Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8

Department of Molecular Genetics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2011; 108(51):20503-8. DOI: 10.1073/pnas.1112831108
Source: PubMed


Accumulation of sterols in membranes of the endoplasmic reticulum (ER) leads to the accelerated ubiquitination and proteasomal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids. This degradation results from sterol-induced binding of reductase to the Insig-1 or Insig-2 proteins of ER membranes. We previously reported that in immortalized human fibroblasts (SV-589 cells) Insig-1, but not Insig-2, recruits gp78, a membrane-bound RING-finger ubiquitin ligase. We now report that both Insig-1 and Insig-2 bind another membrane-bound RING-finger ubiquitin ligase called Trc8. Knockdown of either gp78 or Trc8 in SV-589 cells through RNA interference (RNAi) inhibited sterol-induced ubiquitination of reductase and inhibited sterol-induced degradation by 50-60%. The combined knockdown of gp78 and Trc8 produced a more complete inhibition of degradation (> 90%). Knockdown of gp78 led to a three to fourfold increase in levels of Trc8 and Insig-1 proteins, which opposed the inhibitory action of gp78. In contrast, knockdown of Trc8 had no effect on gp78 or Insig-1. The current results suggest that sterol-induced ubiquitination and proteasomal degradation of reductase is dictated by the complex interplay of at least four proteins: Insig-1, Insig-2, gp78, and Trc8. Variations in the concentrations of any one of these proteins may account for differences in cell- and/or tissue-specific regulation of reductase degradation.

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    • "We presume that the ERAD phenotypes observed in gp78 knockdown cells are not caused by an unknown off-target effect, because they were observed using siRNA/shRNAs with different targeting sequences. In addition, siRNA-mediated knockdown of each component of the gp78 complex (including gp78, UbxD8, and UBAC2) inhibited ERAD in all instances (Fang et al., 2001; Song et al., 2005; Shen et al., 2006; Tsai et al., 2007; Morito et al., 2008; Christianson et al., 2011; Jo et al., 2011a,b; Liu et al., 2014). By contrast, CRISPR cells lacking these proteins are functionally competent in ERAD, at least FIGURE 7: The functional relationship between gp78 and Hrd1. "
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    ABSTRACT: Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD). Central regulators of the ERAD system are membrane-bound ubiquitin ligases, which are thought to channel misfolded proteins through the ER membrane during retrotranslocation. Hrd1 and gp78 are mammalian ubiquitin ligases homologous to Hrd1p, an ubiquitin ligase essential for ERAD in S. cerevisiae. However, the functional relevance of these proteins to Hrd1p is unclear. Here we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that transient inactivation of the gp78 complex by short hairpin RNA-mediated gene silencing causes significant stabilization of both luminal and membrane ERAD substrates, but unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of these ERAD substrates, knockdown of gp78 does not affect either of these processes. Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation.
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    • "c o m / l o c a t e / b b a d i s TRC8 is an ER-resident E3 ligase and its gene disruption has been associated with hereditary renal cancer [10]. In addition to its tumor suppressive function [11] [12], TRC8 has been shown to regulate cholesterol and fatty acid biosynthesis by stimulating the destabilization of the ER-bound sterol regulatory element-binding proteins (SREBPs) [13], hindering ER to Golgi transport of SREBP-2 [14], and mediating the degradation of HMG-CoA reductase [15]. Moreover, studies in Drosophila and mammalian cells revealed that TRC8 suppresses protein translation through targeting eIF3f and eIF3h for degradation [11]. "
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    ABSTRACT: Endoplasmic reticulum (ER) stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). TRC8 is an ER-resident E3 ligase with roles in modulating lipid and protein biosynthesis. In this study we showed that TRC8 expression was downregulated in steatotic livers of patients and mice fed with high fat diet (HFD) or methionine and choline deficient (MCD) diet. To investigate the impact of TRC8 downregulation on steatosis and the progression to non-alcoholic steatohepatitis (NASH), we placed TRC8 knockout (KO) mice and wild type (WT) controls on HFD or MCD diet and the severities of steatosis and NASH developed were compared. We found that TRC8 deficiency did not significantly affect diet-induced steatosis. Nevertheless, MCD diet-induced NASH as characterized by hepatocyte death, inflammation and fibrosis were exacerbated in TRC8-KO mice. The hepatic ER stress response, as evidenced by increased eIF2α phosphorylation and expression of ATF4 and CHOP, and the level of activated caspase 3, an apoptosis indicator, were augmented by TRC8 deficiency. The hepatic ER stress and NASH induced in mice could be ameliorated by adenovirus-mediated hepatic TRC8 overexpression. Mechanistically, we found that TRC8 deficiency augmented lipotoxic-stress-induced unfolded protein response in hepatocytes by attenuating the arrest of protein translation and the misfolded protein degradation. These findings disclose a crucial role of TRC8 in the maintenance of ER protein homeostasis and its downregulation in steatotic liver contributes to the progression of NAFLD. Copyright © 2015. Published by Elsevier B.V.
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    • "TRC8 function likely extends beyond the degradation of TA proteins. Its sterol sensing domain suggests an involvement in membrane lipid homeostasis, though the role of TRC8 in the processing of SREBP transcription factors and sterol-induced degradation of HMG-CoA reductase remains controversial (Irisawa et al., 2009; Jo et al., 2011; Lee et al., 2010; Tsai et al., 2012). The study of US2 has therefore uncovered a novel branch of mammalian ERAD with an E3 ligase involved in channel-independent degradation and lipid homeostasis. "
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