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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Available from: Youngah Jo, Sep 29, 2015
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    • "AMFR/gp78 is an E3 ubiquitin ligase and INSIG is an insulin signaling factor (Flury et al., 2005; Chen et al., 2012). Both are localized to the ER membrane and when activated function together to degrade HMG COA reductase, a cholesterol synthesis enzyme (Jo et al., 2011; Tsai et al., 2012). "
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    ABSTRACT: Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.
    Frontiers in Microbiology 07/2014; 5:322. DOI:10.3389/fmicb.2014.00322 · 3.99 Impact Factor
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    • "Besides gp78, Jo and his colleagues [18] have found that another ubiquitin ligase translocation in renal carcinoma on chromosome 8 protein (TRC8) may also be involved in the regulation of reductase stability (discussed below). "
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    ABSTRACT: To maintain cholesterol homeostasis, the processes of cholesterol metabolism are regulated at multiple levels including transcription, translation, and enzymatic activity. Recently, the regulation of protein stability of some key players in cholesterol metabolism has been characterized. More and more ubiquitin ligases have been identified including gp78, Hrd1, TRC8, TEB4, Fbw7, and inducible degrader of low density lipoprotein receptor. Their working mechanisms and physiological functions are becoming revealed. Here, we summarize the structure, substrates and function of these ubiquitin ligases. Their potential application in drug discovery is also discussed.
    Diabetes & metabolism journal 06/2014; 38(3):171-80. DOI:10.4093/dmj.2014.38.3.171
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    • "This is not surprising, as emerging evidence demonstrates that two, or even more, E3 ligases might assist the processing of certain ERAD substrates. For example, HRD1 cooperates with gp78 in the degradation of mutant neuroserpin (41), whereas gp78 together with Trc8 operates the sterol-mediated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (42), and the orphan CD3-δ subunit is client of both gp78 and RFP2 E3 ligases (34). The latter ligase also mediates the degradation of CaV 1.2 channels (35) and regulates the expression of the p53 tumor suppressor by degrading MDM2 (43). "
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    ABSTRACT: Many membrane and secretory proteins that fail to pass quality control in the endoplasmic reticulum (ER) are dislocated into the cytosol and degraded by the proteasome. In applying rigid rules, however, quality control sometimes discharges proteins that, even though defective, retain their function. The unnecessary removal of such proteins represents the pathogenetic hallmark of diverse genetic diseases, with the case of ΔF508 mutant of CFTR being probably the best known example. Recently, the inappropriate proteasomal degradation of skeletal muscle sarcoglycans (α, β, γ and δ) with missense mutation has been proposed to be at the bases of mild-to-severe forms of limb girdle muscular dystrophy (LGMD) known as type 2D, 2E, 2C and 2F, respectively. The quality control pathway responsible for sarcoglycan mutant disposal, however, is so far unexplored. Here we reveal key components of the degradative route of V247M α-sarcoglycan mutant, the second most frequently reported mutation in LGMD-2D.The disclosure of the pathway, which is led by the E3 ligases HRD1 and RFP2, permits to identify new potential druggable targets of a disease for which no effective therapy is at present available. Notably, we show that the pharmacological inhibition of HRD1 activity rescues the expression of V247-α-sarcoglycan both in a heterologous cell model and in myotubes derived from a LGMD-2D patient carrying the L31P/V247M mutations. This represents the first evidence that the activity of E3 ligases, the enzymes in charge of mutant fate, can be eligible for drug interventions to treat sarcoglycanopathy.
    Human Molecular Genetics 02/2014; 23(14). DOI:10.1093/hmg/ddu088 · 6.39 Impact Factor
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