FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2118838599> ?p ?o ?g. }

• W2118838599 endingPage "1420" @default.
• W2118838599 startingPage "1410" @default.
• W2118838599 abstract "Linkage-specific ubiquitination often leads to distinct cellular events. It has been difficult to establish definitively the requirement for a particular linkage in mammalian degradation pathways due to the inability to deplete endogenous ubiquitin while maintaining cell viability. The E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) targets the low density lipoprotein receptor (LDLR) for degradation. The nature of the linkages employed to signal lysosomal degradation of the LDLR, and to signal proteasomal autodegradation of IDOL, have not been determined. We used an inducible RNAi strategy to replace endogenous ubiquitin with mutants lacking K48 or K63. We found that IDOL catalyzes the transfer of ubiquitin chains to itself and to the LDLR that do not contain exclusively K48 or K63 linkages. Thus, LDLR can be targeted to the lysosome by either K48 or K63 linkages. We further demonstrate that although both ubiquitin conjugating enzyme E2 (UBE2)Ds and UBE2N/V1 can catalyze LDLR ubiquitination in a cell-free system, UBE2Ds appear to be the major E2 enzymes employed by IDOL in cells, consistent with their ability to catalyze both K48 and K63 linkages. The results reveal mechanistic insight into the posttranscriptional control of lipoprotein uptake and provide a test of the requirement of linkage-specific ubiquitination for specific lysosomal and proteasomal degradation pathways in mammalian cells. Linkage-specific ubiquitination often leads to distinct cellular events. It has been difficult to establish definitively the requirement for a particular linkage in mammalian degradation pathways due to the inability to deplete endogenous ubiquitin while maintaining cell viability. The E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) targets the low density lipoprotein receptor (LDLR) for degradation. The nature of the linkages employed to signal lysosomal degradation of the LDLR, and to signal proteasomal autodegradation of IDOL, have not been determined. We used an inducible RNAi strategy to replace endogenous ubiquitin with mutants lacking K48 or K63. We found that IDOL catalyzes the transfer of ubiquitin chains to itself and to the LDLR that do not contain exclusively K48 or K63 linkages. Thus, LDLR can be targeted to the lysosome by either K48 or K63 linkages. We further demonstrate that although both ubiquitin conjugating enzyme E2 (UBE2)Ds and UBE2N/V1 can catalyze LDLR ubiquitination in a cell-free system, UBE2Ds appear to be the major E2 enzymes employed by IDOL in cells, consistent with their ability to catalyze both K48 and K63 linkages. The results reveal mechanistic insight into the posttranscriptional control of lipoprotein uptake and provide a test of the requirement of linkage-specific ubiquitination for specific lysosomal and proteasomal degradation pathways in mammalian cells. Ubiquitination is one of the most universal posttranslational protein modifications occurring in the cell (1Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6880) Google Scholar). Ubiquitin, an evolutionarily highly conserved 76 amino acid polypeptide, is covalently attached to lysine residues of target proteins through a highly organized and hierarchical group of enzymes (2Dye B.T. Schulman B.A. Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins.Annu. Rev. Biophys. Biomol. Struct. 2007; 36: 131-150Crossref PubMed Scopus (212) Google Scholar). Ubiquitin activating enzyme (E1) activates ubiquitin, forming a high-energy thioester bond between the C terminus of ubiquitin and E1. The activated ubiquitin is then transferred to a ubiquitin conjugating enzyme (E2). Following that, a large group of ubiquitin ligases (E3s) facilitate the transfer of ubiquitin from E2 to the lysine residues on the substrate proteins, either directly, or by forming an E3-ubiquitin intermediate (3Pickart C.M. Mechanisms underlying ubiquitination.Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2909) Google Scholar). As there are seven lysine residues on ubiquitin, it is possible that during ubiquitination, new ubiquitin molecules could be added to one of the seven lysine residues of the previously conjugated ubiquitin (4Komander D. Rape M. The ubiquitin code.Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (2227) Google Scholar). This process, called polyubiquitination, is a common cellular signal. Depending on the usage of the lysine residues on ubiquitin, various linkage-specific ubiquitinations could happen. K48 and K63 linkage-specific ubiquitinations are the most predominant forms of polyubiquitination, accounting for 52 and 38% of all ubiquitination events respectively, in HEK293 cells (5Dammer E.B. Na C.H. Xu P. Seyfried N.T. Duong D.M. Cheng D. Gearing M. Rees H. Lah J.J. Levey A.I. et al.Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease.J. Biol. Chem. 2011; 286: 10457-10465Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Because ubiquitination via different lysine linkages would result in distinct conformations of ubiquitin and thereby restrict the accessible lysines (6Datta A.B. Hura G.L. Wolberger C. The structure and conformation of Lys63-linked tetraubiquitin.J. Mol. Biol. 2009; 392: 1117-1124Crossref PubMed Scopus (95) Google Scholar, 7Eddins M.J. Varadan R. Fushman D. Pickart C.M. Wolberger C. Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH.J. Mol. Biol. 2007; 367: 204-211Crossref PubMed Scopus (133) Google Scholar), alternate linkage in polyubiquitination is relatively rare, but has nonetheless been reported (8Boname J.M. Thomas M. Stagg H.R. Xu P. Peng J. Lehner P.J. Efficient internalization of MHC I requires lysine-11 and lysine-63 mixed linkage polyubiquitin chains.Traffic. 2010; 11: 210-220Crossref PubMed Scopus (104) Google Scholar, 9Dynek J.N. Goncharov T. Dueber E.C. Fedorova A.V. Izrael-Tomasevic A. Phu L. Helgason E. Fairbrother W.J. Deshayes K. Kirkpatrick D.S. et al.c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling.EMBO J. 2010; 29: 4198-4209Crossref PubMed Scopus (276) Google Scholar, 10Gerlach B. Cordier S.M. Schmukle A.C. Emmerich C.H. Rieser E. Haas T.L. Webb A.I. Rickard J.A. Anderton H. Wong W.W. et al.Linear ubiquitination prevents inflammation and regulates immune signalling.Nature. 2011; 471: 591-596Crossref PubMed Scopus (683) Google Scholar). In recent years, the role of different linkage-specific polyubiquitinations has begun to be elucidated. A K48-linked polyubiquitin chain has been shown to be sufficient to target a model substrate to the 26S proteasome, and has been proved to be a principal proteasome delivery signal for multiple short-lived proteins in the cell (11Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1116) Google Scholar, 12Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.Mol. Cell. Biol. 1994; 14: 5501-5509Crossref PubMed Scopus (303) Google Scholar). In contrast to the proteolytic role of the K48-specific linkage, the K63-specific linkage has been demonstrated to regulate a variety of nonproteolytic cellular functions, including DNA damage repair (13Spence J. Sadis S. Haas A.L. Finley D. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination.Mol. Cell. Biol. 1995; 15: 1265-1273Crossref PubMed Google Scholar), stress responses (14Arnason T. Ellison M.J. Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain.Mol. Cell. Biol. 1994; 14: 7876-7883Crossref PubMed Scopus (195) Google Scholar), and inflammatory pathways (15Sun L. Deng L. Ea C.K. Xia Z.P. Chen Z.J. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes.Mol. Cell. 2004; 14: 289-301Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). Importantly, K63-specific ubiquitination has also been shown to facilitate the endocytosis of membrane proteins (16Mukhopadhyay D. Riezman H. Proteasome-independent functions of ubiquitin in endocytosis and signaling.Science. 2007; 315: 201-205Crossref PubMed Scopus (956) Google Scholar). In yeast strains in which the endogenous ubiquitin was depleted either by eliminating all ubiquitin-coding genes (12Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.Mol. Cell. Biol. 1994; 14: 5501-5509Crossref PubMed Scopus (303) Google Scholar, 13Spence J. Sadis S. Haas A.L. Finley D. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination.Mol. Cell. Biol. 1995; 15: 1265-1273Crossref PubMed Google Scholar), or by mutating the genes essential for free ubiquitin recycling (17Papa F.R. Hochstrasser M. The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene.Nature. 1993; 366: 313-319Crossref PubMed Scopus (340) Google Scholar), definitive evidence has been obtained that K63-specific polyubiquitination is required for the endocytosis, the vacuole sorting, and the degradation of membrane proteins such as uracil permease (18Galan J.M. Haguenauer-Tsapis R. Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein.EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (322) Google Scholar), Gap1p permease (19Springael J.Y. Galan J.M. Haguenauer-Tsapis R. Andre B. NH4+-induced down-regulation of the Saccharomyces cerevisiae Gap1p permease involves its ubiquitination with lysine-63-linked chains.J. Cell Sci. 1999; 112: 1375-1383Crossref PubMed Google Scholar), and carboxypeptidase S (20Lauwers E. Jacob C. Andre B. K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway.J. Cell Biol. 2009; 185: 493-502Crossref PubMed Scopus (187) Google Scholar). Studies in mammalian cells suggest that K63-specific polyubiquitination is involved in various stages of the internalization, the lysosome sorting, and the degradation processes of membrane proteins, including the epidermal growth factor receptor (EGFR) (21Huang F. Kirkpatrick D. Jiang X. Gygi S. Sorkin A. Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain.Mol. Cell. 2006; 21: 737-748Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 22Stang E. Blystad F.D. Kazazic M. Bertelsen V. Brodahl T. Raiborg C. Stenmark H. Madshus I.H. Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits.Mol. Biol. Cell. 2004; 15: 3591-3604Crossref PubMed Scopus (133) Google Scholar), nerve growth factor receptor TrkA (23Geetha T. Jiang J. Wooten M.W. Lysine 63 polyubiquitination of the nerve growth factor receptor TrkA directs internalization and signaling.Mol. Cell. 2005; 20: 301-312Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), major histocompatibility complex class I molecules (24Duncan L.M. Piper S. Dodd R.B. Saville M.K. Sanderson C.M. Luzio J.P. Lehner P.J. Lysine-63-linked ubiquitination is required for endolysosomal degradation of class I molecules.EMBO J. 2006; 25: 1635-1645Crossref PubMed Scopus (212) Google Scholar), and the prolactin receptor (25Varghese B. Barriere H. Carbone C.J. Banerjee A. Swaminathan G. Plotnikov A. Xu P. Peng J. Goffin V. Lukacs G.L. et al.Polyubiquitination of prolactin receptor stimulates its internalization, postinternalization sorting, and degradation via the lysosomal pathway.Mol. Cell. Biol. 2008; 28: 5275-5287Crossref PubMed Scopus (68) Google Scholar). However, due to the technical difficulty in eliminating endogenous ubiquitin genes in mammalian cells, direct evidence is still lacking whether the K63-specific ubiquitin linkage is an indispensible element in the endocytosis and lysosomal degradation of membrane proteins in mammalian cells. We have demonstrated that under conditions of elevated intracellular cholesterol, the liver X receptor (LXR) induces the transcription of the E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL), which ubiquitinates and facilitates the degradation of the low-density lipoprotein receptor (LDLR) (26Hong C. Duit S. Jalonen P. Out R. Scheer L. Sorrentino V. Boyadjian R. Rodenburg K.W. Foley E. Korhonen L. et al.The E3 ubiquitin ligase IDOL induces the degradation of the low density lipoprotein receptor family members VLDLR and ApoER2.J. Biol. Chem. 2010; 285: 19720-19726Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 27Scotti E. Hong C. Yoshinaga Y. Tu Y. Hu Y. Zelcer N. Boyadjian R. de Jong P.J. Young S.G. Fong L.G. et al.Targeted disruption of the idol gene alters cellular regulation of the low-density lipoprotein receptor by sterols and liver x receptor agonists.Mol. Cell. Biol. 2011; 31: 1885-1893Crossref PubMed Scopus (60) Google Scholar, 28Zelcer N. Hong C. Boyadjian R. Tontonoz P. LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor.Science. 2009; 325: 100-104Crossref PubMed Scopus (553) Google Scholar). Typical of an E3 ubiquitin ligase, IDOL also ubiquitinates itself and thereby promotes its own turnover. Interestingly, autodegradation of IDOL appears to occur through the proteasomal pathway, whereas IDOL-dependent degradation of the LDLR occurs via the lysosome (29Zhang L. Fairall L. Goult B.T. Calkin A.C. Hong C. Millard C.J. Tontonoz P. Schwabe J.W. The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.Genes Dev. 2011; 25: 1262-1274Crossref PubMed Scopus (66) Google Scholar). Indirect evidence has suggested that K63-specific ubiquitination could be involved in the degradation of the LDLR (30Sorrentino V. Scheer L. Santos A. Reits E. Bleijlevens B. Zelcer N. Distinct functional domains contribute to degradation of the low density lipoprotein receptor (LDLR) by the E3 ubiquitin ligase inducible degrader of the LDLR (IDOL).J. Biol. Chem. 2011; 286: 30190-30199Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, it has not been rigorously tested whether K48 or K63 linkage-specific ubiquitination is actually required for the ubiquitination and degradation of IDOL or the LDLR. For most genes it is relatively straightforward to generate a stable knockdown cell and then replace a mutant version of the gene of interest with an expression vector. But there are four ubiquitin genes in mammals, and knocking them all down kills the cell. Thus, standard small inhibitory RNA (siRNA) knockdown studies are impractical. The only way to introduce mutant ubiquitins into a null background is to knockdown all 4 ubiquitin genes while simultaneously introducing the expression of a new ubiquitin molecule to keep the cells alive. Recently, a ubiquitin replacement strategy was developed in which a tetracycline-inducible RNA inhibition (RNAi) was used to replace the endogenous ubiquitin proteins with ubiquitin mutants. This strategy was previously employed to determine the requirement for K63-specific ubiquitin chains in IκB kinase (IKK) activation by IL-1β (31Xu M. Skaug B. Zeng W. Chen Z.J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta.Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Here we have employed the ubiquitin replacement strategy of Xu et al. (31Xu M. Skaug B. Zeng W. Chen Z.J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta.Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) to determine the nature of the ubiquitin linkages involved in IDOL-dependent protein degradation. We initially hypothesized that IDOL might employ K48-polyubiquitin chains to target itself for proteasomal degradation, and K63-specific linkages to trigger lysosomal degradation of the LDLR. Contrary to expectations, however, the degradation of neither IDOL nor the LDLR was exclusively mediated by K48- or K63-specific ubiquitination, strongly suggesting that either linkage can signal proteasomal and lysosomal degradation. We also found that ubiquitin conjugating enzyme E2 (UBE2)N/V1, a heterodimeric ubiquitin E2 enzyme that specifically catalyzes K63-specific ubiquitin linkage, is not required for the ubiquitination and degradation of the LDLR. This study provides a test of the requirement of linkage-specific ubiquitination for lysosomal and proteasomal protein degradation pathways in mammalian cells. The synthetic LXR ligand GW3965 was provided by T. Wilson (GlaxoSmithKline). MG132, tetracycline, bafilomycin, and mevalonic acid were purchased from Sigma-Aldrich. Simvastatin sodium salt was purchased from Calbiochem. The pSA2-N-TAP plasmid that contains the 3xFLAG-Strep tag and the pcDNA-V5-DEST plasmid were kind gifts from Dr. E. Saez (Scripps Institute). pDONR221, pET300N-DEST, and pcDNA-DEST47 plasmids were purchased from Invitrogen. The DNA sequence of the human Idol gene was amplified from a pcDNA-V5::hIdol construct as previously reported (28Zelcer N. Hong C. Boyadjian R. Tontonoz P. LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor.Science. 2009; 325: 100-104Crossref PubMed Scopus (553) Google Scholar), and was then subcloned into pSA2-N-TAP plasmid. The IDOL C387A mutation for the pSA2-N-TAP::hIdol constructs was introduced by site-directed mutagenesis. The DNA sequence of the human Ldlr gene was amplified from pCB6-hLdlr (a kind gift from Dr. K. Matter, University College London, UK) with a predesigned primer encoding a V5 tag to the C terminus of the coding sequence of hLdlr. The DNA sequence of the tagged hLdlr was then subcloned into pcDNA-DEST47 using the Gateway technology (Invitrogen). For the GFP-tagged hLDLR, hLdlr was amplified with the stop codon removed and was then subcloned into pcDNA-DEST47 using the Gateway technology. pcDNA3.1-(HA-Ub)6 was a kind gift from Dr. J. Wohlschlegel (University of California at Los Angeles). The K48R and K63R mutations for the pcDNA3.1-(HA-Ub)6 construct were introduced by site-directed mutagenesis. The human E2 genes hUbe2d2, hUbe2n, and hUbe2v1 were cloned from HEK293T cell cDNA and were then sequentially subcloned into pDONR221 and pET300N-DEST using the Gateway technology for Escherichia coli protein expression. In addition, the hUbe2d2 and hUbe2n genes in the pDONR221::hUbe2d2 and pDONR221::hUbe2n constructs were subcloned into pcDNA-V5-DEST plasmid using the Gateway technology. The UBE2D2 C85A and the UBE2N C87A mutations for the pcDNA-V5::hUbe2d2 and the pcDNA-V5::hUbe2n constructs, respectively, were introduced by site-directed mutagenesis. Rabbit anti-hLDLR antibody was purchased from Cayman Chemicals. Rabbit anti-actin and mouse anti-FLAG M2 antibodies were purchased from Sigma-Aldrich. Mouse anti-V5 antibody, HRP-conjugated goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Invitrogen. Rabbit anti-V5 antibody was purchased from Abcam. Rabbit anti-GFP antibody was purchased from Clontech. Mouse anti-HA antibody was purchased from Covance. All commercially available antibodies were used according to the manufacturers’ instructions. HEK293T cells and the engineered U2OS cells for ubiquitin replacement were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Omega), 2 mM l-glutamine (Invitrogen), 50 U/ml penicillin (Invitrogen), and 50 µg/ml streptomycin (Invitrogen). Cells were grown in a humidified incubator at 37°C and 5% CO2 atmosphere. HEK293T cells were transfected using FuGENE 6 reagents (Roche) according to the manufacturer舗s instructions. Ad-mIdol particles were generated as previously described (28Zelcer N. Hong C. Boyadjian R. Tontonoz P. LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor.Science. 2009; 325: 100-104Crossref PubMed Scopus (553) Google Scholar). For the Ad-hLdlr-V5 particle, the DNA sequence of hLdlr was amplified from pCB6-hLdlr with the stop codon removed. The hLdlr sequence was then subcloned sequentially into pDONR221 and pAd-CMV-V5-DEST (Invitrogen) with the Gateway technology. Viruses were amplified, purified, and titered by Viraquest. The in vitro IDOL autoubiquitination assay and LDLR ubiquitination assay were carried out as previously described (29Zhang L. Fairall L. Goult B.T. Calkin A.C. Hong C. Millard C.J. Tontonoz P. Schwabe J.W. The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.Genes Dev. 2011; 25: 1262-1274Crossref PubMed Scopus (66) Google Scholar). Proteins were resolved on 4–12% gradient SDS-PAGE (Invitrogen) using standard protocols. The protein was electrophoretically transferred to nitrocellulose membranes (Amersham Biosciences) and blocked with milk solution (150 mM NaCl, 20 mM Tris, 5% milk, 0.2% Tween, pH 7.5) to quench nonspecific protein binding. The blocked membranes were probed sequentially with primary and secondary antibodies diluted in the milk solution, and the bands were visualized with the ECL kit (Amersham Biosciences). To examine whether the ubiquitination of IDOL is mediated exclusively by K48- or K63-linked ubiquitin, we transfected HEK293T cells with FLAG-tagged IDOL, together with either HA-tagged wild-type ubiquitin, or ubiquitin mutants harboring lysine to arginine mutations at the K48 or K63 residues. We hypothesized that if the ubiquitination of IDOL was exclusively mediated by K48 or K63 linkage, mutations on these residues would prevent the HA-tagged mutant ubiquitin from participating in the elongation of polyubiquitin chains, and therefore would reduce the ubiquitination revealed by the HA tag. As we previously reported, IDOL undergoes active autoubiquitination and autodegradation (29Zhang L. Fairall L. Goult B.T. Calkin A.C. Hong C. Millard C.J. Tontonoz P. Schwabe J.W. The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.Genes Dev. 2011; 25: 1262-1274Crossref PubMed Scopus (66) Google Scholar). Therefore, in order to better demonstrate the ubiquitination of IDOL, we treated these transfected cells with proteasome inhibitor MG132 prior to harvest. As expected, this treatment led to the accumulation of IDOL protein (Fig. 1A). We did not observe any difference in IDOL ubiquitination between cells transfected with wild-type ubiquitin and cells transfected with either K48R or K63R mutant ubiquitin, as revealed by the HA tag (Fig. 1A). This result suggested that IDOL ubiquitination does not depend exclusively on the K48 or K63 linkage. We also examined whether the ubiquitination of the LDLR was mediated exclusively by K48- or K63-specific linkages in this system. Because the degradation of the LDLR is lysosome dependent, we treated transfected cells prior to harvest with bafilomycin to preserve ubiquitinated LDLR. This treatment resulted in the accumulation of the LDLR but had little effect on IDOL (Fig. 1B). As revealed by the HA tag, there was no discernible difference in LDLR ubiquitination between cells transfected with wild-type ubiquitin and cells transfected with either K48R or K63R mutant ubiquitin (Fig. 1B). This result suggested that, similar to IDOL autoubiquitination, the ubiquitination of the LDLR does not exclusively depend on K48- or K63-linked ubiquitin. Because the liver is an important organ contributing to cholesterol homeostasis, we next sought to investigate lysine-specific ubiquitination of IDOL and the LDLR in two different hepatocyte cell lines, Hep3B and HepG2. We transfected these two cell lines with IDOL and/or the LDLR, together with HA-tagged wild-type ubiquitin, or HA-tagged ubiquitin mutants harboring lysine to arginine mutations at the K48 or K63 residues. In both hepatocyte cell lines we readily observed the formation of polyubiquitin chains from the transfected wild-type, K48R, and K63R ubiquitin on IDOL (Fig. 2A) and the LDLR (Fig. 2B). The levels of IDOL and LDLR ubiquitination observed were similar between cells transfected with wild-type ubiquitin and cells transfected with either K48R or K63R mutant ubiquitin. These results suggest that the ubiquitination of IDOL and the LDLR does not exclusively depend on K48- or K63-linked ubiquitin in hepatocytes. To further investigate whether the ubiquitination and the degradation of IDOL and the LDLR are exclusively dependent on K48- or K63-specific ubiquitin linkage, we made use of a previously described inducible ubiquitin replacement system (31Xu M. Skaug B. Zeng W. Chen Z.J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta.Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). In this system, endogenous ubiquitin in stable U2OS cell lines is inducibly eliminated by shRNA (shUb) and replaced by exogenously expressed HA-tagged wild-type ubiquitin (shUb+WT) or ubiquitin mutants harboring point mutations on the K48 (shUb+K48R) or K63 residues (shUb+K63R). Both the shRNA and the replacement ubiquitin are under the control of a tetracycline-activated promoter. This system has been successfully utilized to differentiate the distinct ubiquitin linkages involved in IKK activation induced by tumor necrosis factor-α versus IL-1β (31Xu M. Skaug B. Zeng W. Chen Z.J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta.Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). We initially set out to validate the efficacy of the system using antibodies recognizing K48- or K63-specific ubiquitination. After induction with tetracycline for 48 h, we found that although the formation of K48-specific ubiquitin linkages was intact in shUb+WT cells and shUb+K63R cells, it was markedly inhibited in shUb+K48R cells (Fig. 3A). Similarly, the formation of K63-specific ubiquitin linkages was inhibited in shUb+K63R cells, but not in shUb+WT cells or shUb+K48R cells (Fig. 3B). In addition, consistent with previous work (31Xu M. Skaug B. Zeng W. Chen Z.J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta.Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), shUb cells and shUb+K48R cells were viable for only 72 h, likely because the lack of K48-specific ubiquitination is detrimental to vital cellular functions. In contrast, shUb+K63R cells were viable for 120 h, and shUb+WT cells are viable for a much longer period (data not shown). To explore the requirement for K48 and K63 ubiquitination in the autoubiquitination and autodegradation of IDOL using this replacement system, we cultured the four lines of engineered U2OS cells (shUb, shUb+WT, shUb+K48R, and shUb+K63R) in the absence or presence of tetracycline for 48 h, then infected them with adenovirus-encoding IDOL for 24 h. In all four cells lines grown in the absence of tetracycline, IDOL underwent active autodegradation and did not accumulate (Fig. 4A). However, in cells growing in the presence of tetracycline, where ubiquitin replacement took place, the autodegradation of IDOL was severely inhibited in shUb cells, as evidenced by the accumulation of IDOL. In contrast, we did not observe IDOL accumulation in shUb+WT, shUb+K48R, or shUb+K63R cells, indicating that the lack of the K48 or the K63 residues of ubiquitin did not prevent the autodegradation of IDOL (Fig. 4A). Meanwhile, we also examined IDOL autoubiquitination in these cells. In order to reveal the ubiquitination of IDOL, we treated the cells with proteasome inhibitor MG132. With MG132 treatment, there was no difference in the amounts of IDOL observed in the four cell lines, because ubiquitinated IDOL could not be degraded under this circumstance (Fig. 4B). We observed that in shUb+WT cells, IDOL was polyubiquitinated as expected with the replaced wild-type ubiquitin (Fig. 4B). Importantly, IDOL was also polyubiquitinated in shUb+K48R and shUb+K63R cells with the replaced mutant ubiquitin (Fig. 4B), indicating that the K48 and the K63 residues of ubiquitin are not required for the assembly of polyubiquitin chains on IDOL. Furthermore, we sought to compare the overall ubiquitination levels of IDOL before and after the endogenous ubiquitin was replaced. To this end, we treated U2OS cells growing in the absence or presence of tetracycline with MG132. Under these conditions, the autodegradation of IDOL was inhibited and ubiquitin conjugation to expressed IDOL could be directly revealed by an antibody against IDOL. We observed IDOL ubiquitination in all cell lines grown in the absence of tetracycline (Fig. 4C). In cells grown in the presence of tetracycline to replace endogenous ubiquitin, we still observed similar levels of IDOL ubiquitination in shUb+WT, shUb+K48R, and shUb+K63R cells (Fig. 4C). There was no IDOL ubiquitination in shUb cells grown in the presence of tetracycline, probably because the endogenous ubiquitin was depleted in these cells without any exogenous repletion (Fig. 4C). Because IDOL autodegradation was severely inhibited only in shUb cells, these results further confirm the functional connection between the ubiquitination and the degradation of IDOL. Taken together, these results indicate that the autoubiquitination and autodegradation of IDOL do not require the K48 or th" @default.
• W2118838599 created "2016-06-24" @default.
• W2118838599 creator A5000591490 @default.
• W2118838599 creator A5011050795 @default.
• W2118838599 creator A5020062473 @default.
• W2118838599 creator A5029940065 @default.
• W2118838599 creator A5066716873 @default.
• W2118838599 date "2013-05-01" @default.
• W2118838599 modified "2023-10-15" @default.
• W2118838599 title "Both K63 and K48 ubiquitin linkages signal lysosomal degradation of the LDL receptor" @default.
• W2118838599 cites W1832030339 @default.
• W2118838599 cites W1963967499 @default.
• W2118838599 cites W1966223027 @default.
• W2118838599 cites W1966460916 @default.
• W2118838599 cites W1971145588 @default.
• W2118838599 cites W1976169230 @default.
• W2118838599 cites W1988617344 @default.
• W2118838599 cites W1991258715 @default.
• W2118838599 cites W2012917547 @default.
• W2118838599 cites W2013339424 @default.
• W2118838599 cites W2016236499 @default.
• W2118838599 cites W2017357821 @default.
• W2118838599 cites W2024812887 @default.
• W2118838599 cites W2030341081 @default.
• W2118838599 cites W2031699509 @default.
• W2118838599 cites W2059680729 @default.
• W2118838599 cites W2061050157 @default.
• W2118838599 cites W2069788390 @default.
• W2118838599 cites W2069862589 @default.
• W2118838599 cites W2073669171 @default.
• W2118838599 cites W2075110858 @default.
• W2118838599 cites W2078709018 @default.
• W2118838599 cites W2080182587 @default.
• W2118838599 cites W2082315732 @default.
• W2118838599 cites W2083148004 @default.
• W2118838599 cites W2083498707 @default.
• W2118838599 cites W2087330517 @default.
• W2118838599 cites W2097724407 @default.
• W2118838599 cites W2100041351 @default.
• W2118838599 cites W2101667070 @default.
• W2118838599 cites W2103210708 @default.
• W2118838599 cites W2104176053 @default.
• W2118838599 cites W2110543371 @default.
• W2118838599 cites W2112385479 @default.
• W2118838599 cites W2117396050 @default.
• W2118838599 cites W2121615969 @default.
• W2118838599 cites W2125905711 @default.
• W2118838599 cites W2134320577 @default.
• W2118838599 cites W2135332272 @default.
• W2118838599 cites W2144091858 @default.
• W2118838599 cites W2147506734 @default.
• W2118838599 cites W2147917253 @default.
• W2118838599 cites W2154395038 @default.
• W2118838599 cites W2158588008 @default.
• W2118838599 cites W2164276007 @default.
• W2118838599 cites W2166987747 @default.
• W2118838599 doi "https://doi.org/10.1194/jlr.m035774" @default.
• W2118838599 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3653405" @default.
• W2118838599 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23419260" @default.
• W2118838599 hasPublicationYear "2013" @default.
• W2118838599 type Work @default.
• W2118838599 sameAs 2118838599 @default.
• W2118838599 citedByCount "48" @default.
• W2118838599 countsByYear W21188385992013 @default.
• W2118838599 countsByYear W21188385992014 @default.
• W2118838599 countsByYear W21188385992015 @default.
• W2118838599 countsByYear W21188385992016 @default.
• W2118838599 countsByYear W21188385992017 @default.
• W2118838599 countsByYear W21188385992018 @default.
• W2118838599 countsByYear W21188385992019 @default.
• W2118838599 countsByYear W21188385992020 @default.
• W2118838599 countsByYear W21188385992021 @default.
• W2118838599 countsByYear W21188385992022 @default.
• W2118838599 countsByYear W21188385992023 @default.
• W2118838599 crossrefType "journal-article" @default.
• W2118838599 hasAuthorship W2118838599A5000591490 @default.
• W2118838599 hasAuthorship W2118838599A5011050795 @default.
• W2118838599 hasAuthorship W2118838599A5020062473 @default.
• W2118838599 hasAuthorship W2118838599A5029940065 @default.
• W2118838599 hasAuthorship W2118838599A5066716873 @default.
• W2118838599 hasBestOaLocation W21188385991 @default.
• W2118838599 hasConcept C104317684 @default.
• W2118838599 hasConcept C170493617 @default.
• W2118838599 hasConcept C185592680 @default.
• W2118838599 hasConcept C25602115 @default.
• W2118838599 hasConcept C2779679103 @default.
• W2118838599 hasConcept C41008148 @default.
• W2118838599 hasConcept C55493867 @default.
• W2118838599 hasConcept C76155785 @default.
• W2118838599 hasConcept C86803240 @default.
• W2118838599 hasConcept C95444343 @default.
• W2118838599 hasConceptScore W2118838599C104317684 @default.
• W2118838599 hasConceptScore W2118838599C170493617 @default.
• W2118838599 hasConceptScore W2118838599C185592680 @default.
• W2118838599 hasConceptScore W2118838599C25602115 @default.
• W2118838599 hasConceptScore W2118838599C2779679103 @default.
• W2118838599 hasConceptScore W2118838599C41008148 @default.
• W2118838599 hasConceptScore W2118838599C55493867 @default.

• next