Abstract
The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome complex (CSN) through both enzymatic (deneddylation) and nonenzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.
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References
Hershko A, Ciechanover A, Varshavsky A. Basic medical research award. The ubiquitin system. Nat Med. 2000;6:1073–81.
Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu Rev Biochem. 2009;78:399–434.
Enchev RI, Schulman BA, Peter M. Protein neddylation: beyond cullin-RING ligases. Nat Rev Mol Cell Biol. 2015;16:30–44.
Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009;458:732–6.
Skaar JR, Pagan JK, Pagano M. Mechanisms and function of substrate recruitment by F-box proteins. Nat Rev Mol Cell Biol. 2013;14:369–81.
Cope GA, Suh GS, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, et al. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science. 2002;298:608–11.
Enchev RI, Scott DC, da Fonseca PC, Schreiber A, Monda JK, Schulman BA, et al. Structural basis for a reciprocal regulation between SCF and CSN. Cell Rep. 2012;2:616–27.
Emberley ED, Mosadeghi R, Deshaies RJ. Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate, and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism. J Biol Chem. 2012;287:29679–89.
Fischer ES, Scrima A, Bohm K, Matsumoto S, Lingaraju GM, Faty M, et al. The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation. Cell. 2011;147:1024–39.
Mosadeghi R, Reichermeier KM, Winkler M, Schreiber A, Reitsma JM, Zhang Y, et al. Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle. eLife. 2016;5;e12102.
Reitsma JM, Liu X, Reichermeier KM, Moradian A, Sweredoski MJ, Hess S, et al. Composition and regulation of the cellular repertoire of SCF ubiquitin ligases. Cell. 2017;171:1326–39 e14.
Liu X, Reitsma JM, Mamrosh JL, Zhang Y, Straube R, Deshaies RJ. Cand1-mediated adaptive exchange mechanism enables variation in F-Box protein expression. Mol Cell. 2018;69:773–86 e6.
Wei N, Serino G, Deng XW. The COP9 signalosome: more than a protease. Trends Biochem Sci. 2008;33:592–600.
Wei N, Deng XW. The COP9 signalosome. Annu Rev Cell Dev Biol. 2003;19:261–86.
Glickman MH, Rubin DM, Coux O, Wefes I, Pfeifer G, Cjeka Z, et al. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell. 1998;94:615–23.
Hofmann K, Bucher P. The PCI domain: a common theme in three multiprotein complexes. Trends Biochem Sci. 1998;23:204–5.
Rozen S, Fuzesi-Levi MG, Ben-Nissan G, Mizrachi L, Gabashvili A, Levin Y, et al. CSNAP Is a stoichiometric subunit of the COP9 signalosome. Cell Rep. 2015;13:585–98.
Udeshi ND, Mertins P, Svinkina T, Carr SA. Large-scale identification of ubiquitination sites by mass spectrometry. Nat Protoc. 2013;8:1950–60.
Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 2002;1:376–86.
Emanuele MJ, Elia AE, Xu Q, Thoma CR, Izhar L, Leng Y, et al. Global identification of modular cullin-RING ligase substrates. Cell. 2011;147:459–74.
Yen HC, Elledge SJ. Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science. 2008;322:923–9.
Zheng N, Zhou Q, Wang Z, Wei W. Recent advances in SCF ubiquitin ligase complex: clinical implications. Biochim Biophys Acta. 2016;1866:12–22.
Koren I, Timms RT, Kula T, Xu Q, Li MZ, Elledge SJ. The eukaryotic proteome is shaped by E3 ubiquitin ligases targeting C-terminal degrons. Cell. 2018;173:1622–35 e14.
Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1:2315–9.
Shalit T, Elinger D, Savidor A, Gabashvili A, Levin Y. MS1-based label-free proteomics using a quadrupole orbitrap mass spectrometer. J Proteome Res. 2015;14:1979–86.
Meir M, Galanty Y, Kashani L, Blank M, Khosravi R, Fernandez-Avila MJ, et al. The COP9 signalosome is vital for timely repair of DNA double-strand breaks. Nucleic Acids Res. 2015;43:4517–30.
Dubois EL, Gerber S, Kisselev A, Harel-Bellan A, Groisman R. UV-dependent phosphorylation of COP9/signalosome in UV-induced apoptosis. Oncol Rep. 2016;35:3101–5.
Hannss R, Dubiel W. COP9 signalosome function in the DDR. FEBS Lett. 2011;585:2845–52.
Fuzesi-Levi MG, Ben-Nissan G, Bianchi E, Zhou H, Deery MJ, Lilley KS, et al. Dynamic regulation of the COP9 signalosome in response to DNA damage. Mol Cell Biol. 2014;34:1066–76.
Marti TM, Hefner E, Feeney L, Natale V, Cleaver JE. H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks. Proc Natl Acad Sci USA. 2006;103:9891–6.
Olive PL, Banath JP. The comet assay: a method to measure DNA damage in individual cells. Nat Protoc. 2006;1:23–9.
Gentile M, Latonen L, Laiho M. Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses. Nucleic Acids Res. 2003;31:4779–90.
Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993;53:3976–85.
Soldani C, Scovassi AI. Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis. 2002;7:321–8.
Chaitanya GV, Steven AJ, Babu PP. PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal. 2010;8:31.
Groisman R, Polanowska J, Kuraoka I, Sawada J, Saijo M, Drapkin R, et al. The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell. 2003;113:357–67.
Pierce NW, Lee JE, Liu X, Sweredoski MJ, Graham RL, Larimore EA, et al. Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell. 2013;153:206–15.
Schmidt MW, McQuary PR, Wee S, Hofmann K, Wolf DA. F-box-directed CRL complex assembly and regulation by the CSN and CAND1. Mol Cell. 2009;35:586–97.
Liu Q, Zhou Y, Tang R, Wang X, Hu Q, Wang Y, et al. Increasing the unneddylated Cullin1 portion rescues the CSN phenotypes by stabilizing adaptor modules to drive SCF assembly. Mol Cell Biol. 2017;37:e00109–17.
Bennett EJ, Rush J, Gygi SP, Harper JW. Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell. 2010;143:951–65.
Scott DC, Schulman BA. SCF E3 ligase substrates switch from CAN-D to can-ubiquitylate. Mol Cell. 2018;69:721–3.
Scherer PC, Ding Y, Liu Z, Xu J, Mao H, Barrow JC, et al. Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function. Proc Natl Acad Sci USA. 2016;113:3503–8.
Lingaraju GM, Bunker RD, Cavadini S, Hess D, Hassiepen U, Renatus M, et al. Crystal structure of the human COP9 signalosome. Nature. 2014;512:161–5.
Cavadini S, Fischer ES, Bunker RD, Potenza A, Lingaraju GM, Goldie KN, et al. Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome. Nature. 2016;531:598–603.
Zhao Y, Sun Y. Cullin-RING ligases as attractive anti-cancer targets. Curr Pharm Des. 2013;19:3215–25.
Wang Z, Liu P, Inuzuka H, Wei W. Roles of F-box proteins in cancer. Nat Rev Cancer. 2014;14:233–47.
Kitagawa K, Kitagawa M. The SCF-type E3 ubiquitin ligases as cancer targets. Curr Cancer Drug Targets. 2016;16:119–29.
Lu G, Middleton RE, Sun H, Naniong M, Ott CJ, Mitsiades CS, et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science. 2014;343:305–9.
Schlierf A, Altmann E, Quancard J, Jefferson AB, Assenberg R, Renatus M, et al. Targeted inhibition of the COP9 signalosome for treatment of cancer. Nat Commun. 2016;7:13166.
Lee YH, Judge AD, Seo D, Kitade M, Gomez-Quiroz LE, Ishikawa T, et al. Molecular targeting of CSN5 in human hepatocellular carcinoma: a mechanism of therapeutic response. Oncogene. 2011;30:4175–84.
Pulvino M, Chen L, Oleksyn D, Li J, Compitello G, Rossi R, et al. Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline. Oncotarget. 2015;6:14796–813.
Altmann E, Erbel P, Renatus M, Schaefer M, Schlierf A, Druet A, et al. Azaindoles as zinc-binding small-molecule inhibitors of the JAMM Protease CSN5. Angew Chem Int Ed Engl. 2017;56:1294–7.
Cope GA, Deshaies RJ. Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels. BMC Biochem. 2006;7:1.
Lauinger L, Li J, Shostak A, Cemel IA, Ha N, Zhang Y, et al. Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases. Nat Chem Biol. 2017;13:709–14.
Essletzbichler P, Konopka T, Santoro F, Chen D, Gapp BV, Kralovics R, et al. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 2014;24:2059–65.
Udeshi ND, Svinkina T, Mertins P, Kuhn E, Mani DR, Qiao JW, et al. Refined preparation and use of anti-diglycine remnant (K-epsilon-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments. Mol Cell Proteomics. 2013;12:825–31.
Ben-Nissan G, Belov ME, Morgenstern D, Levin Y, Dym O, Arkind G, et al. Triple-stage mass spectrometry unravels the heterogeneity of an endogenous protein complex. Anal Chem. 2017;89:4708–15.
Kirshenbaum N, Michaelevski I, Sharon M Analyzing large protein complexes by structural mass spectrometry. J Vis Exp. 2010;e1954.
Acknowledgements
We thank Dieter A. Wolf for comments on the paper, and Dr. Ron Rotkopf for the statistical analysis. MS is grateful for the financial support of the US National Institutes of Health, grant no. GM121834 and for an Israel Science Foundation (ISF) grant 300/17. MS is the incumbent of the Aharon and Ephraim Katzir Memorial Professorial Chair. GF is the Incumbent of the David and Stacey Cynamon Research fellow Chair in Genetics and Personalized Medicine
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MGF-L, GB-N, RIE, MP and MS designed the experiments and analyzed the data. MGF-L and IF performed the cell biology and biochemistry experiments. MGF-L and TMS performed the flow cytometry, and MGF-L and RN performed the microscopy experiments. GB-N performed the mass spectrometry and RIE the Kd measurement experiments. YL and MK performed the SILAC and label-free proteomics analysis. GF performed the bioinformatics analysis of the proteomics data. MGF-L, GB-N and MS wrote the paper.
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Füzesi-Levi, M.G., Fainer, I., Ivanov Enchev, R. et al. CSNAP, the smallest CSN subunit, modulates proteostasis through cullin-RING ubiquitin ligases. Cell Death Differ 27, 984–998 (2020). https://doi.org/10.1038/s41418-019-0392-8
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DOI: https://doi.org/10.1038/s41418-019-0392-8
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