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• W2148532207 abstract "The neddylation-cullin-RING E3 ligase (CRL) pathway has recently been identified as a potential oncogenic event and attractive anticancer target; however, its underlying mechanisms have not been well elucidated. In this study, RhoB, a well known tumor suppressor, was identified and validated with an iTRAQ-based quantitative proteomic approach as a new target of this pathway in liver cancer cells. Specifically, cullin 2-RBX1 E3 ligase, which requires NEDD8 conjugation for its activation, interacted with RhoB and promoted its ubiquitination and degradation. In human liver cancer tissues, the neddylation-CRL pathway was overactivated and reversely correlated with RhoB levels. Moreover, RhoB accumulation upon inhibition of the neddylation-CRL pathway for anticancer therapy contributed to the induction of tumor suppressors p21 and p27, apoptosis, and growth suppression. Our findings highlight the degradation of RhoB via the neddylation-CRL pathway as an important molecular event that drives liver carcinogenesis and RhoB itself as a pivotal effector for anticancer therapy targeting this oncogenic pathway. The neddylation-cullin-RING E3 ligase (CRL) pathway has recently been identified as a potential oncogenic event and attractive anticancer target; however, its underlying mechanisms have not been well elucidated. In this study, RhoB, a well known tumor suppressor, was identified and validated with an iTRAQ-based quantitative proteomic approach as a new target of this pathway in liver cancer cells. Specifically, cullin 2-RBX1 E3 ligase, which requires NEDD8 conjugation for its activation, interacted with RhoB and promoted its ubiquitination and degradation. In human liver cancer tissues, the neddylation-CRL pathway was overactivated and reversely correlated with RhoB levels. Moreover, RhoB accumulation upon inhibition of the neddylation-CRL pathway for anticancer therapy contributed to the induction of tumor suppressors p21 and p27, apoptosis, and growth suppression. Our findings highlight the degradation of RhoB via the neddylation-CRL pathway as an important molecular event that drives liver carcinogenesis and RhoB itself as a pivotal effector for anticancer therapy targeting this oncogenic pathway. Post-translational protein neddylation is a process of the covalent attachment of NEDD8, 1The abbreviations used are:NEDD8neural precursor cell-expressed developmentally down-regulated 8NAE1NEDD8-activating enzyme 1UBC12ubiquitin-conjugating enzyme E2MCRLcullin-RING E3 ligaseMdm2mouse double minute 2 homologHuRhuman antigen RRBX1RING box protein-1iTRAQisobaric tag for relative and absolute quantitationHUVEChuman umbilical vein endothelial cellDMSOdimethyl sulfoxide. 1The abbreviations used are:NEDD8neural precursor cell-expressed developmentally down-regulated 8NAE1NEDD8-activating enzyme 1UBC12ubiquitin-conjugating enzyme E2MCRLcullin-RING E3 ligaseMdm2mouse double minute 2 homologHuRhuman antigen RRBX1RING box protein-1iTRAQisobaric tag for relative and absolute quantitationHUVEChuman umbilical vein endothelial cellDMSOdimethyl sulfoxide. a ubiquitin-like small molecule, to lysine residues of substrate proteins and thus regulates their function through modulating their conformation, stability or subcellular localization (1Xirodimas D.P. Novel substrates and functions for the ubiquitin-like molecule NEDD8.Biochem. Soc. Trans. 2008; 36: 802-806Crossref PubMed Scopus (149) Google Scholar, 2Rabut G. Peter M. Function and regulation of protein neddylation. “Protein modifications: beyond the usual suspects” review series.EMBO Rep. 2008; 9: 969-976Crossref PubMed Scopus (262) Google Scholar). NEDD8 conjugation to substrates is catalyzed by a three-step enzymatic cascade mediated by NEDD8-activating enzyme (E1, NAE1, and UBA3 form a heterodimer), NEDD8-conjugating enzyme (E2, UBC12 or UBE2F), and NEDD8 E3 ligases sequentially (1Xirodimas D.P. Novel substrates and functions for the ubiquitin-like molecule NEDD8.Biochem. Soc. Trans. 2008; 36: 802-806Crossref PubMed Scopus (149) Google Scholar, 2Rabut G. Peter M. Function and regulation of protein neddylation. “Protein modifications: beyond the usual suspects” review series.EMBO Rep. 2008; 9: 969-976Crossref PubMed Scopus (262) Google Scholar, 3Lee I. Schindelin H. Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes.Cell. 2008; 134: 268-278Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). So far, the well recognized neddylation substrates are the cullin family, and they serve as the fundamental components of multiunit cullin-RING E3 ligase (CRL) as well as the potential anticancer target (4Wu J.T. Lin H.C. Hu Y.C. Chien C.T. Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation.Nat. Cell Biol. 2005; 7: 1014-1020Crossref PubMed Scopus (139) Google Scholar, 5Sakata E. Yamaguchi Y. Miyauchi Y. Iwai K. Chiba T. Saeki Y. Matsuda N. Tanaka K. Kato K. Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity.Nat. Struct. Mol. Biol. 2007; 14: 167-168Crossref PubMed Scopus (96) Google Scholar). Functionally, NEDD8 conjugation to cullins changes the conformation of CRL and leads to its activation for protein ubiquitination and degradation (4Wu J.T. Lin H.C. Hu Y.C. Chien C.T. Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation.Nat. Cell Biol. 2005; 7: 1014-1020Crossref PubMed Scopus (139) Google Scholar, 5Sakata E. Yamaguchi Y. Miyauchi Y. Iwai K. Chiba T. Saeki Y. Matsuda N. Tanaka K. Kato K. Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity.Nat. Struct. Mol. Biol. 2007; 14: 167-168Crossref PubMed Scopus (96) Google Scholar). In addition, NEDD8 conjugation has been reported to stabilize oncoproteins Mdm2 (6Xirodimas D.P. Saville M.K. Bourdon J.C. Hay R.T. Lane D.P. Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar) and HuR (7Embade N. Fernández-Ramos D. Varela-Rey M. Beraza N. Sini M. Gutiérrez de Juan V. Woodhoo A. Martínez-López N. Rodríguez-Iruretagoyena B. Bustamante F.J. de la Hoz A.B. Carracedo A. Xirodimas D.P. Rodríguez M.S. Lu S.C. Mato J.M. Martínez-Chantar M.L. Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation.Hepatology. 2012; 55: 1237-1248Crossref PubMed Scopus (90) Google Scholar) while to repress the transcriptional activity of tumor suppressor p53 (6Xirodimas D.P. Saville M.K. Bourdon J.C. Hay R.T. Lane D.P. Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). Most recently, we reported that the entire neddylation pathway, including NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and global neddylation of substrates, is overactivated in human lung cancer and associates with worse overall survival of patients (8Li L. Wang M. Yu G. Chen P. Li H. Wei D. Zhu J. Xie L. Jia H. Shi J. Li C. Yao W. Wang Y. Gao Q. Jeong L.S. Lee H.W. Yu J. Hu F. Mei J. Wang P. Chu Y. Qi H. Yang M. Dong Z. Sun Y. Hoffman R.M. Jia L. Overactivated neddylation pathway as a therapeutic target in lung cancer.J. Natl. Cancer Inst. 2014; 106: dju083Crossref PubMed Scopus (131) Google Scholar). These findings highlight a pivotal role of neddylation in carcinogenesis and tumor progression; however, whether neddylation is overactivated in other types of tumors, such as liver cancer, and how it facilitates tumor development remain elusive. neural precursor cell-expressed developmentally down-regulated 8 NEDD8-activating enzyme 1 ubiquitin-conjugating enzyme E2M cullin-RING E3 ligase mouse double minute 2 homolog human antigen R RING box protein-1 isobaric tag for relative and absolute quantitation human umbilical vein endothelial cell dimethyl sulfoxide. neural precursor cell-expressed developmentally down-regulated 8 NEDD8-activating enzyme 1 ubiquitin-conjugating enzyme E2M cullin-RING E3 ligase mouse double minute 2 homolog human antigen R RING box protein-1 isobaric tag for relative and absolute quantitation human umbilical vein endothelial cell dimethyl sulfoxide. Inhibition of the neddylation-CRL pathway has recently emerged as a promising anticancer strategy (9Soucy T.A. Dick L.R. Smith P.G. Milhollen M.A. Brownell J.E. The NEDD8 conjugation pathway and its relevance in cancer biology and therapy.Genes Cancer. 2010; 1: 708-716Crossref PubMed Scopus (166) Google Scholar). MLN4924, a small molecule inhibitor of NAE, has been discovered as a first-in-class anticancer agent by high throughput screening and has been advanced into several Phase I clinical trials due to its significant anticancer efficacy and tolerated toxicity in preclinical studies (9Soucy T.A. Dick L.R. Smith P.G. Milhollen M.A. Brownell J.E. The NEDD8 conjugation pathway and its relevance in cancer biology and therapy.Genes Cancer. 2010; 1: 708-716Crossref PubMed Scopus (166) Google Scholar, 10Tanaka T. Nakatani T. Kamitani T. Negative regulation of NEDD8 conjugation pathway by novel molecules and agents for anticancer therapy.Curr. Pharm. Des. 2013; 19: 4131-4139Crossref PubMed Scopus (15) Google Scholar, 11Tanaka T. Nakatani T. Kamitani T. Inhibition of NEDD8-conjugation pathway by novel molecules: potential approaches to anticancer therapy.Mol. Oncol. 2012; 6: 267-275Crossref PubMed Scopus (48) Google Scholar, 12Nawrocki S.T. Griffin P. Kelly K.R. Carew J.S. MLN4924: a novel first-in-class inhibitor of NEDD8-activating enzyme for cancer therapy.Expert Opin. Investig. Drugs. 2012; 21: 1563-1573Crossref PubMed Scopus (134) Google Scholar). Mechanistically, MLN4924 blocks cullin neddylation, inactivates CRL, induces the accumulation of CRL substrates and eventually causes DNA damage, cell cycle defects, senescence, apoptosis, and autophagy (13Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1356) Google Scholar, 14Luo Z. Yu G. Lee H.W. Li L. Wang L. Yang D. Pan Y. Ding C. Qian J. Wu L. Chu Y. Yi J. Wang X. Sun Y. Jeong L.S. Liu J. Jia L. The Nedd8-activating enzyme inhibitor MLN4924 induces autophagy and apoptosis to suppress liver cancer cell growth.Cancer Res. 2012; 72: 3360-3371Crossref PubMed Scopus (181) Google Scholar, 15Jia L. Li H. Sun Y. Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression.Neoplasia. 2011; 13: 561-569Crossref PubMed Scopus (122) Google Scholar, 16Milhollen M.A. Traore T. Adams-Duffy J. Thomas M.P. Berger A.J. Dang L. Dick L.R. Garnsey J.J. Koenig E. Langston S.P. Manfredi M. Narayanan U. Rolfe M. Staudt L.M. Soucy T.A. Yu J. Zhang J. Bolen J.B. Smith P.G. MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-κB-dependent lymphoma.Blood. 2010; 116: 1515-1523Crossref PubMed Scopus (261) Google Scholar). So far, several classes of CRL substrates have been reported to accumulate upon neddylation inhibition and mediate MLN4924-induced cellular responses in a cell context-dependent manner. They include but are not limited to 1) DNA replication licensing proteins CDT1 and ORC1, inducing DNA rereplication stress and DNA damage (13Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1356) Google Scholar, 15Jia L. Li H. Sun Y. Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression.Neoplasia. 2011; 13: 561-569Crossref PubMed Scopus (122) Google Scholar); 2) cell cycle inhibitors such as p21, p27, and Wee1, inducing cell cycle arrest and senescence (13Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1356) Google Scholar, 15Jia L. Li H. Sun Y. Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression.Neoplasia. 2011; 13: 561-569Crossref PubMed Scopus (122) Google Scholar, 17Mackintosh C. García-Domínguez D.J. Ordóñez J.L. Ginel-Picardo A. Smith P.G. Sacristán M.P. de Álava E. WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells.Oncogene. 2013; 32: 1441-1451Crossref PubMed Scopus (54) Google Scholar); 3) NF-κB inhibitor IκB-α, resulting in inhibition of NF-κB activity (16Milhollen M.A. Traore T. Adams-Duffy J. Thomas M.P. Berger A.J. Dang L. Dick L.R. Garnsey J.J. Koenig E. Langston S.P. Manfredi M. Narayanan U. Rolfe M. Staudt L.M. Soucy T.A. Yu J. Zhang J. Bolen J.B. Smith P.G. MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-κB-dependent lymphoma.Blood. 2010; 116: 1515-1523Crossref PubMed Scopus (261) Google Scholar); and 4) mTOR inhibitory protein Deptor, leading to inactivation of the mammalian target of rapamycin and induction of autophagic responses (18Zhao Y. Xiong X. Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCFβTrCP E3 ubiquitin ligase and regulates survival and autophagy.Mol. Cell. 2011; 44: 304-316Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 19Luo Z. Pan Y. Jeong L.S. Liu J. Jia L. Inactivation of the Cullin (CUL)-RING E3 ligase by the NEDD8-activating enzyme inhibitor MLN4924 triggers protective autophagy in cancer cells.Autophagy. 2012; 8: 1677-1679Crossref PubMed Scopus (43) Google Scholar). Given that CRL, the largest multiunit ubiquitin ligase family in cells, is in charge of degradation of ∼20% of ubiquitinated cellular proteins (13Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1356) Google Scholar), the identification of novel CRL substrates will not only broaden our understanding of how the neddylation-CRL pathway regulates tumor development, but also provide a new insight into how cancer cells respond to anticancer therapy targeting this oncogenic pathway. RhoB, a member of the Rho family of small GTPases, participates in the modulation of numerous essential cellular processes, including actin organization, gene transcription, cell adhesion, proliferation, apoptosis and vesicle traffic (20Bishop A.L. Hall A. Rho GTPases and their effector proteins.Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1676) Google Scholar, 21Prendergast G.C. Actin' up: RhoB in cancer and apoptosis.Nat. Rev. Cancer. 2001; 1: 162-168Crossref PubMed Scopus (189) Google Scholar, 22Liu A. Cerniglia G.J. Bernhard E.J. Prendergast G.C. RhoB is required to mediate apoptosis in neoplastically transformed cells after DNA damage.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6192-6197Crossref PubMed Scopus (132) Google Scholar). Moreover, RhoB controls a variety of important cancer-associated signaling pathways through modulation of many downstream effectors. For example, RhoB triggers cell cycle arrest or apoptosis through inducing the expression of p21 and p27 (23Marlow L.A. Reynolds L.A. Cleland A.S. Cooper S.J. Gumz M.L. Kurakata S. Fujiwara K. Zhang Y. Sebo T. Grant C. McIver B. Wadsworth J.T. Radisky D.C. Smallridge R.C. Copland J.A. Reactivation of suppressed RhoB is a critical step for the inhibition of anaplastic thyroid cancer growth.Cancer Res. 2009; 69: 1536-1544Crossref PubMed Scopus (59) Google Scholar, 24Mazzocca A. Giusti S. Hamilton A.D. Sebti S.M. Pantaleo P. Carloni V. Growth inhibition by the farnesyltransferase inhibitor FTI-277 involves Bcl-2 expression and defective association with Raf-1 in liver cancer cell lines.Mol. Pharmacol. 2003; 63: 159-166Crossref PubMed Scopus (20) Google Scholar). RhoB also regulates the traffic of several critical cancer-associated molecules, such as epidermal growth factor receptor, platelet-derived growth factor receptor, chemokine receptor, PDK1, Akt, and Src (25Gampel A. Parker P.J. 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Expression loss and revivification of RhoB gene in ovary carcinoma carcinogenesis and development.PLoS ONE. 2013; 8: e78417Crossref PubMed Scopus (17) Google Scholar). Moreover, RhoB expression is further decreased or lost in invasive tumors and poorly differentiated tumors (35Liu Y. Song N. Ren K. Meng S. Xie Y. Long Q. Chen X. Zhao X. Expression loss and revivification of RhoB gene in ovary carcinoma carcinogenesis and development.PLoS ONE. 2013; 8: e78417Crossref PubMed Scopus (17) Google Scholar, 36Bousquet E. Mazières J. Privat M. Rizzati V. Casanova A. Ledoux A. Mery E. Couderc B. Favre G. Pradines A. Loss of RhoB expression promotes migration and invasion of human bronchial cells via activation of AKT1.Cancer Res. 2009; 69: 6092-6099Crossref PubMed Scopus (59) Google Scholar). In contrast, the revivification of RhoB expression inhibits the malignant phenotypes of cancer cells, such as cell proliferation, migration, and invasion (33Zhou J. Zhu Y. Zhang G. Liu N. Sun L. Liu M. Qiu M. Luo D. Tang Q. Liao Z. Zheng Y. Bi F. A distinct role of RhoB in gastric cancer suppression.Int. J. Cancer. 2011; 128: 1057-1068Crossref PubMed Scopus (55) Google Scholar, 37Couderc B. Pradines A. Rafii A. Golzio M. Deviers A. Allal C. Berg D. Penary M. Teissie J. Favre G. In vivo restoration of RhoB expression leads to ovarian tumor regression.Cancer Gene Ther. 2008; 15: 456-464Crossref PubMed Scopus (46) Google Scholar). These findings highlight that down-regulation of RhoB is an important molecular event that drives carcinogenesis; however, the molecular mechanisms by which RhoB expression is regulated during the process are largely unknown. In this study, RhoB was identified and validated via iTRAQ-based quantitative proteomic analysis as a new substrate of the neddylation-CRL pathway. Moreover, the expression of this pathway, mediating the degradation of RhoB, was overactivated in human liver cancer. Inhibition of this pathway induced RhoB accumulation, which led to tumor-suppressive cellular responses. Our studies revealed a previously unknown regulatory mechanism of RhoB loss in liver cancer and validated RhoB as a novel target of the neddylation-CRL pathway in human liver carcinoma. Human hepatocellular carcinoma cell lines HepG2 and Huh7, lung cancer cell line A549, breast cancer cell line MCF-7, pancreatic cancer cell line MiaPaCa-2, colon cancer cell line HCT116, and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection, and routinely cultured. MLN4924 was synthesized and used for in vitro studies as described previously (38Lee H.W. Nam S.K. Choi W.J. Kim H.O. Jeong L.S. Stereoselective synthesis of MLN4924, an inhibitor of NEDD8-activating enzyme.J. Org. Chem. 2011; 76: 3557-3561Crossref PubMed Scopus (27) Google Scholar). For in vitro studies, MLN4924 was dissolved in dimethyl sulfoxide (DMSO) and kept at −20°C. HUVEC extracts were prepared following our published methods (39Ding C. Jiang J. Wei J. Liu W. Zhang W. Liu M. Fu T. Lu T. Song L. Ying W. Chang C. Zhang Y. Ma J. Wei L. Malovannaya A. Jia L. Zhen B. Wang Y. He F. Qian X. Qin J. A fast workflow for identification and quantification of proteomes.Mol. Cell. Proteomics. 2013; 12: 2370-2380Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Briefly, proteins of HUVECs were extracted with 8 m urea and 50 mm NH4HCO3, and 200 μg of protein was reduced by adding 2.96 μl of 0.1 m dithiotheitol for 4 h at 37 °C and then alkylated by adding 3.29 μl of 0.5 m iodoacetamide for 60 min at room temperature in the dark. The protein sample was diluted to 1 m urea with 50 mm NH4HCO3 in water and digested with trypsin at a mass ratio of 1:50 enzyme/protein overnight at 37 °C, followed by termination with 1% formic acid. Peptide mixtures (80 μg) from each condition were labeled with iTRAQ 4plex reagent (Applied Biosystems, Foster City, CA), with tag 114 for the DMSO condition and tag 116 for the MLN4924 condition (40Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3687) Google Scholar). The reaction was terminated by adding an equal volume of distilled water (41Narumi R. Murakami T. Kuga T. Adachi J. Shiromizu T. Muraoka S. Kume H. Kodera Y. Matsumoto M. Nakayama K. Miyamoto Y. Ishitobi M. Inaji H. Kato K. Tomonaga T. A strategy for large-scale phosphoproteomics and SRM-based validation of human breast cancer tissue samples.J. Proteome Res. 2012; 11: 5311-5322Crossref PubMed Scopus (77) Google Scholar). The labeled peptides were combined, desalted with a C18 solid-phase extraction column (Waters Associates, Milford, MA), and lyophilized for further analysis. Peptide mixtures were separated by off-line high-pH reversed phase chromatography first following the parameters, and 19 fractions were generated for further LC-MS/MS analysis, for which a nanoflow HPLC instrument (EASY-nLC 1000 system, Thermo Fisher Scientific, Waltham, MA) coupled to an on-line Q Exactive mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ion source (Thermo Fisher Scientific) was used. The chromatography columns were packed in-house with Ultimate XB-C18 3-μm resin (Welch Materials, Shanghai, China). The peptide mixtures were loaded onto the C18 reversed phase column (10-cm length, 75-μm inner diameter) with buffer A (99.5% water and 0.5% formic acid) and separated with a 75-min linear gradient of 5–100% buffer B (99.5% acetonitrile and 0.5% formic acid) at a flow rate of 300 nl/min. Including the loading and washing steps, the total time for an LC-MS/MS run was ∼90 min. The electrospray voltage was 2.0 kV. Peptides were analyzed by data-dependent MS/MS acquisition with dynamic exclusion duration of 18 s. In MS1, the resolution was 70,000, the automatic gain control (AGC) target was 3e6, and the maximum injection time was 20 ms. In MS2, the resolution was 17,500, the automatic gain control target was 1e6, and the maximum injection time was 60 ms. The scan range was 300–1400 m/z, and the top 75 intensive precursor ions were selected for MS/MS analysis. The raw data were processed using the proteomic workflow of Proteome Discoverer 1.3. The fragmentation spectra were searched against the UniProt reviewed human database (20130415, 20268 sequences) using the Mascot search engine (version 2.2.06) with the precursor and fragment mass tolerances set to 15 ppm and 20 milli-mass units (mmu), respectively. Two missed cleavage sites were allowed. The fixed modification was carbamidomethylation (Cysteine), and the variable modifications were oxidation (Methionine), acetylation (protein N terminus), and iTRAQ labeling (Tyrosine and Lysine, N-terminal residue). Peptide ions were filtered using the cutoff scores of Percolator based on p < .01. The false discovery rate was set to 1% for peptide identifications. An additional filter was applied with the removal of spectrum matches with scores lower than 10. The iTRAQ quantization values were automatically calculated on the basis of the intensity of the iTRAQ reporter ions in the dissociation scans with higher collision energy using Proteome Discoverer (41Narumi R. Murakami T. Kuga T. Adachi J. Shiromizu T. Muraoka S. Kume H. Kodera Y. Matsumoto M. Nakayama K. Miyamoto Y. Ishitobi M. Inaji H. Kato K. Tomonaga T. A strategy for large-scale phosphoproteomics and SRM-based validation of human breast cancer tissue samples.J. Proteome Res. 2012; 11: 5311-5322Crossref PubMed Scopus (77) Google Scholar). All protein iTRAQ ratios were exported to an Excel file, the Gaussian distribution of ratios of 116:114 was recalculated manually, and all ratios were transformed to base 10 logarithm values. A confidence interval of 99% was used to determine the cutoff values for statistically significant changes (42Unwin R.D. Griffiths J.R. Whetton A.D. Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC-MS/MS.Nat. Protoc. 2010; 5: 1574-1582Crossref PubMed Scopus (206) Google Scholar). HepG2 or Huh7 cells were transfected with siRNA oligonucleotides using Lipofectamine 2000 reagent (Life Technologies, Invitrogen, CA) according to the manufacturer's instructions. The sequences of siRNAs are as follows: for RBX1 (43Jia L. Soengas M.S. Sun Y. ROC1/RBX1 E3 ubiquitin ligase silencing suppresses tumor cell growth via sequential induction of G2-M arrest, apoptosis, and senescence.Cancer Res. 2009; 69: 4974-4982Crossref PubMed Scopus (99) Google Scholar), 5′-GACUUUCCCUGCUGUUACCUAATT-3′ and 5′-GGACAACAGAGAGUGGGAATT-3′; for RBX2 (44Li H. Tan M. Jia L. Wei D. Zhao Y. Chen G. Xu J. Zhao L. Thomas D. Beer D.G. Sun Y. Inactivation of SAG/RBX2 E3 ubiquitin ligase suppresses KrasG12D-driven lung tumorigenesis.J. Clin. Invest. 2014; 124: 835-846Crossref PubMed Scopus (62) Google Scholar), 5′-GAGGACUGUGUUGUGGUCUTT-3′; for NAE1 (45Luo J. Emanuele M.J. Li D. Creighton C.J. Schlabach M.R. Westbrook T.F. Wong K.K. Elledge S.J. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene.Cel" @default.
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• W2148532207 title "The Neddylation-Cullin 2-RBX1 E3 Ligase Axis Targets Tumor Suppressor RhoB for Degradation in Liver Cancer" @default.
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