sequestered in the ER to block reductase ERAD. Using lines of genetically manipulated cells, we now examine consequences of UBIAD1 deficiency and SCD-associated UBIAD1 on reductase ERAD and cholesterol synthesis. Our results indicated that reductase becomes destabilized in the absence of UBIAD1, resulting in reduced cholesterol synthesis and intracellular accumulation. In contrast, an SCD-associated UBIAD1 variant inhibited reductase ERAD, thereby stabilizing the enzyme and contributing to enhanced synthesis and intracellular accumulation of cholesterol. Finally, we present evidence that GGpp-regulated, ER-to-Golgi transport enables UBIAD1 to modulate reductase ERAD such that synthesis of nonsterol isoprenoids is maintained in sterol-replete cells. These findings further establish UBIAD1 as a central player in the reductase ERAD pathway and regulation of isoprenoid synthesis. They also indicate that UBIAD1-mediated inhibition of reductase ERAD underlies cholesterol accumulation associated with SCD.

Highlights

  • A marked accumulation of reductase was observed in SV-589 (⌬UBIAD1)/pMyc-UBIAD1 (N102S) cells, which express the Schnyder corneal dystrophy (SCD)-associated variant of UBIAD1 that harbors substitution of serine for asparagine-102 (N102S)

  • Considering these results together with the previous finding that overexpression of UBIAD1 (N102S) and other SCD-associated UBIAD1 variants blocks ER-associated degradation (ERAD) of endogenous reductase (16), we conclude that changes in levels of reductase protein observed here were attributable to modulation of its sterol-accelerated ERAD

  • Expression of SCD-associated UBIAD1 (N102S) led to the overaccumulation of neutral lipids (Fig. 4, C and D), because of the inhibition of reductase ERAD (Fig. 1A) that led to enhanced synthesis of cholesterol (Fig. 3A) and cholesteryl esters (Fig. 4B)

Read more

Summary

Results

The results show that when wild-type SV-589 cells were cultured in sterol-replete medium containing FCS, a low but detectable level of reductase protein was observed by immunoblot analysis of isolated membrane fractions (Fig. 1A, lane 1). Immunoblot analysis of membrane fractions from SV-589 (⌬UBIAD1)/pMyc-UBIAD1 cells revealed that expression of Myc-UBIAD1 restored expression of reductase protein to approximately wild-type levels (compare lanes 1 and 3). When SV-589 and SV-589 (⌬UBIAD1) cells were cultured in sterol-depleted lipoprotein-deficient serum (LPDS)-containing medium, a condition that enhances transcription of the reductase gene and slows reductase ERAD (1), similar levels of reductase protein were observed (lanes 5 and 6). The results show that incorporation of [3H]mevalonate into cholesterol was reduced 3– 4-fold in SV-589 (⌬UBIAD1) cells cultured in FCS compared with that in SV-589 cells (Fig. 2B). The incorporation of [14C]acetate was markedly enhanced (Ͼ5-fold) in SV-589 (⌬UBIAD1) cells express-

50 ACAT-1
Discussion
Experimental procedures
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call