Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
17 01 2020
17 01 2020
Historique:
received:
29
08
2019
accepted:
13
12
2019
entrez:
19
1
2020
pubmed:
19
1
2020
medline:
20
11
2020
Statut:
epublish
Résumé
Early in vivo studies demonstrated the involvement of a tumor-suppressing transcription factor, p53, into cellular droplets such as Cajal and promyelocytic leukemia protein bodies, suggesting that the liquid-liquid phase separation (LLPS) might be involved in the cellular functions of p53. To examine this possibility, we conducted extensive investigations on the droplet formation of p53 in vitro. First, p53 itself was found to form liquid-like droplets at neutral and slightly acidic pH and at low salt concentrations. Truncated p53 mutants modulated droplet formation, suggesting the importance of multivalent electrostatic interactions among the N-terminal and C-terminal domains. Second, FRET efficiency measurements for the dimer mutants of p53 revealed that distances between the core domains and between the C-terminal domains were modulated in an opposite manner within the droplets. Third, the molecular crowding agents were found to promote droplet formation, whereas ssDNA, dsDNA, and ATP, to suppress it. Finally, the p53 mutant mimicking posttranslational phosphorylation did not form the droplets. We conclude that p53 itself has a potential to form droplets that can be controlled by cellular molecules and by posttranslational modifications, suggesting that LLPS might be involved in p53 function.
Identifiants
pubmed: 31953488
doi: 10.1038/s41598-020-57521-w
pii: 10.1038/s41598-020-57521-w
pmc: PMC6969132
doi:
Substances chimiques
TP53 protein, human
0
Tumor Suppressor Protein p53
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
580Subventions
Organisme : MEXT/JSPS KAKENHI
ID : JP16K07313
Pays : International
Références
Kamagata, K., Murata, A., Itoh, Y. & Takahashi, S. Characterization of facilitated diffusion of tumor suppressor p53 along DNA using single-molecule fluorescence imaging. J. Photochem. Photobiol. C Photochem. Reviews 30, 36–50 (2017).
doi: 10.1016/j.jphotochemrev.2017.01.004
Tafvizi, A. et al. Tumor suppressor p53 slides on DNA with low friction and high stability. Biophys. J. 95, L01–03 (2008).
doi: 10.1529/biophysj.108.134122
pubmed: 18424488
pmcid: 2426630
Tafvizi, A., Huang, F., Fersht, A. R., Mirny, L. A. & van Oijen, A. M. A single-molecule characterization of p53 search on DNA. Proc. Natl. Acad. Sci. USA 108, 563–568 (2011).
doi: 10.1073/pnas.1016020107
pubmed: 21178072
Leith, J. S. et al. Sequence-dependent sliding kinetics of p53. Proc. Natl. Acad. Sci. USA 109, 16552–16557 (2012).
doi: 10.1073/pnas.1120452109
pubmed: 23012405
Murata, A. et al. One-dimensional sliding of p53 along DNA is accelerated in the presence of Ca(2+) or Mg(2+) at millimolar concentrations. J. Mol. Biol. 427, 2663–2678 (2015).
doi: 10.1016/j.jmb.2015.06.016
pubmed: 26143716
Murata, A. et al. One-dimensional search dynamics of tumor suppressor p53 regulated by a disordered C-terminal domain. Biophys. J. 112, 2301–2314 (2017).
doi: 10.1016/j.bpj.2017.04.038
pubmed: 28591603
pmcid: 5474741
Subekti, D. R. G. et al. The disordered linker in p53 participates in nonspecific binding to and one-dimensional sliding along DNA revealed by single-molecule fluorescence measurements. Biochemistry 56, 4134–4144 (2017).
doi: 10.1021/acs.biochem.7b00292
pubmed: 28718283
Itoh, Y. et al. Activation of p53 facilitates the target search in DNA by enhancing the target recognition probability. J. Mol. Biol. 428, 2916–2930 (2016).
doi: 10.1016/j.jmb.2016.06.001
pubmed: 27291286
Itoh, Y., Murata, A., Takahashi, S. & Kamagata, K. Intrinsically disordered domain of tumor suppressor p53 facilitates target search by ultrafast transfer between different DNA strands. Nucleic Acids Res. 46, 7261–7269 (2018).
doi: 10.1093/nar/gky586
pubmed: 29986056
pmcid: 6101536
Khazanov, N. & Levy, Y. Sliding of p53 along DNA can be modulated by its oligomeric state and by cross-talks between its constituent domains. J. Mol. Biol. 408, 335–355 (2011).
doi: 10.1016/j.jmb.2011.01.059
pubmed: 21338609
Terakawa, T., Kenzaki, H. & Takada, S. p53 searches on DNA by rotation-uncoupled sliding at C-terminal tails and restricted hopping of core domains. J. Am. Chem. Soc. 134, 14555–14562 (2012).
doi: 10.1021/ja305369u
pubmed: 22880817
Terakawa, T. & Takada, S. p53 dynamics upon response element recognition explored by molecular simulations. Sci. Rep. 5, 17107 (2015).
doi: 10.1038/srep17107
pubmed: 26596470
pmcid: 4656996
Hainaut, P., Butcher, S. & Milner, J. Temperature sensitivity for conformation is an intrinsic property of wild-type p53. Br. J. Cancer 71, 227–231 (1995).
doi: 10.1038/bjc.1995.48
pubmed: 7841034
pmcid: 2033583
Ghosh, S. et al. Investigating the intrinsic aggregation potential of evolutionarily conserved segments in p53. Biochemistry 53, 5995–6010 (2014).
doi: 10.1021/bi500825d
pubmed: 25181279
Wang, G. & Fersht, A. R. Mechanism of initiation of aggregation of p53 revealed by Phi-value analysis. Proc. Natl. Acad. Sci. USA 112, 2437–2442 (2015).
doi: 10.1073/pnas.1500243112
pubmed: 25675526
Wang, G. & Fersht, A. R. Propagation of aggregated p53: Cross-reaction and coaggregation vs. seeding. Proc. Natl. Acad. Sci. USA 112, 2443–2448 (2015).
doi: 10.1073/pnas.1500262112
pubmed: 25675527
Cino, E. A., Soares, I. N., Pedrote, M. M., de Oliveira, G. A. & Silva, J. L. Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds. Sci. Rep. 6, 32535 (2016).
doi: 10.1038/srep32535
pubmed: 27600721
pmcid: 5013286
Pedrote, M. M. et al. Aggregation-primed molten globule conformers of the p53 core domain provide potential tools for studying p53C aggregation in cancer. J. Biol. Chem. 293, 11374–11387 (2018).
doi: 10.1074/jbc.RA118.003285
pubmed: 29853637
pmcid: 6065177
Bullock, A. N. et al. Thermodynamic stability of wild-type and mutant p53 core domain. Proc. Natl. Acad. Sci. USA 94, 14338–14342 (1997).
doi: 10.1073/pnas.94.26.14338
pubmed: 9405613
Friedler, A., Veprintsev, D. B., Hansson, L. O. & Fersht, A. R. Kinetic instability of p53 core domain mutants: implications for rescue by small molecules. J. Biol. Chem. 278, 24108–24112 (2003).
doi: 10.1074/jbc.M302458200
pubmed: 12700230
Higashimoto, Y. et al. Unfolding, aggregation, and amyloid formation by the tetramerization domain from mutant p53 associated with lung cancer. Biochemistry 45, 1608–1619 (2006).
doi: 10.1021/bi051192j
pubmed: 16460008
pmcid: 2536691
Herzog, G. et al. Evaluating Drosophila p53 as a model system for studying cancer mutations. J. Biol. Chem. 287, 44330–44337 (2012).
doi: 10.1074/jbc.M112.417980
pubmed: 23135266
pmcid: 3531747
Wang, G. & Fersht, A. R. First-order rate-determining aggregation mechanism of p53 and its implications. Proc. Natl. Acad. Sci. USA 109, 13590–13595 (2012).
doi: 10.1073/pnas.1211557109
pubmed: 22869710
Wilcken, R., Wang, G., Boeckler, F. M. & Fersht, A. R. Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition. Proc. Natl. Acad. Sci. USA 109, 13584–13589 (2012).
doi: 10.1073/pnas.1211550109
pubmed: 22869713
Butler, J. S. & Loh, S. N. Structure, function, and aggregation of the zinc-free form of the p53 DNA binding domain. Biochemistry 42, 2396–2403 (2003).
doi: 10.1021/bi026635n
pubmed: 12600206
Xu, J. et al. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat. Chem. Biol. 7, 285–295 (2011).
doi: 10.1038/nchembio.546
pubmed: 21445056
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
doi: 10.1038/nrm.2017.7
pubmed: 28225081
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357 (2017).
Fogal, V. et al. Regulation of p53 activity in nuclear bodies by a specific PML isoform. EMBO J. 19, 6185–6195 (2000).
doi: 10.1093/emboj/19.22.6185
pubmed: 11080164
pmcid: 305840
Guo, A. et al. The function of PML in p53-dependent apoptosis. Nat. Cell Biol. 2, 730–736 (2000).
doi: 10.1038/35036365
pubmed: 11025664
Cioce, M. & Lamond, A. I. Cajal bodies: a long history of discovery. Annu. Rev. Cell Dev. Biol. 21, 105–131 (2005).
doi: 10.1146/annurev.cellbio.20.010403.103738
pubmed: 16212489
de Stanchina, E. et al. PML is a direct p53 target that modulates p53 effector functions. Mol. Cell 13, 523–535 (2004).
doi: 10.1016/S1097-2765(04)00062-0
pubmed: 14992722
Bao-Lei, T. et al. Knocking down PML impairs p53 signaling transduction pathway and suppresses irradiation induced apoptosis in breast carcinoma cell MCF-7. J. Cell. Biochem. 97, 561–571 (2006).
doi: 10.1002/jcb.20584
pubmed: 16215989
Ding, W., Tong, Y., Zhang, X., Pan, M. & Chen, S. Study of Arsenic Sulfide in Solid Tumor Cells Reveals Regulation of Nuclear Factors of Activated T-cells by PML and p53. Sci. Rep. 6, 19793 (2016).
doi: 10.1038/srep19793
pubmed: 26795951
pmcid: 4726130
Bernardi, R. et al. PML regulates p53 stability by sequestering Mdm2 to the nucleolus. Nat. Cell Biol. 6, 665–672 (2004).
doi: 10.1038/ncb1147
pubmed: 15195100
Alsheich-Bartok, O. et al. PML enhances the regulation of p53 by CK1 in response to DNA damage. Oncogene 27, 3653–3661 (2008).
doi: 10.1038/sj.onc.1211036
pubmed: 18246126
Young, P. J. et al. A direct interaction between the survival motor neuron protein and p53 and its relationship to spinal muscular atrophy. J. Biol. Chem. 277, 2852–2859 (2002).
doi: 10.1074/jbc.M108769200
pubmed: 11704667
Pampin, M., Simonin, Y., Blondel, B., Percherancier, Y. & Chelbi-Alix, M. K. Cross talk between PML and p53 during poliovirus infection: implications for antiviral defense. J. Virol. 80, 8582–8592 (2006).
doi: 10.1128/JVI.00031-06
pubmed: 16912307
pmcid: 1563870
Rokudai, S. et al. MOZ increases p53 acetylation and premature senescence through its complex formation with PML. Proc. Natl. Acad. Sci. USA 110, 3895–3900 (2013).
doi: 10.1073/pnas.1300490110
pubmed: 23431171
Ivanschitz, L. et al. PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence. Proc. Natl. Acad. Sci. USA 112, 14278–14283 (2015).
doi: 10.1073/pnas.1507540112
pubmed: 26578773
Fukuda, T. et al. CACUL1/CAC1 attenuates p53 activity through PML post-translational modification. Biochem. Biophys. Res. Commun. 482, 863–869 (2017).
doi: 10.1016/j.bbrc.2016.11.125
pubmed: 27889610
Selivanova, G., Ryabchenko, L., Jansson, E., Iotsova, V. & Wiman, K. G. Reactivation of mutant p53 through interaction of a C-terminal peptide with the core domain. Mol. Cell. Biol. 19, 3395–3402 (1999).
doi: 10.1128/MCB.19.5.3395
pubmed: 10207063
pmcid: 84132
Huang, F. et al. Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 106, 20758–20763 (2009).
doi: 10.1073/pnas.0909644106
pubmed: 19933326
Retzlaff, M. et al. The regulatory domain stabilizes the p53 tetramer by intersubunit contacts with the DNA binding domain. J. Mol. Biol. 425, 144–155 (2013).
doi: 10.1016/j.jmb.2012.10.015
pubmed: 23103206
Natan, E. et al. Interaction of the p53 DNA-binding domain with its n-terminal extension modulates the stability of the p53 tetramer. J. Mol. Biol. 409, 358–368 (2011).
doi: 10.1016/j.jmb.2011.03.047
pubmed: 21457718
pmcid: 3176915
Krois, A. S., Dyson, H. J. & Wright, P. E. Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain. Proc. Natl. Acad. Sci. USA 115, E11302–e11310 (2018).
doi: 10.1073/pnas.1814051115
pubmed: 30420502
Safari, M. S. et al. Anomalous Dense Liquid Condensates Host the Nucleation of Tumor Suppressor p53 Fibrils. iScience 12, 342–355 (2019).
doi: 10.1016/j.isci.2019.01.027
pubmed: 30739016
pmcid: 6369220
Igarashi, C. et al. DNA garden: a simple method for producing arrays of stretchable DNA for single-molecule fluorescence imaging of DNA binding proteins. Bull. Chem. Soc. Jpn. 90, 34–43 (2017).
doi: 10.1246/bcsj.20160298
Molliex, A. et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123–133 (2015).
doi: 10.1016/j.cell.2015.09.015
pubmed: 26406374
pmcid: 5149108
Boehning, M. et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 25, 833–840 (2018).
doi: 10.1038/s41594-018-0112-y
pubmed: 30127355
Kitayner, M. et al. Structural basis of DNA recognition by p53 tetramers. Mol. Cell 22, 741–753 (2006).
doi: 10.1016/j.molcel.2006.05.015
pubmed: 16793544
Tidow, H. et al. Quaternary structures of tumor suppressor p53 and a specific p53 DNA complex. Proc. Natl. Acad. Sci. USA 104, 12324–12329 (2007).
doi: 10.1073/pnas.0705069104
pubmed: 17620598
Malecka, K. A., Ho, W. C. & Marmorstein, R. Crystal structure of a p53 core tetramer bound to DNA. Oncogene 28, 325–333 (2009).
doi: 10.1038/onc.2008.400
pubmed: 18978813
Chen, Y., Dey, R. & Chen, L. Crystal structure of the p53 core domain bound to a full consensus site as a self-assembled tetramer. Structure 18, 246–256 (2010).
doi: 10.1016/j.str.2009.11.011
pubmed: 20159469
pmcid: 2824536
Emamzadah, S., Tropia, L. & Halazonetis, T. D. Crystal structure of a multidomain human p53 tetramer bound to the natural CDKN1A (p21) p53-response element. Mol. Cancer Res. 9, 1493–1499 (2011).
doi: 10.1158/1541-7786.MCR-11-0351
pubmed: 21933903
Gaglia, G., Guan, Y., Shah, J. V. & Lahav, G. Activation and control of p53 tetramerization in individual living cells. Proc. Natl. Acad. Sci. USA 110, 15497–15501 (2013).
doi: 10.1073/pnas.1311126110
pubmed: 24006363
Sun, Z. et al. Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS. PLoS Biol. 9, e1000614 (2011).
doi: 10.1371/journal.pbio.1000614
pubmed: 21541367
pmcid: 3082519
Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl. Acad. Sci. USA 112, 7189–7194 (2015).
doi: 10.1073/pnas.1504822112
pubmed: 26015579
Maharana, S. et al. RNA buffers the phase separation behavior of prion-like RNA binding proteins. Science 360, 918–921 (2018).
doi: 10.1126/science.aar7366
pubmed: 29650702
pmcid: 6091854
Qamar, S. et al. FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-pi Interactions. Cell 173, 720–734.e715 (2018).
doi: 10.1016/j.cell.2018.03.056
pubmed: 5927716
pmcid: 5927716
Wang, J. et al. A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins. Cell 174, 688–699.e616 (2018).
doi: 10.1016/j.cell.2018.06.006
pubmed: 29961577
pmcid: 6063760
Vernon, R. M., et al. Pi-Pi contacts are an overlooked protein feature relevant to phase separation. eLife 7 (2018).
Pak, C. W. et al. Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein. Mol. Cell 63, 72–85 (2016).
doi: 10.1016/j.molcel.2016.05.042
pubmed: 27392146
pmcid: 4973464
Kim, S. et al. Complexation and coacervation of like-charged polyelectrolytes inspired by mussels. Proc. Natl. Acad. Sci. USA 113, E847–853 (2016).
doi: 10.1073/pnas.1521521113
pubmed: 26831090
Cummings, C. S. & Obermeyer, A. C. Phase Separation Behavior of Supercharged Proteins and Polyelectrolytes. Biochemistry 57, 314–323 (2018).
doi: 10.1021/acs.biochem.7b00990
pubmed: 29210575
Vieregg, J. R. et al. Oligonucleotide-Peptide Complexes: Phase Control by Hybridization. J. Am. Chem. Soc. 140, 1632–1638 (2018).
doi: 10.1021/jacs.7b03567
pubmed: 29314832
Zhou, H. et al. Mechanism of DNA-Induced Phase Separation for Transcriptional Repressor VRN1. Angew. Chem. Int. Ed. Engl. 58, 4858–4862 (2019).
doi: 10.1002/anie.201810373
pubmed: 30762296
Wang, Y. V. et al. Quantitative analyses reveal the importance of regulated Hdmx degradation for P53 activation. Proc. Natl. Acad. Sci. USA 104, 12365–12370 (2007).
doi: 10.1073/pnas.0701497104
pubmed: 17640893
Pearson, M. et al. PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Nature 406, 207–210 (2000).
doi: 10.1038/35018127
pubmed: 10910364
Lahav, G. et al. Dynamics of the p53-Mdm2 feedback loop in individual cells. Nat. Genet. 36, 147–150 (2004).
doi: 10.1038/ng1293
pubmed: 14730303
Tasdemir, E. et al. Regulation of autophagy by cytoplasmic p53. Nat. Cell Biol. 10, 676–687 (2008).
doi: 10.1038/ncb1730
pubmed: 18454141
pmcid: 2676564
Kruger, T. & Scheer, U. p53 localizes to intranucleolar regions distinct from the ribosome production compartments. J. Cell Sci. 123, 1203–1208 (2010).
doi: 10.1242/jcs.062398
pubmed: 20332106
Tendler, Y., Pokroy, R., Panshin, A. & Weisinger, G. p53 protein subcellular localization and apoptosis in rodent corneal epithelium cell culture following ultraviolet irradiation. Int. J. Mol. Med. 31, 540–546 (2013).
doi: 10.3892/ijmm.2013.1247
pubmed: 23338225
Oikawa, H., Kamagata, K., Arai, M. & Takahashi, S. Complexity of the folding transition of the B domain of protein A revealed by the high-speed tracking of single-molecule fluorescence time series. J. Phys. Chem. B 119, 6081–6091 (2015).
doi: 10.1021/acs.jpcb.5b00414
pubmed: 25938341