The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor - PubMed (original) (raw)

The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor

Li Zhang et al. Genes Dev. 2011.

Abstract

We previously identified the E3 ubiquitin ligase IDOL as a sterol-dependent regulator of the LDL receptor (LDLR). The molecular pathway underlying IDOL action, however, remains to be determined. Here we report the identification and biochemical and structural characterization of an E2-E3 ubiquitin ligase complex for LDLR degradation. We identified the UBE2D family (UBE2D1-4) as E2 partners for IDOL that support both autoubiquitination and IDOL-dependent ubiquitination of the LDLR in a cell-free system. NMR chemical shift mapping and a 2.1 Å crystal structure of the IDOL RING domain-UBE2D1 complex revealed key interactions between the dimeric IDOL protein and the E2 enzyme. Analysis of the IDOL-UBE2D1 interface also defined the stereochemical basis for the selectivity of IDOL for UBE2Ds over other E2 ligases. Structure-based mutations that inhibit IDOL dimerization or IDOL-UBE2D interaction block IDOL-dependent LDLR ubiquitination and degradation. Furthermore, expression of a dominant-negative UBE2D enzyme inhibits the ability of IDOL to degrade the LDLR in cells. These results identify the IDOL-UBE2D complex as an important determinant of LDLR activity, and provide insight into molecular mechanisms underlying the regulation of cholesterol uptake.

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Figures

Figure 1.

Figure 1.

The UBE2Ds are specific partners for IDOL autoubiquitination. (A) Immunoblot of a panel of 19 human UBE2 enzymes that were expressed in E. coli as fusion proteins with 6xHis tags on their N termini. (B) Autoubiquitination of IDOL induced by UBE2D family proteins in an in vitro autoubiquitination assay. Immunoprecipitated TAP-IDOL was incubated with UBE1, HA-ubiquitin, and the indicated UBE2 proteins. IDOL ubiquitination was detected by immunoblotting for HA-tagged ubiquitin associated with IDOL. (C) Autoubiquitination of IDOL induced by UBE2D requires an active RING domain. Immunoprecipitated TAP-IDOL and TAP-IDOL C387A were incubated with UBE1, HA-ubiquitin, and the indicated UBE2 proteins. IDOL ubiquitination was detected by immunoblotting for HA-tagged ubiquitin associated with IDOL. (D) Autoubiquitination induced by UBE2D is specific to IDOL. Immunoprecipitated TAP-EGFP, TAP-IDOL, and TAP-IDOL C387A were incubated with UBE1, HA-ubiquitin, and UBE2D proteins. IDOL ubiquitination was detected by immunoblotting for HA-tagged ubiquitin associated with IDOL. The amounts of TAP-tagged proteins in the in vitro autoubiquitination assay were determined by immunoblotting using anti-Flag antibody. (E) UBE2D family proteins have similar capacity for inducing IDOL autoubiquitination. UBED1–4 protein levels used in the IDOL autoubiquitination assay were normalized by Coomassie staining of SDS-PAGE gels. Immunoprecipitated TAP-IDOL and TAP-IDOL C387A were incubated with UBE1, HA-ubiquitin, and the indicated UBE2D family proteins. IDOL ubiquitination was detected by immunoblotting for HA-tagged ubiquitin associated with IDOL. (F) IDOL autoubiquitination is not exclusively dependent on the Lys11, Lys48, or the Lys63 linkage of ubiquitin. Immunoprecipitated TAP-IDOL and TAP-IDOL C387A were incubated with UBE1, UBE2D2, and HA-tagged ubiquitin with the indicated lysine mutations. IDOL ubiquitination was detected by immunoblotting.

Figure 2.

Figure 2.

UBE2D family proteins are the E2 enzymes for LDLR ubiquitination. (A) Ubiquitination of the LDLR by UBE2D and IDOL in an in vitro ubiquitination assay. Membrane preparations of 293 cells expressing LDLR-GFP or GFP alone were incubated with UBE1, UBE2D2, tandem affinity–purified IDOL or IDOL C387A, and HA-ubiquitin. LDLR was then immunoprecipitated with an anti-GFP antibody. The ubiquitination of LDLR was detected by immunoblotting for HA-tagged ubiquitin associated with LDLR. (B) Expression of dominant-negative UBE2D2 inhibits the degradation of LDLR. Immunoblot analysis of protein levels in 293 cells transfected with wild-type (WT) or dominant-negative UBE2D or UBE2H, in addition to LDLR and IDOL. (C) Immunoblot analysis of IDOL and LDLR expression in response to inhibition of proteasomal or lysosomal degradation pathways. 293 cells were transfected for 24 h with expression vectors for wild-type or C387A mutant IDOL and LDLR. Cells were treated with MG-132 (25 μM) or bafilomycin (BFL; 100 nM), as indicated, 4 h prior to harvest.

Figure 3.

Figure 3.

NMR chemical shift mapping of the IDOL RING domain with UBE2D1. (A) 1H,15N HSQC spectra of 150 μM 15N-labeled IDOL RING domain in the absence (blue) and presence (green) of UBE2D1 at an equimolar ratio. (B) Weighted shift map obtained from the [1H,15N]-HSQC spectra of the IDOL RING domain with the addition of UBE2D1. (C) Ribbon representation of the crystal structure of the IDOL RING domain. (D) Surface representation of the IDOL RING domain, with the most significant shifts (>0.05 ppm) shown in orange and smaller perturbations (>0.025 ppm) shown in yellow.

Figure 4.

Figure 4.

Crystal structure of the IDOL RING domain dimer complexed with UBE2D1. (A) Cartoon representation with the IDOL RING is shown in purple, and the UBE2D1 is shown in gray. (B) The RING domain dimer interface. (C) Close-up of the RING domain dimer interface. (D) The interface of the IDOL RING domain with UBE2D1. (E) Close-up of the IDOL RING–UBE2D1 complex interface.

Figure 5.

Figure 5.

Specificity determinants for the IDOL RING:UBE2D interaction. (A) Electrostatic potential of the interface between the IDOL RING domain (left) and UBE2D1 (right). Note that the main interaction surface on the E2 is highly basic, and the complementary surface on the E3 is acidic. Arg15 in UBE2D1 provides a basic pocket to accommodate Glu383 from IDOL. (Insert) In noncomplementary E2s such as UBE2E3, the residue in this position is neutral or acidic and disfavors interaction. (B) Some E2s that are noncomplementary with IDOL have a basic residue in position 15, but an important serine at the interface (Ser94 in UBE2D1) is substituted with other amino acids, such as lysine in UBE2L3. The serine makes an important backbone contact that could not be formed by the alternative residues. (C) Alignment of key regions of various E2 ligases. Only members of the UBE2D family have both a basic residue and a serine to support appropriate interactions with the IDOL RING.

Figure 6.

Figure 6.

Disruption of the IDOL–UBE2D interaction blocks LDLR degradation. (A) Mutations in the IDOL RING domain–UBE2D interaction interface inhibit LDLR degradation. Immunoblot analysis of protein levels in 293 cells transfected with LDLR and wild-type (WT) or mutant IDOL expression vectors. (B) UBE2D is unable to catalyze the autoubiquitination of mutant IDOL with a disrupted IDOL RING domain–UBE2D interaction. Immunoprecipitated TAP-IDOL, TAP-IDOL C387A, and TAP-IDOL V389R were incubated with UBE1, UBE2D2, and HA-ubiquitin. IDOL ubiquitination was detected by Western blot for HA-tagged ubiquitin associated with IDOL. (C) UBE2D2 mutated at the interface with IDOL is unable to catalyze IDOL autoubiquitination. Immunoprecipitated TAP-IDOL and TAP-IDOL C387A were incubated with UBE1, wild-type, or P61A/F62R UBE2D2 and HA-ubiquitin. IDOL ubiquitination was detected by Western blot for HA-tagged ubiquitin associated with IDOL. (D) Mutation of UBE2D2 residues predicted to be involved in IDOL specificity determination reduces the ability of UBE2D2 to support IDOL autoubiquitination. Immunoprecipitated TAP-IDOL and TAP-IDOL C387A were incubated with UBE1, wild-type, R15E, S94K, or K8E UBE2D2 and HA-ubiquitin. IDOL ubiquitination was detected by Western blot for HA-tagged ubiquitin associated with IDOL. (E) IDOL forms a dimer in vivo. 293 cells were transfected with vectors expressing TAP-IDOL and V5-IDOL. V5-IDOL in the cell lysate was immunoprecipitated with an anti-V5 antibody. The TAP-IDOL that coimmunoprecipitated with V5-IDOL was detected by immunoblotting using an anti-Flag antibody. (F) Structure-based mutations predicted to disrupt dimer formation prevent the coimmunoprecipitation of TAP-IDOL and V5-IDOL. 293 cells were transfected with the indicated combination of expression vectors. V5-IDOL and V5-mutant IDOL in the cell lysate were immunoprecipitated with an anti-V5 antibody. The coimmunoprecipitated TAP-IDOL was detected by immunoblotting using anti-Flag. (G) A dimer-defective IDOL mutant is unable to induce LDLR degradation. Immunoblot analysis of protein levels in 293 cells transfected with LDLR and wild-type or mutant IDOL expression vectors. (H) IDOL harboring a mutation in the IDOL RING domain–UBE2D interaction interface functions as a dominant negative in LDLR degradation assays. Immunoblot analysis of protein levels in 293 cells transfected with increasing amounts of V5-tagged wild-type or mutant IDOL, in addition to constant levels of LDLR and TAP-IDOL.

Figure 7.

Figure 7.

IDOL is an iron-binding protein. (A) Schematic diagram of the domain structure of IDOL. (B) Alignment of IDOL sequences: (hs) Homo sapiens; (cf) Canis familiaris; (mm) Mus musculus; (xl) Xenopus laevis; (gg) Gallus gallus. The three conserved Cys residues N-terminal to the RING domain are highlighted in yellow. The Cys zinc ligands are highlighted in orange, and the His zinc ligand is highlighted in blue. (C) Results from atomic absorption spectroscopy. (D) Photograph of protein samples of IDOL constructs eluted from glutathione Sepharose by cleavage with TEV protease. (E) Coomassie-stained SDS-PAGE gel of IDOL constructs eluted from glutathione Sepharose by cleavage with TEV protease. (F,G) Disruption of the putative iron-binding cysteine residues alters IDOL stability and LDLR degradation. Immunoblot analysis of protein levels in 293 cells transfected with LDLR and wild-type (WT) or mutant IDOL expression vectors. (H) Effect of IDOL interaction mutants on the ability of IDOL to inhibit LDL uptake. 293 cells were transfected with LDLR and wild-type or mutant IDOL expression vectors and then incubated for 4 h with DiI-labeled LDL. Cells were washed and associated cellular LDL were quantified by fluorescence. Results are presented as percent wild-type (% WT) IDOL inhibitory activity in LDL uptake assays. The inhibitory activity of wild-type IDOL was defined as 100%, and that of the inactive RING mutant (C387A) was defined as 0. (**) P < 0.001; (*) P < 0.05.

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References

    1. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al. 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66: 213–221 - PMC - PubMed
    1. Barlow PN, Luisi B, Milner A, Elliott M, Everett R 1994. Structure of the C3HC4 domain by 1H-nuclear magnetic resonance spectroscopy. A new structural class of zinc-finger. J Mol Biol 237: 201–211 - PubMed
    1. Brown MS, Goldstein JL 1986. A receptor-mediated pathway for cholesterol homeostasis. Science 232: 34–47 - PubMed
    1. Bruford EA, Lush MJ, Wright MW, Sneddon TP, Povey S, Birney E 2008. The HGNC Database in 2008: a resource for the human genome. Nucleic Acids Res 36: D445–D448 doi: 10.1093/nar/gkm881 - PMC - PubMed
    1. Brzovic PS, Keeffe JR, Nishikawa H, Miyamoto K, Fox D III, Fukuda M, Ohta T, Klevit R 2003. Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex. Proc Natl Acad Sci 100: 5646–5651 - PMC - PubMed

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