SUMOylation of the ubiquitin ligase IDOL decreases LDL receptor levels and is reversed by SENP1
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015420
ISSN1083-351X
AutoresJu-Qiong Wang, Zi‐Cun Lin, Liang-Liang Li, Shao‐Fang Zhang, Weihui Li, Wei Liu, Bao‐Liang Song, Jie Luo,
Tópico(s)14-3-3 protein interactions
ResumoInducible degrader of the low-density lipoprotein receptor (IDOL) is an E3 ubiquitin ligase mediating degradation of low-density lipoprotein (LDL) receptor (LDLR). IDOL also controls its own stability through autoubiquitination, primarily at lysine 293. Whether IDOL may undergo other forms of posttranslational modification is unknown. In this study, we show that IDOL can be modified by small ubiquitin-like modifier 1 at the K293 residue at least. The SUMOylation of IDOL counteracts its ubiquitination and augments IDOL protein levels. SUMOylation and the associated increase of IDOL protein are effectively reversed by SUMO-specific peptidase 1 (SENP1) in an activity-dependent manner. We further demonstrate that SENP1 affects LDLR protein levels by modulating IDOL. Overexpression of SENP1 increases LDLR protein levels and enhances LDL uptake in cultured cells. On the contrary, loss of SENP1 lowers LDLR levels in an IDOL-dependent manner and reduces LDL endocytosis. Collectively, our results reveal SUMOylation as a new regulatory posttranslational modification of IDOL and suggest that SENP1 positively regulates the LDLR pathway via deSUMOylation of IDOL and may therefore be exploited for the treatment of cardiovascular disease. Inducible degrader of the low-density lipoprotein receptor (IDOL) is an E3 ubiquitin ligase mediating degradation of low-density lipoprotein (LDL) receptor (LDLR). IDOL also controls its own stability through autoubiquitination, primarily at lysine 293. Whether IDOL may undergo other forms of posttranslational modification is unknown. In this study, we show that IDOL can be modified by small ubiquitin-like modifier 1 at the K293 residue at least. The SUMOylation of IDOL counteracts its ubiquitination and augments IDOL protein levels. SUMOylation and the associated increase of IDOL protein are effectively reversed by SUMO-specific peptidase 1 (SENP1) in an activity-dependent manner. We further demonstrate that SENP1 affects LDLR protein levels by modulating IDOL. Overexpression of SENP1 increases LDLR protein levels and enhances LDL uptake in cultured cells. On the contrary, loss of SENP1 lowers LDLR levels in an IDOL-dependent manner and reduces LDL endocytosis. Collectively, our results reveal SUMOylation as a new regulatory posttranslational modification of IDOL and suggest that SENP1 positively regulates the LDLR pathway via deSUMOylation of IDOL and may therefore be exploited for the treatment of cardiovascular disease. The low-density lipoprotein (LDL) receptor (LDLR)-mediated uptake of circulating LDL particles is a prototype of receptor-mediated endocytosis and plays a key role in regulating cholesterol homeostasis at both cellular and whole-body levels (1Luo J. Yang H. Song B.L. Mechanisms and regulation of cholesterol homeostasis.Nat. Rev. Mol. Cell Biol. 2020; 21: 225-245Crossref PubMed Scopus (135) Google Scholar). The LDLR pathway begins with LDL binding to LDLR, followed by clathrin-dependent internalization of the LDL–LDLR complex. In the acidic endosomes, LDLR is induced to dissociate from LDL, allowing the latter to be further delivered to late endosomes/lysosomes, where LDL-carried cholesteryl esters are hydrolyzed to release cholesterol (2Goldstein J.L. Brown M.S. The LDL receptor.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 431-438Crossref PubMed Scopus (719) Google Scholar). LDLR is either directed to the cell surface for reutilization or targeted to lysosome for degradation. Mutations in LDLR as well as genes encoding apolipoprotein B and LDLR adaptor protein 1 (also known as autosomal recessive hypercholesterolemia), which are involved in LDL–LDLR binding and LDL–LDLR complex endocytosis, respectively, confer elevated levels of plasma LDL-cholesterol (LDL-C) that eventually increase the risk for cardiovascular disease (3Henderson R. O'Kane M. McGilligan V. Watterson S. The genetics and screening of familial hypercholesterolaemia.J. Biomed. Sci. 2016; 23: 39Crossref PubMed Scopus (72) Google Scholar, 4Chen L. Chen X.W. Huang X. Song B.L. Wang Y. Wang Y. Regulation of glucose and lipid metabolism in health and disease.Sci. China Life Sci. 2019; 62: 1420-1458Crossref PubMed Scopus (38) Google Scholar). The gain-of-function mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9)—which binds and targets LDLR for lysosomal degradation—also underlie a subclass of familial hypercholesterolemia. Blocking PCSK9-mediated LDLR degradation using the anti-PCSK9 monoclonal antibodies has emerged as an effective strategy to lower LDL-C levels in familial hypercholesterolemia patients or those intolerant to statins (5Sabatine M.S. PCSK9 inhibitors: clinical evidence and implementation.Nat. Rev. Cardiol. 2019; 16: 155-165Crossref PubMed Scopus (48) Google Scholar). Inducible degrader of the LDLR (IDOL, also known as myosin regulatory light-chain interacting protein) is another critical regulator of the LDLR pathway. As an E3 ubiquitin ligase, IDOL binds the cytoplasmic tail of LDLR via the N-terminal FERM 3b subdomain and triggers K48- and K63-linked polyubiquitination via the C-terminal really interesting new gene (RING) domain (6Calkin A.C. Goult B.T. Zhang L. Fairall L. Hong C. Schwabe J.W.R. Tontonoz P. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 20107-20112Crossref PubMed Scopus (35) Google Scholar, 7Sorrentino 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 (37) Google Scholar, 8Zelcer 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 (494) Google Scholar). The ubiquitinated LDLR is internalized in an epsin-dependent manner, sorted to multivesicular bodies by the endosomal sorting complexes required for transport complexes, and finally degraded in lysosomes (9Sorrentino V. Nelson J.K. Maspero E. Marques A.R.A. Scheer L. Polo S. Zelcer N. The LXR-IDOL axis defines a clathrin-, caveolae-, and dynamin-independent endocytic route for LDLR internalization and lysosomal degradation.J. Lipid Res. 2013; 54: 2174-2184Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 10Scotti E. Calamai M. Goulbourne C.N. Zhang L. Hong C. Lin R.R. Choi J. Pilch P.F. Fong L.G. Zou P. Ting A.Y. Pavone F.S. Young S.G. Tontonoz P. IDOL stimulates clathrin-independent endocytosis and multivesicular body-mediated lysosomal degradation of the low-density lipoprotein receptor.Mol. Cell Biol. 2013; 33: 1503-1514Crossref PubMed Scopus (52) Google Scholar). Aside from LDLR, IDOL can modulate its own stability through formation of a homodimer followed by autoubiquitination and degradation in proteasomes (6Calkin A.C. Goult B.T. Zhang L. Fairall L. Hong C. Schwabe J.W.R. Tontonoz P. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 20107-20112Crossref PubMed Scopus (35) Google Scholar, 8Zelcer 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 (494) Google Scholar, 11Zhang L. Fairall L. Goult B.T. Calkin A.C. Hong C. Millard C.J. Tontonoz P. Schwabe J.W.R. The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.Genes Dev. 2011; 25: 1262-1274Crossref PubMed Scopus (58) Google Scholar). Disruption of IDOL dimerization between the RING domain and deubiquitination of IDOL by ubiquitin-specific protease (USP) 2 prevent IDOL degradation as well as abolish its ability to degrade LDLR (11Zhang L. Fairall L. Goult B.T. Calkin A.C. Hong C. Millard C.J. Tontonoz P. Schwabe J.W.R. The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.Genes Dev. 2011; 25: 1262-1274Crossref PubMed Scopus (58) Google Scholar, 12Nelson J.K. Sorrentino V. Avagliano Trezza R. Heride C. Urbe S. Distel B. Zelcer N. The deubiquitylase USP2 regulates the LDLR pathway by counteracting the E3-ubiquitin ligase IDOL.Circ. Res. 2016; 118: 410-419Crossref PubMed Scopus (30) Google Scholar). Another dimerization-defective IDOL G51S mutation that has been associated with high blood LDL-C levels in humans can increase IDOL protein abundance and, notably, accelerate IDOL-induced LDLR degradation (13Adi D. Lu X.Y. Fu Z.Y. Wei J. Baituola G. Meng Y.J. Zhou Y.X. Hu A. Wang J.K. Lu X.F. Wang Y. Song B.L. Ma Y.T. Luo J. IDOL G51S variant is associated with high blood cholesterol and increases low-density lipoprotein receptor degradation.Arterioscler. Thromb. Vasc. Biol. 2019; 39: 2468-2479Crossref PubMed Scopus (9) Google Scholar). These results highlight the importance of IDOL stabilization in modulating LDLR expression and blood LDL-C levels. SUMOylation resembles ubiquitination in that substrate proteins are covalently modified with small molecules in single entities or polymeric chains via the E1-E2-E3 enzymatic cascade (14Denuc A. Marfany G. SUMO and ubiquitin paths converge.Biochem. Soc. Trans. 2010; 38: 34-39Crossref PubMed Scopus (63) Google Scholar, 15Wilkinson K.A. Henley J.M. Mechanisms, regulation and consequences of protein SUMOylation.Biochem. J. 2010; 428: 133-145Crossref PubMed Scopus (433) Google Scholar). However, unlike simple addition of ubiquitin to target proteins, small ubiquitin-like modifier (SUMO, also called sentrin)—which comprises five paralogues in mammals—is initially synthesized as a precursor and undergoes proteolytic processing to become active (16Flotho A. Melchior F. Sumoylation: a regulatory protein modification in health and disease.Annu. Rev. Biochem. 2013; 82: 357-385Crossref PubMed Scopus (612) Google Scholar, 17Liang Y.C. Lee C.C. Yao Y.L. Lai C.C. Schmitz M.L. Yang W.M. SUMO5, a novel poly-SUMO isoform, regulates PML nuclear bodies.Sci. Rep. 2016; 6: 26509Crossref PubMed Scopus (82) Google Scholar). The maturation of SUMO1–3 requires a C-terminal cleavage mediated by SUMO-specific peptidases (SENPs) (18Hickey C.M. Wilson N.R. Hochstrasser M. Function and regulation of SUMO proteases.Nat. Rev. Mol. Cell Biol. 2012; 13: 755-766Crossref PubMed Scopus (360) Google Scholar). Humans have six SENPs that exhibit distinct substrate preferences and subcellular distributions (19Nayak A. Muller S. SUMO-specific proteases/isopeptidases: SENPs and beyond.Genome Biol. 2014; 15: 422Crossref PubMed Scopus (93) Google Scholar, 20Kunz K. Piller T. Muller S. SUMO-specific proteases and isopeptidases of the SENP family at a glance.J. Cell Sci. 2018; 131jcs211904Crossref PubMed Scopus (58) Google Scholar). These cysteine proteases can also reverse SUMOylation by deconjugating SUMO from substrates. The SUMOylation–deSUMOylation cycle governs many biological processes by affecting protein stability, activity, localization as well as their interaction with other proteins (21Hendriks I.A. Vertegaal A.C. A comprehensive compilation of SUMO proteomics.Nat. Rev. Mol. Cell Biol. 2016; 17: 581-595Crossref PubMed Scopus (219) Google Scholar). Deregulation of SUMOylation or deSUMOylation has been implicated in various cancers and neurodegenerative diseases (22Sarge K.D. Park-Sarge O.K. Sumoylation and human disease pathogenesis.Trends Biochem. Sci. 2009; 34: 200-205Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 23Yang Y. He Y. Wang X. Liang Z. He G. Zhang P. Zhu H. Xu N. Liang S. Protein SUMOylation modification and its associations with disease.Open Biol. 2017; 7: 170167Crossref PubMed Scopus (67) Google Scholar). However, there has been a paucity of reports on how SUMO modification modulates cholesterol metabolism. The nuclear form of sterol regulatory element-binding protein (SREBP) 2, the master transcriptional regulator of cholesterol biosynthesis and uptake, can be SUMOylated at the K464 residue for decreased transcriptional activity (24Hirano Y. Murata S. Tanaka K. Shimizu M. Sato R. Sterol regulatory element-binding proteins are negatively regulated through SUMO-1 modification independent of the ubiquitin/26 S proteasome pathway.J. Biol. Chem. 2003; 278: 16809-16819Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). SUMOylation is also reported to promote the nuclear receptor liver receptor homolog 1 to interact with its co-repressor prosperso homeobox protein 1, leading to reduced transcription of the target genes involved in reverse cholesterol transport (25Stein S. Oosterveer M.H. Mataki C. Xu P. Lemos V. Havinga R. Dittner C. Ryu D. Menzies K.J. Wang X. Perino A. Houten S.M. Melchior F. Schoonjans K. SUMOylation-dependent LRH-1/PROX1 interaction promotes atherosclerosis by decreasing hepatic reverse cholesterol transport.Cell Metab. 2014; 20: 603-613Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Whether SUMO can modify and regulate other players of the cholesterol pathway is largely unknown. In the current study, we report for the first time that IDOL is a SUMO1 target protein. SUMOylation occurs at multiple lysine residues including K293, which is also a key ubiquitination site. SUMOylation stabilizes IDOL by competing against its autoubiquitination, thus increasing IDOL protein level and its potency in degrading LDLR. Moreover, we show that SENP1 can deSUMOylate and destabilize IDOL. Overexpression of SENP1 increases LDLR protein level and LDL uptake, whereas knockdown or knockout of SENP1 has opposite effects. Together, these results reveal SUMOylation as a new posttranslational modification that modulates IDOL abundance and suggest a role of SENP1 in regulating the LDLR pathway. SUMOylation can occur on a lysine residue within the inverted SUMOylation consensus motif E/DxKψ (where x stands for any amino acid and ψ for a hydrophobic amino acid) (26Matic I. Schimmel J. Hendriks I.A. van Santen M.A. van de Rijke F. van Dam H. Gnad F. Mann M. Vertegaal A.C. Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif.Mol. Cell. 2010; 39: 641-652Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Analysis of the human IDOL sequence revealed EAK20A and DLK293G, both of which are highly conserved in vertebrates, that matched the criteria (Fig. 1A). To examine whether IDOL is modified by SUMO, we transiently transfected human hepatocellular carcinoma Huh7 cells with the plasmid expressing Flag-tagged IDOL alone or together with the plasmids expressing Myc-tagged UBC9, the SUMO-conjugating enzyme E2, and His-tagged SUMO1. Lysates were pulled down by the nickel beads and examined for the presence of IDOL. We detected a dose-dependent increase in the high-molecular-weight smears when SUMOylation machinery was present (Fig. 1B). However, substitution of SUMO2 or SUMO3 for SUMO1 yielded negative results (Fig. 1C). These data suggest that IDOL is specifically conjugated with SUMO1. To determine the potential SUMOylation site(s) on IDOL, we performed the SUMOylation assay using IDOL mutants in which K20 and K293 were individually or simultaneously replaced by arginine. Contrasting to the wild-type (WT) form of IDOL that was extensively modified by SUMO1, the K293R and K20R/K293R mutants were apparently less SUMOylated, whereas the K20R mutant had a similar level of SUMO1 conjugates (Fig. 1D). Quantitative analysis showed that the K293R mutation indeed caused significant decreases in IDOL SUMOylation (Fig. 1E). These results suggest that IDOL is SUMOylated by SUMO1 mainly at the K293 residue. However, other SUMOylation site(s) might also exist because the K293R mutation did not completely abolish SUMO1-conjugation of IDOL. IDOL is unstable, and the overexpressed IDOL protein has a half-life of about 2 h (8Zelcer 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 (494) Google Scholar, 13Adi D. Lu X.Y. Fu Z.Y. Wei J. Baituola G. Meng Y.J. Zhou Y.X. Hu A. Wang J.K. Lu X.F. Wang Y. Song B.L. Ma Y.T. Luo J. IDOL G51S variant is associated with high blood cholesterol and increases low-density lipoprotein receptor degradation.Arterioscler. Thromb. Vasc. Biol. 2019; 39: 2468-2479Crossref PubMed Scopus (9) Google Scholar). We next sought to investigate whether IDOL SUMOylation may affect its protein abundance. Because there have been no commercial antibodies that can effectively detect endogenous IDOL (10Scotti E. Calamai M. Goulbourne C.N. Zhang L. Hong C. Lin R.R. Choi J. Pilch P.F. Fong L.G. Zou P. Ting A.Y. Pavone F.S. Young S.G. Tontonoz P. IDOL stimulates clathrin-independent endocytosis and multivesicular body-mediated lysosomal degradation of the low-density lipoprotein receptor.Mol. Cell Biol. 2013; 33: 1503-1514Crossref PubMed Scopus (52) Google Scholar, 12Nelson J.K. Sorrentino V. Avagliano Trezza R. Heride C. Urbe S. Distel B. Zelcer N. The deubiquitylase USP2 regulates the LDLR pathway by counteracting the E3-ubiquitin ligase IDOL.Circ. Res. 2016; 118: 410-419Crossref PubMed Scopus (30) Google Scholar), we examined the level of transfected protein in the absence or presence of UBC9 and SUMO1 in Huh7 cells. The expression of IDOL protein was proportionally elevated with increasing concentrations of SUMO1 (Fig. 2A). Single mutation of the K20 residue failed to alter the responsiveness of IDOL to UBC9 and SUMO1, whereas arginine substitution of the K293 residue, which is also the primary ubiquitination site (6Calkin A.C. Goult B.T. Zhang L. Fairall L. Hong C. Schwabe J.W.R. Tontonoz P. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 20107-20112Crossref PubMed Scopus (35) Google Scholar), resulted in an increased basal level of IDOL protein that was resistant to further augmentation by UBC9 and SUMO1 (Fig. 2, B–C). We next treated Huh7 cells transfected with IDOL and UBC9 plus SUMO1 with the proteasome inhibitor MG132 to exclude the possibility that enhanced IDOL protein expression may result from reduced ubiquitination. MG132 caused parallel increases in the amounts of SUMOylated (Fig. 2D) and total (Fig. 2E) IDOL proteins. SENP1 that can deconjugate SUMO1-modified proteins (20Kunz K. Piller T. Muller S. SUMO-specific proteases and isopeptidases of the SENP family at a glance.J. Cell Sci. 2018; 131jcs211904Crossref PubMed Scopus (58) Google Scholar, 27Sharma P. Yamada S. Lualdi M. Dasso M. Kuehn M.R. Senp1 is essential for desumoylating Sumo1-modified proteins but dispensable for Sumo2 and Sumo3 deconjugation in the mouse embryo.Cell Rep. 2013; 3: 1640-1650Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) effectively eliminated the bands higher than the predicted size of unmodified IDOL protein (Fig. 2D). Deletion of the entire RING domain, which contributes to IDOL autoubiquitination and degradation via several mechanisms (6Calkin A.C. Goult B.T. Zhang L. Fairall L. Hong C. Schwabe J.W.R. Tontonoz P. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 20107-20112Crossref PubMed Scopus (35) Google Scholar, 8Zelcer 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 (494) Google Scholar, 11Zhang L. Fairall L. Goult B.T. Calkin A.C. Hong C. Millard C.J. Tontonoz P. Schwabe J.W.R. The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.Genes Dev. 2011; 25: 1262-1274Crossref PubMed Scopus (58) Google Scholar), elevated IDOL SUMOylation as well (Fig. 2F). These results imply a possible competition between the two posttranslational modifications. To directly demonstrate that IDOL SUMOylation can antagonize its ubiquitination, we co-expressed IDOL and ubiquitin in the absence or presence of UBC9 plus SUMO1 in Huh7 cells. As shown in Figure 2G, ubiquitination of IDOL was greatly reduced when SUMOylation components were provided. These results suggest that SUMOylation and ubiquitination regulate IDOL protein levels in a competitive manner. To further investigate the effects of SENP1 on IDOL, we prepared the recombinant SENP1-Flag protein from HEK293T cells (Fig. 3A). The deubiquitinating enzyme USP19 that deconjugates ubiquitin but not SUMO1 was included as a control. The Flag-tagged SENP1 displayed a high potency in hydrolyzing SUMO1-AMC (7-amino-4-methylcoumarin) as the substrate (Fig. 3B). As expected, USP19 but not SENP1 was able to hydrolyze ubiquitin-AFC (7-amino-4-trifluoromethylcoumarin) (Fig. 3C). We next applied increasing concentrations of WT or catalytically inactive (C603S) SENP1 in the IDOL SUMOylation assay. The SUMO1-modified species of IDOL were dose-dependently reduced by the active SENP1 but not the C603S mutant (Fig. 3D). The level of IDOL protein was significantly reduced by higher concentrations of WT SENP1 but remained unresponsive to the inactive SENP1 (Fig. 3, E–F). So far, our results suggest that SUMO1-mediated SUMOylation and SENP1-mediated deSUMOylation are two opposing processes regulating IDOL protein levels. IDOL negatively modulates LDLR stability by targeting its degradation in lysosomes (6Calkin A.C. Goult B.T. Zhang L. Fairall L. Hong C. Schwabe J.W.R. Tontonoz P. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 20107-20112Crossref PubMed Scopus (35) Google Scholar, 7Sorrentino 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 (37) Google Scholar, 8Zelcer 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 (494) Google Scholar). We next sought to determine whether SENP1 affects IDOL-mediated degradation of LDLR. USP2 can counteract the degradative effect of IDOL on LDLR (12Nelson J.K. Sorrentino V. Avagliano Trezza R. Heride C. Urbe S. Distel B. Zelcer N. The deubiquitylase USP2 regulates the LDLR pathway by counteracting the E3-ubiquitin ligase IDOL.Circ. Res. 2016; 118: 410-419Crossref PubMed Scopus (30) Google Scholar) and was used as a control. As shown in Figure 4, A–B, SENP1 prevented degradation of the transfected LDLR protein induced by IDOL to a similar extent as USP2. Inactivation of the protease activity completely abolished the stimulatory effect of SENP1 on LDLR (Fig. 4C). We also generated two individual lines of CRL1601 cells stably expressing SENP1 (hereinafter refer to as the stable cells). We found these cells had elevated levels of endogenous LDLR protein compared with the parental cells (Fig. 4D). The mRNA abundance of Ldlr and Idol was not altered by SENP1 overexpression (Fig. 4E). PCSK9 is another potent LDLR degrader aside from IDOL (28Horton J.D. Cohen J.C. Hobbs H.H. PCSK9: a convertase that coordinates LDL catabolism.J. Lipid Res. 2009; 50: S172-S177Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 29Lagace T.A. PCSK9 and LDLR degradation: regulatory mechanisms in circulation and in cells.Curr. Opin. Lipidol. 2014; 25: 387-393Crossref PubMed Scopus (137) Google Scholar). To rule out the possibility that PCSK9 was involved in LDLR elevation upon SENP1 overexpression, CRL1601 and stable cells were incubated with the purified PCSK9 protein at various concentrations in cholesterol-depleting medium containing lipoprotein-deficient serum, lovastatin and minimal amount of mevalonate, a condition in which the SREBP pathway and thus LDLR expression are activated (2Goldstein J.L. Brown M.S. The LDL receptor.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 431-438Crossref PubMed Scopus (719) Google Scholar). Cells grown under normal cholesterol-rich conditions were used as controls. Despite higher basal levels of LDLR in the stable cells, PCSK9 was capable of inducing LDLR degradation at a similar rate as in WT cells (Fig. S1). We next examined the effects of SENP1 on LDLR-mediated uptake of DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-labeled LDL. CRL1601 and stable cells were deprived of cholesterol and pulsed with DiI-LDL for 1 h. Cells were then shifted to 37 °C to allow DiI-LDL endocytosis to occur. We observed time-dependent increases of DiI-positive puncta in the cytosol of both control and stable cells (Fig. 4F). However, the internalization of DiI-LDL was much faster in the stable cells than that in control cells (Fig. 4G). To corroborate the positive role of SENP1 on LDLR levels and LDL uptake, we knocked down SENP1 in Huh7 cells with two different small interfering RNA duplexes (siSENP1-1, siSENP1-2). Contrary to the findings under the conditions where SENP1 was overexpressed (Fig. 4), silencing of SENP1 decreased the endogenous LDLR protein level (Fig. 5A) without affecting the mRNA expression of LDLR or IDOL (Fig. 5B). Of note, IDOL knockout cells (Fig. 5C) had constitutively high levels of LDLR protein even when SENP1 was depleted (Fig. 5D). These results suggest an absolute requirement of IDOL for LDLR downregulation upon SENP1 deficiency. Consistently, in CRISPR/Cas9-mediated SENP1 knockout cells, we detected profoundly reduced expression of LDLR protein but not that of LDLR or IDOL mRNA (Fig. 5, E–F). The pulse-chase experiment showed that in SENP1 knockout cells, the internalized DiI-LDL was about half of that in control cells following a 1-h pulse (Fig. 5, G–H). Altogether, these results demonstrate that SENP1 increases LDLR protein and LDL uptake through deSUMOylating IDOL. IDOL mediates ubiquitination and degradation of LDLR, and its mutations are tightly associated with abnormal plasma LDL-C levels in human populations (13Adi D. Lu X.Y. Fu Z.Y. Wei J. Baituola G. Meng Y.J. Zhou Y.X. Hu A. Wang J.K. Lu X.F. Wang Y. Song B.L. Ma Y.T. Luo J. IDOL G51S variant is associated with high blood cholesterol and increases low-density lipoprotein receptor degradation.Arterioscler. Thromb. Vasc. Biol. 2019; 39: 2468-2479Crossref PubMed Scopus (9) Google Scholar, 30Weissglas-Volkov D. Calkin A.C. Tusie-Luna T. Sinsheimer J.S. Zelcer N. Riba L. Tino A.M.V. Ordoriez-Sanchez M.L. Cruz-Bautista I. Aguilar-Salinas C.A. Tontonoz P. Pajukanta P. The N342S MYLIP polymorphism is associated with high total cholesterol and increased LDL receptor degradation in humans.J. Clin. Invest. 2011; 121: 3062-3071Crossref PubMed Scopus (47) Google Scholar, 31Sorrentino V. Fouchier S.W. Motazacker M.M. Nelson J.K. Defesche J.C. Dallinga-Thie G.M. Kastelein J.J.P. Hovingh G.K. Zelcer N. Identification of a loss-of-function inducible degrader of the low-density lipoprotein receptor variant in individuals with low circulating low-density lipoprotein.Eur. Heart J. 2013; 34: 1292-1297Crossref PubMed Scopus (42) Google Scholar). So far, self-catalyzed ubiquitination has been the only known posttranslational modification of IDOL. In this study, we present evidence for the first time that IDOL can be conjugated by SUMO1 at several lysine residues including the major autoubiquitination site K293 (Fig. 1). SUMOylation elevates IDOL protein levels by competing against its autoubiquitination (Fig. 2). SENP1 can reverse IDOL SUMOylation and reduce IDOL protein abundance (Fig. 3). This SENP1-mediated deSUMOylation of IDOL attenuates its potency in degrading LDLR and, consequently, increases LDLR expression and LDL endocytosis (Figs. 4 and 5). The proposed model for SUMOylation and SENP1-mediated deSUMOylation of IDOL in regulating the LDLR pathway is depicted in Figure 6. Our findings position SENP1 as a potential regulator of the LDLR pathway and suggest that overexpression of SENP1 may serve as a therapeutic strategy for the treatment of hypercholesterolemia. IDOL is a well-characterized transcriptional target of the liver X receptors (LXRs) (8Zelcer 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 (494) Google Scholar, 32Zhang L. Reue K. Fong L.G. Young S.G. Tontonoz P. Feedback regulation of cholesterol uptake by the LXR-IDOL-LDLR axis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2541-2546Crossref PubMed Scopus (77) Google Scholar) and, as shown here, can be modified by SUMO. Interestingly, LXRs as the oxysterol-sensitive nuclear receptors become SUMOylated in the presence of endogenous and/or synthetic ligands, thereby repressing the transcription of inflammatory target genes in multiple cell types (33Lee J.H. Park S.M. Kim O.S. Lee C.S. Woo J.H. Park S.J. Joe E.H. Jou I. Differential SUMOylation of LXRalpha and LXRbeta mediates transrepression of STAT1 inflammatory signaling in IFN-gamma-stimulated brain astrocytes.Mol. Cell. 2009; 35: 806-817Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 34Venteclef N. Jakobsson T. Ehrlund A. Damdimopoulos A. Mikkonen L. Ellis E. Nilsson L.M. Parini P. Janne O.A. Gustafsson J.A. Steffensen K.R. Treuter E. GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRbeta in the hepatic acute phase response.Genes Dev. 2010; 24: 381-395Crossref PubMed Scopus (132) Google Scholar, 35Le
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