The stress protein Nupr1 is a highly basic, multifunctional, intrinsically disordered protein (IDP). MSL1 is a histone acetyl transferase-associated protein, known to intervene in the dosage compensation complex (DCC). In this work, we show that both Nupr1 and MSL1 proteins were recruited and formed a complex into the nucleus in response to DNA-damage, which was essential for cell survival in reply to cisplatin damage. We studied the interaction of Nupr1 and MSL1, and their binding affinities to DNA by spectroscopic and biophysical methods. The MSL1 bound to Nupr1, with a moderate affinity (2.8 μM) in an entropically-driven process. MSL1 did not bind to non-damaged DNA, but it bound to chemically-damaged-DNA with a moderate affinity (1.2 μM) also in an entropically-driven process. The Nupr1 protein bound to chemically-damaged-DNA with a slightly larger affinity (0.4 μM), but in an enthalpically-driven process. Nupr1 showed different interacting regions in the formed complexes with Nupr1 or DNA; however, they were always disordered (“fuzzy”), as shown by NMR. These results underline a stochastic description of the functionality of the Nupr1 and its other interacting partners.
References
[1]
Smith ER, Cayrou C, Huang R, Lane WS, Coté J, et al. (2005) A human protein complex homologous to the Drosophila MSL complex is responsible for the majority of histone H4 acetylation at lysine 16. Mol Cell Biol 25: 9175–9188.
[2]
Mendjan S, Akhtar A (2007) The right dose for every sex. Chromosoma 116: 95–106.
[3]
Gelbart ME, Kuroda MI (2009) Drosophila dosage compensation: A complex voyage to the X- chromosome. Development 136: 1399–1410.
[4]
Hilfiker A, Hilfiker-Kleiner D, Pannuti A, Lucchesi JC (1997) MOF, a putative acetyl transferase gene related to the Tip60 and MOZ human genes and to the SAS genes of yeast, is required for dosage compensation in Drosophila. EMBO J 16: 2054–2060.
[5]
Morales V, Straub T, Neumann MF, Mengus G, Akhtar A, et al. (2004) Functional integration of the histone acetyltransferase MOF into the dosage compensation complex. EMBO J 23: 2258–2268.
[6]
Füllgrabe J, Lynch-Day MA, Heldring N, Li W, Struijk RB, et al. (2013) The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy. Nature 500: 468–471.
[7]
Marín I (2003) Evolution of chromatin-remodelling complexes: comparative genomics reveals the ancient origin of “novel” compensasome genes. J Mol Evol 56: 527–539.
[8]
Wu L, Zee BM, Wang Y, García BA, Dou Y (2011) The RING finger protein MSL2 in the MOF complex is an E3 ubiquitin ligase for HB2 K34 and is involved in cross-talk with H3 K4 and K79 methylation. Mol Cell 43: 132–144.
[9]
Li X, Wu L, Corsa CA, Kunkel S, Dou Y (2009) Two mammalian MOF complexes regulate transcription activation by distinct mechanisms. Mol Cell 36: 290–301.
[10]
Li X, Corsa CA, Pan PW, Wu L, Ferguson D, et al. (2010) MOF and H4 K16 acetylation play important roles in DNA damage repair by modulating recruitment of DNA damage repair protein Mdc1. Mol Cell Biol 30: 5335–5347.
[11]
Li X, Dou Y (2010) New perspectives for the regulation of acetyltransferase MOF. Epigenetics 5: 185–188.
[12]
Dmitriev RI, Korneenko TV, Bessonov AA, Shakhparonov MI, Modyanov NN, et al. (2007) Characterization of hampin/MSL1 as a node in the nuclear interactome. Biochem Biophys Res Commun 355: 1051–1057.
[13]
Cai Y, Jin J, Swanson SK, Colle MD, Choi SH, et al. (2010) Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex. J Biol Chem 285: 4268–4272.
[14]
Raja SJ, Charapitsa I, Conrad T, Vaquerizas JM, Gebhardt P, et al. (2010) The nonspecific lethal complex is a transcriptional regulator in Drosophila. Mol Cell 38: 827–841.
[15]
Huang J, Wan B, Wu L, Yang Y, Dou Y, et al. (2012) Structural insight into the regulation of MOF in the male-specific lethal complex and the non-specific lethal complex. Cell Res 22: 1078–1081.
[16]
Kadlec J, Hallacli E, Lipp M, Holz H, Sánchez-Wetherby J, et al. (2011) Structural basis for MOF and MSL3 recruitment into the dosage compensation complex by MSL1. Nature Struct Mol Biol 18: 142–149.
[17]
Hallacli E, Lipp M, Georgiev P, Spieleman C, Cusack S, et al. (2012) MSL1-mediated dimerization of the dosage compensation complex is essential for male X-chromosome regulation in Drosophila. Mol Cell 48: 587–600.
[18]
Mallo GV, Fieldler F, Calvo EL, Ortiz EM, Vasseur S, et al. (1997) Cloning an expression of the rat Nupr1 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development and regeneration, and which promotes cellular growth. J Biol Chem 272: 32360–32369.
[19]
Encinar JA, Mallo GV, Mizyrycki C, Giono L, González-Ros JM, et al. (2001) Human Nupr1 is a HMG-I/Y-like protein with DNA binding activity enhanced by phosphorylation. J Biol Chem 276: 2742–2751.
Hamidi T, Algül H, Cano CE, Sandi MJ, Molejon MI, et al. (2012) Nuclear protein 1 promotes pancreatic cancer development and protects cells from stress by inhibiting apoptosis. J Clin Invest 122: 2092–2103.
[22]
Cano CE, Hamidi T, Sandi MJ, Iovanna JL (2011) Nupr1: the Swiss-knife to cancer. J Cell Physiol 226: 1439–1443.
[23]
Goruppi S, Iovanna JL (2010) Stress-inducible protein Nupr1 in several physiological and pathological processes. J Biol Chem 285: 1577–1581.
[24]
Gironella M, Malicet C, Cano CE, Sandi MJ, Hamidi T, et al. (2009) p8/Nupr1 regulates DNA-repair activity after double-strand gamma irradiation induced DNA damage. J Cell Physiol 221: 594–602.
[25]
Carracedo A, Gironella M, Lorente M, García S, Guzmán M, et al. (2006) Cannabinoids induce apoptosis of pancreatic tumour cells via endoplasmic reticulum stress-related genes. Cancer Res 66: 6748–6755.
[26]
Neira JL, Sandi MJ, Bacarizo J, Archange C, Cámara-Artigas A, et al. (2013) An N-terminally truncated mutant of human chemokine CXCL14 has biological activity. Protein Pep Letters 20: 955–967.
[27]
Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260: 289–298.
[28]
Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182: 319–326.
[29]
Bodenhausen G, Ruben D (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69: 185–189.
[30]
Piotto M, Saudek V, Sklenar V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2: 661–665.
[31]
Cavanagh J, Fairbrother WJ, Palmer AG 3rd, Skelton N (1996) Protein NMR spectroscopy. Principles and Practice (1st ed), Academic Press, NY.
[32]
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, et al. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6: 277–293.
[33]
Johnson BA, Blevins RA (1994) NMR view: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4: 603.
[34]
Pantoja-Uceda D, Santoro J (2008) Amino acid type identification in NMR spectra of proteins via beta- and gamma-carbon edited experiments. J Magn Reson 195: 187–195.
[35]
Pantoja-Uceda D, Santoro J (2012) New amino acid residue type identification experiments valid for protonated and deuterated proteins. J Biomol NMR 54: 145–153.
[36]
Wishart DS, Bigam CG, Yao J, Abildgaard F, Dyson HJ, et al. (1995) 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR 6: 135–140.
[37]
Aguado-Llera D, Martínez-Gómez AI, Prieto J, Marenchino M, Traverso JA, et al. (2011) The conformational stability and biophysical properties of the eukaryotic thioredoxins of Pisum sativum are not family-conserved. PLoS One 6: e17068.
[38]
Dyson HJ (2011) Expanding the proteome: disordered or alternatively folded proteins. Q Rev Biophys 44: 467–518.
[39]
Khan H, Cino EA, Brickenden A, Fan J, Yand D, et al. (2013) Fuzzy complex formation between the intrinsically disordered prothymosin α and the kelch domain of Keap1 involved in the oxidative stress response. J Mol Biol 425: 1011–1027.
[40]
Fuxreiter M, Tompa P (2012) Fuzzy complexes: a more stcohastic view of protein function. Adv Exp Med Biol 725: 1–14.
[41]
Tzeng SR, Kalodimos CG (2009) Dynamic activation of an allosteric regulator protein. Nature 462: 368–372.
[42]
Fuxreiter M, Simon I, Bondos S (2011) Dynamic protein-DNA recognition: beyond what can be seen. Trends Biochem Sci 36: 415–423.
[43]
Savvides SN, Raghunathan S, Fütterer K, Kozlov AG, Lohman TM, et al. (2004) The C-terminal domain of full-length E. coli SSB is disordered even when bound to DNA. Protein Sci 13: 1942–1947.
[44]
Malicet C, Giroux V, Vasseur S, Dagorn JC, Neira JL, et al. (2006) Regulation of apoptosis by the p8/prothymosin alpha complex. Proc Natl Acad Sci USA 103: 2671–2676.
[45]
Bravo J, Neira JL (2010) Folded and unfolded conformations of proteins involved in pancreatic cancer: a layman’s guide. The Scientific World Journal 10: 1613–1633.
[46]
Berjanski MV, Wishart DS (2005) A simple method to predict protein flexibility using secondary chemical shifts. J Am Chem Soc 127: 14970–14971.
