Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

DAXX represents a new type of protein-folding enabler

Abstract

Protein quality control systems are crucial for cellular function and organismal health. At present, most known protein quality control systems are multicomponent machineries that operate via ATP-regulated interactions with non-native proteins to prevent aggregation and promote folding1, and few systems that can broadly enable protein folding by a different mechanism have been identified. Moreover, proteins that contain the extensively charged poly-Asp/Glu (polyD/E) region are common in eukaryotic proteomes2, but their biochemical activities remain undefined. Here we show that DAXX, a polyD/E protein that has been implicated in diverse cellular processes3,4,5,6,7,8,9,10, possesses several protein-folding activities. DAXX prevents aggregation, solubilizes pre-existing aggregates and unfolds misfolded species of model substrates and neurodegeneration-associated proteins. Notably, DAXX effectively prevents and reverses aggregation of its in vivo-validated client proteins, the tumour suppressor p53 and its principal antagonist MDM2. DAXX can also restore native conformation and function to tumour-associated, aggregation-prone p53 mutants, reducing their oncogenic properties. These DAXX activities are ATP-independent and instead rely on the polyD/E region. Other polyD/E proteins, including ANP32A and SET, can also function as stand-alone, ATP-independent molecular chaperones, disaggregases and unfoldases. Thus, polyD/E proteins probably constitute a multifunctional protein quality control system that operates via a distinctive mechanism.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: DAXX prevents protein misfolding and aggregation.
Fig. 2: DAXX dissolves protein aggregates and unfolds misfolded species.
Fig. 3: Other polyD/E proteins can function as molecular chaperones, disaggregases, and unfoldases.
Fig. 4: DAXX maintains and restores the native conformation of p53 and MDM2.

Data availability

All data supporting the findings of this study are provided within the manuscript and its Supplementary InformationSource data are provided with this paper.

Code availability

Source code and datasets for polyD/E protein analysis are available in GitHub: https://github.com/SunmoonTao/de-enrichemnt-analysis.

References

  1. 1.

    Balchin, D., Hayer-Hartl, M. & Hartl, F. U. In vivo aspects of protein folding and quality control. Science 353, aac4354 (2016).

    Article  Google Scholar 

  2. 2.

    Karlin, S., Brocchieri, L., Bergman, A., Mrazek, J. & Gentles, A. J. Amino acid runs in eukaryotic proteomes and disease associations. Proc. Natl Acad. Sci. USA 99, 333–338 (2002).

    ADS  CAS  Article  Google Scholar 

  3. 3.

    Yang, X., Khosravi-Far, R., Chang, H. Y. & Baltimore, D. Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell 89, 1067–1076 (1997).

    CAS  Article  Google Scholar 

  4. 4.

    Chang, H. Y., Nishitoh, H., Yang, X., Ichijo, H. & Baltimore, D. Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx. Science 281, 1860–1863 (1998).

    ADS  CAS  Article  Google Scholar 

  5. 5.

    Perlman, R., Schiemann, W. P., Brooks, M. W., Lodish, H. F. & Weinberg, R. A. TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation. Nat. Cell Biol. 3, 708–714 (2001).

    CAS  Article  Google Scholar 

  6. 6.

    Zhao, L. Y. et al. Negative regulation of p53 functions by Daxx and the involvement of MDM2. J. Biol. Chem. 279, 50566–50579 (2004).

    CAS  Article  Google Scholar 

  7. 7.

    Tang, J. et al. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. 8, 855–862 (2006).

    CAS  Article  Google Scholar 

  8. 8.

    Lewis, P. W., Elsaesser, S. J., Noh, K. M., Stadler, S. C. & Allis, C. D. Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proc. Natl Acad. Sci. USA 107, 14075–14080 (2010).

    ADS  CAS  Article  Google Scholar 

  9. 9.

    Song, M. S. et al. The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network. Nature 455, 813–817 (2008).

    ADS  CAS  Article  Google Scholar 

  10. 10.

    Mahmud, I. & Liao, D. DAXX in cancer: phenomena, processes, mechanisms and regulation. Nucleic Acids Res. 47, 7734–7752 (2019).

    CAS  Article  Google Scholar 

  11. 11.

    Michaelson, J. S., Bader, D., Kuo, F., Kozak, C. & Leder, P. Loss of Daxx, a promiscuously interacting protein, results in extensive apoptosis in early mouse development. Genes Dev. 13, 1918–1923 (1999).

    CAS  Article  Google Scholar 

  12. 12.

    Jiao, Y. et al. DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 331, 1199–1203 (2011).

    ADS  CAS  Article  Google Scholar 

  13. 13.

    Gopal, R. K. et al. Widespread chromosomal losses and mitochondrial DNA alterations as genetic drivers in hurthle cell carcinoma. Cancer Cell 34, 242–255 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    Knowles, T. P., Vendruscolo, M. & Dobson, C. M. The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 15, 384–396 (2014).

    CAS  Article  Google Scholar 

  15. 15.

    Jackrel, M. E. et al. Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events. Cell 156, 170–182 (2014).

    CAS  Article  Google Scholar 

  16. 16.

    Jucker, M. & Walker, L. C. Self-propagation of pathogenic protein aggregates in neurodegenerative diseases. Nature 501, 45–51 (2013).

    ADS  CAS  Article  Google Scholar 

  17. 17.

    Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489 (2003).

    ADS  CAS  Article  Google Scholar 

  18. 18.

    Saibil, H. Chaperone machines for protein folding, unfolding and disaggregation. Nat. Rev. Mol. Cell Biol. 14, 630–642 (2013).

    CAS  Article  Google Scholar 

  19. 19.

    Glover, J. R. & Lindquist, S. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73–82 (1998).

    CAS  Article  Google Scholar 

  20. 20.

    Sharma, S. K., De los Rios, P., Christen, P., Lustig, A. & Goloubinoff, P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat. Chem. Biol. 6, 914–920 (2010).

    CAS  Article  Google Scholar 

  21. 21.

    Gupta, R. et al. Firefly luciferase mutants as sensors of proteome stress. Nat. Methods 8, 879–884 (2011).

    CAS  Article  Google Scholar 

  22. 22.

    Outeiro, T. F. et al. Formation of toxic oligomeric alpha-synuclein species in living cells. PLoS ONE 3, e1867 (2008).

    ADS  Article  Google Scholar 

  23. 23.

    Rentzeperis, D., Jonsson, T. & Sauer, R. T. Acceleration of the refolding of Arc repressor by nucleic acids and other polyanions. Nat. Struct. Biol. 6, 569–573 (1999).

    CAS  Article  Google Scholar 

  24. 24.

    Walker, J. M., Hastings, J. R. & Johns, E. W. A novel continuous sequence of 41 aspartic and glutamic residues in a non-histone chromosomal protein. Nature 271, 281–282 (1978).

    ADS  CAS  Article  Google Scholar 

  25. 25.

    Adachi, Y., Pavlakis, G. N. & Copeland, T. D. Identification and characterization of SET, a nuclear phosphoprotein encoded by the translocation break point in acute undifferentiated leukemia. J. Biol. Chem. 269, 2258–2262 (1994).

    CAS  Article  Google Scholar 

  26. 26.

    Vaesen, M. et al. Purification and characterization of two putative HLA class II associated proteins: PHAPI and PHAPII. Biol. Chem. Hoppe Seyler 375, 113–126 (1994).

    CAS  Article  Google Scholar 

  27. 27.

    Wang, D. et al. Acetylation-regulated interaction between p53 and SET reveals a widespread regulatory mode. Nature 538, 118–122 (2016).

    ADS  CAS  Article  Google Scholar 

  28. 28.

    Butler, J. S. & Loh, S. N. Folding and misfolding mechanisms of the p53 DNA binding domain at physiological temperature. Protein Sci. 15, 2457–2465 (2006).

    CAS  Article  Google Scholar 

  29. 29.

    Ishimaru, D. et al. Fibrillar aggregates of the tumor suppressor p53 core domain. Biochemistry 42, 9022–9027 (2003).

    CAS  Article  Google Scholar 

  30. 30.

    Ano Bom, A. P. et al. Mutant p53 aggregates into prion-like amyloid oligomers and fibrils: implications for cancer. J. Biol. Chem. 287, 28152–28162 (2012).

    CAS  Article  Google Scholar 

  31. 31.

    Zhang, R. & Monsma, F. Fluorescence-based thermal shift assays. Curr. Opin. Drug Discov. Devel. 13, 389–402 (2010).

    CAS  PubMed  Google Scholar 

  32. 32.

    Muller, P. A. & Vousden, K. H. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell 25, 304–317 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Guo, L. et al. A cellular system that degrades misfolded proteins and protects against neurodegeneration. Mol. Cell 55, 15–30 (2014).

    CAS  Article  Google Scholar 

  34. 34.

    Zhu, G. et al. TRIM11 prevents and reverses protein aggregation and rescues a mouse model of Parkinson’s disease. Cell Rep. 33, 108418 (2020).

    CAS  Article  Google Scholar 

  35. 35.

    Bykov, V. J. N., Eriksson, S. E., Bianchi, J. & Wiman, K. G. Targeting mutant p53 for efficient cancer therapy. Nat. Rev. Cancer 18, 89–102 (2018).

    CAS  Article  Google Scholar 

  36. 36.

    Chu, Y. & Yang, X. SUMO E3 ligase activity of TRIM proteins. Oncogene 30, 1108–1116 (2011).

    CAS  Article  Google Scholar 

  37. 37.

    Chen, L. et al. Enhanced degradation of misfolded proteins promotes tumorigenesis. Cell Rep. 18, 3143–3154 (2017).

    CAS  Article  Google Scholar 

  38. 38.

    Yen, C. F., Harischandra, D. S., Kanthasamy, A. & Sivasankar, S. Copper-induced structural conversion templates prion protein oligomerization and neurotoxicity. Sci. Adv. 2, e1600014 (2016).

    ADS  Article  Google Scholar 

  39. 39.

    Månsson, C. et al. Interaction of the molecular chaperone DNAJB6 with growing amyloid-beta 42 (Aβ42) aggregates leads to sub-stoichiometric inhibition of amyloid formation. J. Biol. Chem. 289, 31066–31076 (2014).

    Article  Google Scholar 

  40. 40.

    Zhang, Y. et al. Upregulation of antioxidant capacity and nucleotide precursor availability suffices for oncogenic transformation. Cell Metab. 33, 94–109 (2021).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank S. K. Sharma and P. Goloubinoff for providing the LucD plasmid, D. Brady for help with protein purification, D. Ricketts and A. Olia for help with the thermal shift assay, J. Huang for technical assistance, and E. Dean for Sf9 protein production. This work was supported by National Institutes of Health (NIH) grants R01CA182675, R01CA184867, R01CA235760, R01CA243520 (X.Y.), P01 AG031862 (R.M.) and R01GM099836 (J.S.); an Alzheimer’s Association Research Fellowship and a Warren Alpert Foundation Distinguished Scholars Fellowship (J.L.); and a Sponsored Research Agreement from Wealth Strategy Holding Limited (X.Y.).

Author information

Affiliations

Authors

Contributions

X.Y. conceived and supervised the study. L.H. and X.Y. designed the experiments. L.H. performed most experiments. T.A. initiated the study and performed part of in vitro assays. G.Z. helped with in vitro assays. S.Y. helped with p53-related experiments. L.T. performed protein sequence analysis. R.M. helped with protein production in insect cells and advised on thermal shift assay. J.S. and J.L. provided HSP104 protein and advised on protein folding assays. X.Y. and L.H. prepared the manuscript with major inputs from J.S. and R.M. and comments from all other authors.

Corresponding author

Correspondence to Xiaolu Yang.

Ethics declarations

Competing interests

X.Y. is a founder and equity holder of Evergreen Therapeutics LLC, which received investments from Wealth Strategy Holding Limited. J.S. is a consultant for Dewpoint Therapeutics, Maze Therapeutics, Vivid Sciences, Korro Bio, and ADRx. The other authors declare no competing financial interests.

Additional information

Peer review information Nature thanks Jerson Silva and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Purification of recombinant DAXX proteins and their ability to prevent luciferase misfolding and aggregation.

a, Scheme for purifying DAXX–6xHis from bacteria BL21(DE3) and insect Sf9 cells. To generate full-length protein, DAXX was fused to GST at the N terminus with a TEV protease cleavage site inserted in between GST and DAXX, and to 6xHis at the C terminus. The fusion protein was first purified with glutathione resins. Beads-bound GST–DAXX–6xHis protein was treated with TEV protease to released DAXX–6xHis, which was subsequently purified with Ni-NTA resins. After elution with imidazole, DAXX-6xHis was further purified with ion-exchange and gel-filtration columns, and concentrated as needed. b, c, DAXX–6xHis purified from bacteria BL21(DE3) cells (b) and insect Sf9 cells (c) were analysed by SDS–PAGE and Coomassie blue staining. Bovine serum albumin (BSA) was used as a protein standard (c). Mass spectrometry analysis indicated that the vast majority of species in the minor bands of the DAXX preps were derived from DAXX. d, Scheme for purifying Flag–DAXX from HEK293T cells. Flag–DAXX was transiently transfected in HEK293T cells and purified using anti-Flag M2 beads. After elution with 3xFlag peptides, Flag–DAXX was further purified with ion-exchange and gel filtration columns, and concentrated as needed. e, Flag–DAXX purified from HEK293T cells was analysed by SDS–PAGE and Coomassie blue staining. Mass spectrometry analysis indicated that the vast majority of species in the minor bands of the DAXX preps were derived from DAXX. fh, DAXX proteins purified from bacteria, insect cells, and mammalian cells protect luciferase from heat-induced inactivation and aggregation. Luciferase (f, g, 5 nM; h, 200 nM) was heated at 42 °C in the presence of indicated concentrations of GST, DAXX–6xHis (from bacteria) or HSP70 (plus HSP40 at a half concentration, same below) for 1 min (f), or in the presence or absence of GST, DAXX–6xHis (from bacteria), DAXX–6xHis (from insect cells), or Flag–DAXX (200 nM each) for the indicated times (g, h). Shown are luciferase activity relative to the native protein (f, g) and relatively turbidity measured at OD600 (h). The DAXX protein purified from HEK293T cells appeared to be more active than those purified from bacteria and insect cells i, j, Protective activity of DAXX for a higher amount of luciferase. Luciferase (50 nM) was heated at 42 °C in the presence of the indicated concentrations of GST, DAXX–6xHis (from Sf9 cells), or HSP70 for 1 min (i), or in the presence of absence of GST, DAXX–6xHis (Sf9 cells) or HSP70–HSP40 (200 nM each) for the indicated times. Luciferase activity was normalized to native protein. RT-CTRL, control luciferase sample kept at room temperature Assays in b, c and e have been performed three times with similar results. Numerical data are mean ± s.d. (n = 3) and are representative of three (f, j) or two (g, h, i) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001,  ns, not significant; unpaired Student’s t-test.

Source data

Extended Data Fig. 2 DAXX prevents α-Syn and Aβ42 aggregation, acts independently of ATP, and probably exists as a monomer.

a, b, PFF-induced aggregation of soluble α-Syn monomers and its inhibition by DAXX. α-Syn monomers (13.3 μM) was incubated alone, together with in α-Syn PFFs (133 nM) (a), or together with in α-Syn PFFs (133 nM) in the presence of GST (0.2 μM), HSPs (0.2 μM HSP70, 0.1 μM HSP40, and 0.4 μM HSP104(A503S)), and DAXX–6xHis (from Sf9 cells, 0.1, 0.2,and 0.4 μM) (b). Aggregation was monitored by real-time quaking-induced conversion (RT-QuIC) assay. ce, DAXX suppresses Aβ42 fibrillization for a prolonged incubation, during which DAXX itself did not form fibrils or other sedimentable aggregates. Aβ42 monomers (10 μM) and DAXX–6xHis (from Sf9 cells, 0.05, 0.1, 0.2, 0.4 and 0.6 μM) were incubated alone (c, e) or together (d) at 37 °C for 120 h. Formation of fibrils was analysed by ThT fluorescence assay (c, d). Solubility of DAXX was analysed by sedimentation assay (e). f, g, DAXX blocks Aβ42 monomers to form PFFs that accelerate aggregation of fresh Aβ42 monomers and Aβ42 PFF-induced aggregation of fresh Aβ42 monomers. Aβ42 monomers (10 μM) were incubated at 37 °C alone, together Aβ42 PFFs (6 nM) (f, g), Aβ42 (6 nM) preincubated with DAXX–6xHis (from Sf9 cells) at a 100:1 molar ratio (Aβ42/DAXX), Aβ42/DAXX plus DAXX–6xHis (0.6 μM) (DAXX) (f), or Aβ42 PFFs (6 nM) in the presence of DAXX (g). Formation of fibrils was analysed by ThT fluorescence assay. Assays in f and g were done at the same time. h, i, The chaperone activity of DAXX is not affected by the addition of ATP or the treatment of apyrase. Luciferase (0.2 μM) was heated at 42 °C in presence of GST (0.2 μM) (h, i), DAXX–6xHis (insect cells, 0.2 μM) with or without ATP (5 mM ATP-Mg2+ plus an ATP-regeneration system) and apyrase as indicated (h), or HSP70–HSP40 (0.2 and 0.1 μM, respectively) with or without apyrase (i). Aggregation formation was monitored by OD at 600 nm. j, DAXX does not bind to ATP. Recombinant DAXX–6xHis and HSP70 were incubated with agarose beads conjugated without ATP (−) or with ATP via the phosphate moiety (AP-ATP), ribose moiety (EDA-ATP), or the adenine base at different positions (6AH-ATP and 8AH-ATP). The input and pulldown samples were analysed by western blot. k, DAXX exists as a homogeneous species of relatively low molecular weights. Recombinant Flag–DAXX protein was analysed by Superdex 200 10/300 GL column. Proteins standards (in kDa) are indicated. l, DAXX likely exists as a monomer. Recombinant Flag–DAXX (1 μM) was crosslinked with indicated concentration of DSS at 25 °C for 30 min and analysed by western blot. Flag–p53 (1 μM), which is expected to be a tetramer, was used as control. Similar results were obtained for DAXX–6xHis. Assays have been performed three (be, k, l) or two (a, h, i, j) times with similar results. Numerical data are mean ± s.d. (n = 3) and are representative of three independent experiments (f, g).

Source data

Extended Data Fig. 3 DAXX can dissolve small luciferase aggregates, but not large luciferase aggregates or α-Syn fibrils.

ac, DAXX dissolves and reactivates heat-denatured luciferase aggregates. Heat-denatured luciferase (5 nM) was incubated at 25 °C with GST (100 nM), DAXX–6xHis (from E. coli, 100 nM), and HSPs (100 nM HSP70, 50 nM HSP40 and 200 nM HSP104(A503S)) for the indicated times (a), or with the indicated amounts of GST or DAXX–6xHis for 90 min (b, c). Shown are luciferase activity relative to that of native luciferase (b), and the amount of luciferase in the SN after sedimentation (a, c). d, Disaggregase activity of DAXX proteins purified from different sources. Relative activity of heat-denatured luciferase (5 nM) that was incubated at 25 °C for 90 min with increasing concentrations of DAXX purified from the indicated sources. e, f, Disaggregase activity of DAXX for a higher amount of denatured luciferase. Relative activity of heat-denatured luciferase (50 nM) that was incubated at 25 °C with the increasing molar ratios of DAXX–6xHis (from Sf9 cells) for the indicated times (e) or 90 min (f). g, DAXX achieves the maximal recovery of luciferase activity at fivefold excess. Heat-denatured luciferase (0.1, 0.2, 0.5, 1 and 2 μM) was incubated with DAXX–6xHis (0.1 μM) at 25 °C for 90 min. Luciferase activity is relative to that of native luciferase. h, DAXX restores the native conformation to denatured luciferase. Native or heat-denatured luciferase (1 μM) incubated alone or together with GST (1 μM) or DAXX–6xHis (0.1×: 0.1 μM; 0.5×: 0.5 μM) for 90 min were examined by circular dichroism spectroscopy. Data were analysed by CAPITO, with the percentages of β-stand shown in Fig. 2b. i, Different sizes of luciferase aggregates generated by heat and urea treatments. Luciferase (1 μM) denatured by heat or urea was fractionated on Superdex 200 10/300 GL column. Fractions were analysed by western blot and the relative abundance of luciferase is indicated. j, DAXX cannot reactive urea-denatured luciferase. Relative activity of urea-denatured luciferase (5 nM) that was incubated with GST (0.2 μM), DAXX (0.2 and 1 μM), or HSPs (0.2 μM HSP70, 0.1 μM HSP40 and 0.4 μM HSP104(A503S)) at 25 °C for 90 min. k, l, Luciferase denatured by heat (k) or urea (l) was fractionated on gel filtration chromatography. Fractions in the range of 44 to 2,000 kDa were incubated with buffer, lysozyme (0.1 μM), DAXX–6xHis (0.1 μM), or HSP70–HSP40–HSP104(A503S) (0.1, 0.05 and 0.2 μM, respectively) at 25 °C for 90 min, and luciferase activity was determined. mp, DAXX is unable to dissolve α-Syn fibrils. Preformed α-Syn fibrils (0.2 μM) were treated with GST (0.2 μM) (mp), DAXX–6xHis at the indicated molar ratios (m, n) or at 0.2 μM (o, p), HSPs (0.2 μM HSP70, 0.1 μM HSP40, and 0.4 μM HSP104(A503S) plus ATP and an ATP regeneration system) (mp), or both DAXX and HSPs (o, p). Reactions mixtures were analysed by dot blot (m, o), and soluble α-Syn relative to total α-Syn was quantified (n, p) (n = 3). Assays in a, c, h, i, kp have been performed three times with similar results. Numerical data are mean ± s.d. (n = 3) and are representative of three independent experiments (b, d-g, j). **P < 0.01, ***P < 0.001; unpaired Student’s t-test.

Source data

Extended Data Fig. 4 DAXX unfolds misfolded LucD and protects against protein aggregation and oligomerization in cells.

a, A schematic representation of compact misfolded LucD monomers, the unfolded intermediates, and the native conformers, as well as their sensitivity to brief trypsin digestion. b, DAXX changes trypsin sensitivity of LucD. LucD (50 nM) was incubated with DAXX-–xHis (100 nM), GST (100 nM), or HSPs (100 nM HSP70, 50 nM HSP40 and 200 nM HSP104(A503S)) at 25 °C. At indicated time points, aliquots of luciferase were incubated with 2.5 μM trypsin at 22 °C for 2 min and were analysed by western blot. Shown is luciferase band intensity. A representative western blot is presented in Fig. 2g. c, DAXX increases the enzymatic activity of LucD. Misfolded LucD monomers (50 nM) were incubated with GST (100 nM) or DAXX–6xHis (100 nM) for indicated durations and assayed for luciferase activity. d, DAXX elevates the levels of nLucDM, but not nLuc, in cells. nLuc or nLucDM was transfected together with empty vector (EV) or Flag–DAXX in HEK293T cells. Cell lysates were analysed by western blot 24 h after transfection. e, f, DAXX reduces aggregation, but not expression, of ATXN1(82Q) in cells. U2OS cells transfected with HA-Atxn1-82Q together with EV, Flag–DAXX (e, f) or GFP–HSP70 (f) were analysed by western blot (e), or immunofluorescence with anti-HA (red) and anti-Flag (green) antibodies (f; scale bar, 10 μm). Quantification of the percentage of cells containing different sizes Atxn1-82Q inclusions is shown in Fig. 2i. g, Schematic representation of BiFC assay based on Venus, an improved version of yellow fluorescent protein (YFP). hj, DAXX inhibits α-Syn oligomerization in cells. HEK293T cells were transfected with V1S and SV2 individually, or together with empty vector (EV) or DAXX. Cells were analysed by western blot for protein expression (h) and by fluorescence microscopy for BiFC signals and Flag–DAXX expression (i; scale bars, 100 μm), with the quantification of BiFC signals shown in j. Assay in d, e, f, h and i have been performed two times with similar results. Numerical data are mean ± s.d. (n = 3) and are representative of three independent experiments (b, c, j). *P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t-test.

Source data

Extended Data Fig. 5 DAXX binds to misfolded proteins and depends on the polyD/E region for its activity.

a, The DAXX-luciferase interaction is increased upon heat shock. HEK293T cells were transfected with HA–DAXX and Flag–nLuc as indicated, and treated with or without heat shock. Cell lysates were immunoprecipitated with anti-Flag mAb (M2) beads. Input and immunoprecipitated samples were analysed by western blot. b, DAXX preferentially binds to heat-denatured luciferase. GST or GST–DAXX (100 nM each) was incubated with native (N) or heat-denatured (D) 6xHis-luciferase immobilized on Ni-NTA agarose. The input and pulldown samples were analysed by western blot. c, Binding of DAXX to cellulose-bound peptide scans derived from luciferase, p53, MDM2, H3.3, H4 and DAXX. Each peptide contained thirteen amino acid residues that overlapped adjacent peptides by ten. d, Protein sequence of DAXX. The poly D/E region is marked in red colour, and the four continuous runs of Asp/Glu (with 14, 11, 7, and 5 residues, respectively) are underlined. e, Schematic presentation of full-length DAXX and its mutants. DAXX(D/E) was fused to GST at the N terminus for protein stabilization. f, DAXX(ΔD/E) and DAXX(D/E) lack unfoldase activity. Misfolded LucD monomers (50 nM) were incubated with DAXX(ΔD/E) or DAXX(D/) (100 nM each) for the indicated times and assayed for luciferase activity (,ean ± s.d., n = 3). g, h, DAXX(ΔD/E) and DAXX(D/E) remain soluble during heat treatment. Recombinant DAXX ΔD/E (g) and D/E (h) proteins were heated at 42 °C for the indicated durations. Luciferase (200 nM) was used as a positive control. Aggregation formation was monitored by OD600 and normalized to the luciferase control. Assays in ac have been performed two times with similar results. Numerical data are mean or mean ± s.d. (n = 3) and are representative of two independent experiments (fh).

Source data

Extended Data Fig. 6 Role of other polyD/E proteins in protein folding.

a, Sequences of human SET and ANP32A proteins. The poly D/E region is marked in red colour, and the continuous runs of Asp/Glu are underlined. b, c, ANP32A and SET does not block α-Syn fibrillization. α-Syn monomers (70 μM) were incubated with SET or ANP32A (0.4 μM each) for 7 days. Samples were analysed by electron microscopy (b) and ThT staining (c). Scale bar, 100 nm. d, SET and ANP32A are unable to solubilize urea-denatured luciferase. Urea-denatured luciferase (5 nM) was incubated with GST, SET, ANP32A (0.2 μM each) or HSPs (0.2 μM HSP70, 0.1 μM HSP40, and 0.4 μM HSP104(A503S)) at 25 °C for 90 min. Activity relative to that of the native control are shown. e, f, Unfoldase activity of ANP32A and SET. Misfolded LucD (50 nM) was incubated with GST, SET or ANP32A (200 nM each) at 25 °C. At indicated time points, aliquots of refolding luciferase were incubated with 2.5 μM trypsin at 22 °C for 2 min, denatured in SDS sample buffer, and analysed by western blot (e), with the quantification showed in (f). g, h, Schematic presentation of SET and its deletion mutants (g), and the numbers of Asp (D) and Glu (E) in each mutant (h). i, Heat-inactivated luciferase (5 nM) was incubated at 25 °C with SET or its deletion mutants (200 nM each) for 90 min. Activity relative to that of native luciferase are shown. j, Number of polyD/E proteins different species. k, l, Gene Ontology analysis of polyD/E proteins in humans. Proteins are classified into pie chart based on their molecular functions (k) and protein classes (l). Assays have been performed two (b) or three (e) times with similar results. Numerical data are mean ± s.d. (n = 3) and are representative of two independent experiments (c, d, f, i). **P < 0.01, unpaired Student’s t-test.

Source data

Extended Data Fig. 7 DAXX maintains the native conformation of both p53 and MDM2.

a, DAXX abrogates p53 fibrillization. Recombinant wild-type p53 and DAXX–6xHis proteins (5 μM each) were incubated alone or together at 37 °C for 2 h in the presence of ThT (25 μM). Formation of amyloid fibrils was assayed by ThT. b, c, DAXX(ΔD/E) and DAXX(D/E) cannot protect p53 from aggregation. Native p53 (n-p53) (b), or denatured p53 aggregates (d-p53) (c), (100 nM each) was incubated with GST, Flag–DAXX, DAXX(D/E) or DAXX(ΔD/E) (200 nM each) at 37 °C (b) or at 25 °C (c) for the indicated times. Samples were partitioned into supernatant (soluble) and pellet (insoluble) fractions via sedimentation, and analysed by western blot. As in Fig. 4a, b except that DAXX(D/E) and DAXX(ΔD/E) samples are included. d, e, g, h, DAXX restore native conformation to denatured p53 and MDM2. Native p53 (d) or native MDM2 (n-MDM2, g), or denatured p53 (e) or MDM2 (d-MDM2, h), (1 μM each) was incubated alone or together with GST or DAXX–6xHis (0.5, 1, 2 μM, from Sf9 cells) at the indicated molar ratios for 3 h and analysed by thermal shift assay. The transition of the unfolding curve represents the temperature at which the protein unfolding occurs (Tm). f, DAXX dissolve preformed MDM2 aggregates. d-MDM2 (100 nM) was incubated with Flag–DAXX (200 nM) at 25 °C for the indicated times. Supernatant (soluble) and pellet (insoluble) fractions after sedimentation were analysed by western blot. i, DAXX enhances MDM2-mediated p53 ubiquitination. Native p53 (20 nM) was incubated with native MDM2 (45 nM) in the presence or absence of DAXX (20 or 100 nM) at 37 °C for 1.5 h. E1, E2 and His-ubiquitin (His-Ub) were then added for in vitro ubiquitination assay. The reaction mixtures were analysed by western blot. j, Native MDM2-mediated ubiquitination of native p53 (20 nM) in the presence or absence of Flag–DAXX (100 nM), or of denatured p53 (20 nM) pre-incubated with or without Flag–DAXX (100 nM) for 3 h at 25 °C. k, l, DAXX reduces p53 levels in cells, but does not alter the largely diffuse nuclear localization pattern of p53. Flag–p53 was transfected into U2OS cells together with empty vector or DAXX. Cells were analysed by immunofluorescence (k) and western blot (l). mo, H1299 cells inducibly expressing wild-type p53 or p53(R175H) were transfected with control vector (−) or HA–DAXX. Upon induction of p53 expression by Dox (1 μg ml−1), cells were analysed for protein levels by western blot with relative p53/GAPDH ratios indicated (m) and for mRNA levels of p53 (n) and p53 target genes (o) by qRT–PCR. Scale bar, 10 μm. Assays in panels have been performed two (d, e, g, h, km) or three (b, c, f, i, j) times with similar results. Numerical data are mean ± s.d. (n = 3) and are representative of two independent experiments (a, n, o). *P < 0.05, **P < 0.01, ns, not significant; unpaired Student’s t-test.

Source data

Extended Data Fig. 8 DAXX restores native conformation and function of mutant p53.

a, DAXX prevents p53(R175H) aggregation. p53(R175H) protein (100 nM) was incubated with GST or Flag–DAXX (200 nM each) at 37 °C for the indicated times. SN and pellet fractions were analysed by western blot. b, DAXX blocks p53(R175H) PFF-induced fibrillization of p53. Wild-type p53 (5 μM) was incubated with or without p53(R175H) PFFs and DAXX as indicated. Fibril formation was assayed by ThT binding. c, DAXX reduces p53(R175H) aggregates in cells. Flag–p53(R175H) was transfected into the U2OS cells together with empty vector (Ctrl) or HA–DAXX. Cells were analysed by immunofluorescence. Scale bar, 10 μm. Part of the images are also shown in Fig. 4i. d, DAXX partially restores the function of mutant p53. H1299 cells inducibly expressing p53(R175H) were transfected with control vector (−) or HA–DAXX. Upon induction of p53 expression by Dox (1 μg ml−1), cells were analysed for the expression of p53 target genes by RT–PCR. eg, Effect of DAXX on aggregation of endogenous mutant p53. MDA-MB-231 cells were transduced with lentiviral vectors expressing control or DAXX shRNA (e, f), or transfected with control siRNA, DAXX siRNA, and/or an siRNA-resistant form of DAXX (Flag–DAXX) as indicated (g). Cells were immunostained with anti-p53 (DO-1) and anti-fibrillar oligomer (A11) antibodies (e, g) and quantified (f). Scale bar, 50 μm. h-j, Knocking down DAXX enhances growth and tumorigenicity of MDA-MB-231 cells. Control and DAXX-knockdown MDA-MB-231 cells were assayed for adherent proliferation, protein expression (h), and soft-agar colony formation (21 days), with number and sizes of colonies (i) and representative images of colonies (j) shown. k, l, Overexpressing DAXX inhibits growth and tumorigenicity of MDA-MB-231 cells. MDA-MB-231 transduced with pBabe or pBabe-Flag-DAXX were assay for adherent proliferation for 5 days (k) and soft-agar colony formation (21 days), with representative images of colonies shown (l). Assays have been performed two (a) or three (c, e, g) times with similar results. Numerical data are mean ± s.d. (n = 3 for b, d, and 6 for i, h, k) and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t-test.

Source data

Extended Data Table 1 Human polyD/E proteins containing 35 or more D or E residues in any 50-residue window

Supplementary information

Supplementary Information

This file contains legends for Supplementary Figure 1 and Supplementary Table 1.

Reporting Summary

Supplementary Figure 1

Unprocessed western blots and/or gels associated with the data presented in the Figures and Extended Data Figures.

Supplementary Table 1

Human polyD/E proteins containing 35 or more D or E residues in any 50-residue window.

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Huang, L., Agrawal, T., Zhu, G. et al. DAXX represents a new type of protein-folding enabler. Nature 597, 132–137 (2021). https://doi.org/10.1038/s41586-021-03824-5

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing