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Hsp27 chaperones FUS phase separation under the modulation of stress-induced phosphorylation

Nature Structural & Molecular Biology volume 27, pages 363–372 (2020)Cite this article

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Abstract

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid−liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.

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Fig. 1: Hsp27 inhibits FUS from associating with SGs.
Fig. 2: Hsp27 inhibits FUS LC LLPS via weak interactions.
Fig. 3: Effect of Hsp27 on the long-range and transient interactions in FUS LC probed by paramagnetic relaxation enhancement.
Fig. 4: Characterization of Hsp27 domains that account for LLPS inhibition.
Fig. 5: Hsp27-3D phase separates with FUS LC and inhibits amyloid fibril formation.
Fig. 6: Phosphorylation of Hsp27 upon FUS P525L aggregation and mitigation of FUS aggregation in HeLa cells.
Fig. 7: Molecular mechanism of Hsp27 inhibits FUS LC amyloid aggregation.
Fig. 8: Schematic illustration of Hsp27 chaperoning FUS phase separation under the regulation of stress-induced post-translation phosphorylation.

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Data Availability Statement

Source data for Fig. 1a,b, 2c, 4b,g, 5c,g,i, 6a,c and 7a,b are available with the paper online.

References

  1. 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).

    CAS  PubMed  Google Scholar 

  2. Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).

    PubMed  Google Scholar 

  3. Boeynaems, S. et al. Protein phase separation: a new phase in cell biology. Trends Cell Biol. 28, 420–435 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Gui, X. et al. Structural basis for reversible amyloids of hnRNPA1 elucidates their role in stress granule assembly. Nat. Commun. 10, 2006 (2019).

    PubMed  PubMed Central  Google Scholar 

  5. Patel, A. et al. A Liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162, 1066–1077 (2015).

    CAS  PubMed  Google Scholar 

  6. Maharana, S. et al. RNA buffers the phase separation behavior of prion-like RNA binding proteins. Science 360, 918–921 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Murakami, T. et al. ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function. Neuron 88, 678–690 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Aguzzi, A. & Altmeyer, M. Phase separation: linking cellular compartmentalization to disease. Trends Cell Biol. 26, 547–558 (2016).

    CAS  PubMed  Google Scholar 

  9. Ramaswami, M., Taylor, J. P. & Parker, R. Altered ribostasis: RNA-protein granules in degenerative disorders. Cell 154, 727–736 (2013).

    CAS  PubMed  Google Scholar 

  10. Tyedmers, J., Mogk, A. & Bukau, B. Cellular strategies for controlling protein aggregation. Nat. Rev. Mol. Cell Biol. 11, 777–788 (2010).

    CAS  PubMed  Google Scholar 

  11. Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

    CAS  Google Scholar 

  12. Guo, L. et al. Nuclear-import receptors reverse aberrant phase transitions of RNA-binding proteins with prion-like domains. Cell 173, 677–692.e20 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Hofweber, M. et al. Phase separation of FUS is suppressed by its nuclear import receptor and arginine methylation. Cell 173, 706–719.e13 (2018).

    CAS  PubMed  Google Scholar 

  14. Qamar, S. et al. FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation-π interactions. Cell 173, 720–734.e15 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Yoshizawa, T. et al. Nuclear import receptor inhibits phase separation of FUS through binding to multiple sites. Cell 173, 693–705.e22 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Mateju, D. et al. An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function. EMBO J. 36, 1669–1687 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Sharp, P. S. et al. Protective effects of heat shock protein 27 in a model of ALS occur in the early stages of disease progression. Neurobiol. Dis. 30, 42–55 (2008).

    CAS  PubMed  Google Scholar 

  19. Jesse, C. M. et al. ALS-associated endoplasmic reticulum proteins in denervated skeletal muscle: implications for motor neuron disease pathology. Brain Pathol. 27, 781–794 (2017).

    CAS  PubMed  Google Scholar 

  20. Dierick, I. et al. Genetic variant in the HSPB1 promoter region impairs the HSP27 stress response. Hum. Mutat. 28, 830 (2007).

    PubMed  Google Scholar 

  21. Benndorf, R., Martin, J. L., Kosakovsky Pond, S. L. & Wertheim, J. O. Neuropathy- and myopathy-associated mutations in human small heat shock proteins: characteristics and evolutionary history of the mutation sites. Mutat. Res. Rev. Mutat. Res. 761, 15–30 (2014).

    CAS  Google Scholar 

  22. Bruey, J. M. et al. Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat. Cell Biol. 2, 645–652 (2000).

    CAS  PubMed  Google Scholar 

  23. Choi, S., Chen, M., Cryns, V. L. & Anderson, R. A. A nuclear phosphoinositide kinase complex regulates p53. Nat. Cell Biol. 21, 462–475 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Haslbeck, M., Franzmann, T., Weinfurtner, D. & Buchner, J. Some like it hot: the structure and function of small heat-shock proteins. Nat. Struct. Mol. Biol. 12, 842–846 (2005).

    CAS  PubMed  Google Scholar 

  25. Freilich, R. et al. Competing protein-protein interactions regulate binding of Hsp27 to its client protein tau. Nat. Commun. 9, 4563 (2018).

    PubMed  PubMed Central  Google Scholar 

  26. van Montfort, R. L., Basha, E., Friedrich, K. L., Slingsby, C. & Vierling, E. Crystal structure and assembly of a eukaryotic small heat shock protein. Nat. Struct. Biol. 8, 1025–1030 (2001).

    PubMed  Google Scholar 

  27. Cox, D. et al. The small heat shock protein Hsp27 binds alpha-synuclein fibrils, preventing elongation and cytotoxicity. J. Biol. Chem. 293, 4486–4497 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Yerbury, J. J. et al. The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro. Cell Stress Chaperones 18, 251–257 (2013).

    CAS  PubMed  Google Scholar 

  29. Baughman, H. E. R., Pham, T. T., Adams, C. S., Nath, A. & Klevit, R. E. Release of a disordered domain enhances HspB1 chaperone activity toward tau. Proc. Natl Acad. Sci. USA 117, 2923–2929 (2020).

    CAS  PubMed  Google Scholar 

  30. Jovcevski, B. et al. Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity. Chem. Biol. 22, 186–195 (2015).

    CAS  PubMed  Google Scholar 

  31. Luo, F. et al. Atomic structures of FUS LC domain segments reveal bases for reversible amyloid fibril formation. Nat. Struct. Mol. Biol. 25, 341–346 (2018).

    CAS  PubMed  Google Scholar 

  32. Hochberg, G. K. et al. The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity. Proc. Natl Acad. Sci. USA 111, E1562–E1570 (2014).

    CAS  PubMed  Google Scholar 

  33. Liu, Z. et al. Mechanistic insights into the switch of αB-crystallin chaperone activity and self-multimerization. J. Biol. Chem. 293, 14880–14890 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Burke, K. A., Janke, A. M., Rhine, C. L. & Fawzi, N. L. Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II. Mol. Cell 60, 231–241 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Dedmon, M. M., Lindorff-Larsen, K., Christodoulou, J., Vendruscolo, M. & Dobson, C. M. Mapping long-range interactions in α-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J. Am. Chem. Soc. 127, 476–477 (2005).

    CAS  PubMed  Google Scholar 

  36. He, L., Sharpe, T., Mazur, A. & Hiller, S. A molecular mechanism of chaperone-client recognition. Sci. Adv. 2, e1601625 (2016).

    PubMed  PubMed Central  Google Scholar 

  37. Bibow, S. et al. Structural impact of proline-directed pseudophosphorylation at AT8, AT100, and PHF1 epitopes on 441-residue tau. J. Am. Chem. Soc. 133, 15842–15845 (2011).

    CAS  PubMed  Google Scholar 

  38. Mainz, A. et al. The chaperone αB-crystallin uses different interfaces to capture an amorphous and an amyloid client. Nat. Struct. Mol. Biol. 22, 898–905 (2015).

    CAS  PubMed  Google Scholar 

  39. Pasta, S. Y., Raman, B., Ramakrishna, T. & Rao, Ch. M. The IXI/V motif in the C-terminal extension of alpha-crystallins: alternative interactions and oligomeric assemblies. Mol. Vis. 10, 655–662 (2004).

    CAS  PubMed  Google Scholar 

  40. Hayes, D., Napoli, V., Mazurkie, A., Stafford, W. F. & Graceffa, P. Phosphorylation dependence of Hsp27 multimeric size and molecular chaperone function. J. Biol. Chem. 284, 18801–18807 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Landry, J. et al. Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II. J. Biol. Chem. 267, 794–803 (1992).

    CAS  PubMed  Google Scholar 

  42. Rouse, J. et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78, 1027–1037 (1994).

    CAS  PubMed  Google Scholar 

  43. Chio, A. et al. Two Italian kindreds with familial amyotrophic lateral sclerosis due to FUS mutation. Neurobiol. Aging 30, 1272–1275 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Conte, A. et al. P525L FUS mutation is consistently associated with a severe form of juvenile amyotrophic lateral sclerosis. Neuromuscul. Disord. 22, 73–75 (2012).

    PubMed  Google Scholar 

  45. Dormann, D. et al. ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import. EMBO J. 29, 2841–2857 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Lo Bello, M. et al. ALS-related mutant FUS protein is mislocalized to cytoplasm and is recruited into stress granules of fibroblasts from asymptomatic FUS P525L mutation carriers. Neurodegener. Dis. 17, 292–303 (2017).

    PubMed  Google Scholar 

  47. Errichelli, L. et al. FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons. Nat. Commun. 8, 14741 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. De Santis, R. et al. Mutant FUS and ELAVL4 (HuD) aberrant crosstalk in amyotrophic lateral sclerosis. Cell Reports 27, 3818–3831.e5 (2019).

    PubMed  PubMed Central  Google Scholar 

  49. Rogalla, T. et al. Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor α by phosphorylation. J. Biol. Chem. 274, 18947–18956 (1999).

    CAS  PubMed  Google Scholar 

  50. Stokoe, D., Engel, K., Campbell, D. G., Cohen, P. & Gaestel, M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 313, 307–313 (1992).

    CAS  PubMed  Google Scholar 

  51. Ludwig, S. et al. 3pK, a novel mitogen-activated protein (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways. Mol. Cell. Biol. 16, 6687–6697 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Rousseau, S., Houle, F., Landry, J. & Huot, J. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 15, 2169–2177 (1997).

    CAS  PubMed  Google Scholar 

  53. Ganassi, M. et al. A surveillance function of the HSPB8-BAG3-HSP70 chaperone complex ensures stress granule integrity and dynamism. Mol. Cell. 63, 796–810 (2016).

    CAS  PubMed  Google Scholar 

  54. Rajagopal, P., Liu, Y., Shi, L., Clouser, A. F. & Klevit, R. E. Structure of the α-crystallin domain from the redox-sensitive chaperone, HSPB1. J. Biomol. NMR 63, 223–228 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Fawzi, N. L., Ying, J., Torchia, D. A. & Clore, G. M. Kinetics of amyloid β monomer-to-oligomer exchange by NMR relaxation. J. Am. Chem. Soc. 132, 9948–9951 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Oroz, J. et al. Structure and pro-toxic mechanism of the human Hsp90/PPIase/Tau complex. Nat. Commun. 9, 4532 (2018).

    PubMed  PubMed Central  Google Scholar 

  57. Peschek, J. et al. Regulated structural transitions unleash the chaperone activity of ɑB-crystallin. Proc. Natl Acad. Sci. USA 110, E3780–E3789 (2013).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank staff members of the National Center for Protein Science Shanghai for assistance in NMR data collection. This work was supported by the National Natural Science Foundation (NSF) of China (91853113 to D.L. and C.L.), the Major State Basic Research Development Program (2016YFA0501902 to C.L.), the Science and Technology Commission of Shanghai Municipality (18JC1420500 to C.L.), Shanghai Pujiang Program (18PJ1404300 to D.L.), the “Eastern Scholar” project supported by Shanghai Municipal Education Commission (to D.L.), Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to C.L.), and Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-02-E00037 to D.L.).

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  1. These authors contributed equally: Zhenying Liu, Shengnan Zhang, Jinge Gu.

Authors and Affiliations

  1. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    Zhenying Liu, Shengnan Zhang, Jinge Gu, Yilun Tong, Xinrui Gui, Houfang Long, Chuchu Wang, Chunyu Zhao, Ying Li & Cong Liu

  2. University of the Chinese Academy of Sciences, Shijingshan District, Beijing, China

    Zhenying Liu, Shengnan Zhang, Jinge Gu, Yilun Tong, Xinrui Gui, Houfang Long, Chuchu Wang, Chunyu Zhao, Ying Li & Cong Liu

  3. Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China

    Yichen Li, Jinxia Lu, Lin He & Dan Li

  4. National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, China

    Zhijun Liu

  5. Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    Dan Li

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Contributions

D.L., C.L. and Zhenying Liu designed the project. Zhenying Liu, Yichen Li, J.L., H.L. and X.G. prepared constructs and purified proteins. Zhenying Liu, J.G., Ying Li and Y.T. performed the biochemical and cellular assays. Zhenying Liu, S.Z., C.W., C.Z. and Zhijun Liu performed the NMR experiments. D.L. wrote the manuscript. D.L., C.L., and L.H. revised the manuscript.

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Correspondence to Dan Li or Cong Liu.

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Extended data

Extended Data Fig. 1 Intracellular localization of endogenous Hsp27.

a, Endogenous Hsp27 (endo-Hsp27, red) of HeLa cells localizes in the cytoplasm under normal condition. As treated with stress (0.5 mM sodium arsenite), endo-Hsp27 condenses in SGs. Cells were immunostained for DAPI (blue). Scale bar, 20 μm. b, HeLa cells expressing RFP-FUS were treated with stress (0.5 mM sodium arsenite). Co-localization of Hsp27 (purple) and overexpressed RFP FUS (red) in SGs are indicated with arrows. Cells were immunostained for G3BP1 (green) to mark SGs. Scale bar, 20 μm.

Extended Data Fig. 2 Establishment of Hsp27 KO HeLa cells and cellular expression levels of FUS, Hsp27 and αBc (control) by western blot.

a, A fragment in Hsp27 exon 1 was deleted by using CRISPR/Cas9. We constructed two KO plasmids—sgHsp27-1 and sgHsp27-2. b, Western blot shows that plasmid sgHsp27-2 is efficient to suppress Hsp27 expression. sgNC is empty plasmid transfected in cells as control. c, A similar expression level of FUS in the Hsp27 KO and WT cells. d, Expression of endogenous (endo-Hsp27) and exogenous (myc-Hsp27) with/without stress (0.5 mM sodium arsenite). e, Expression levels of αBc with or without stress and Hsp27 overexpression. f, A similar expression level of FUS in cells with or without myc-Hsp27 overexpression.

Extended Data Fig. 3 Hsp27 reverses the LLPS of FUS-LC, while exhibits no effect on FUS-RGG LLPS.

a, Visualization of the reversion of Hsp27 to FUS-LC LLPS in test tubes and by DIC and fluorescence imaging. Scale bar: 10 μm. αBc is performed as a negative control. b, The LLPS of 150 μM FUS-LC was induced by decreasing the temperature to 4 °C, followed by the measurement of turbidities at OD600 nm. The molar ratios of Hsp27 are indicated. Error bars correspond to mean ± S.D. with n=3. c, αBc (negative control) exhibits no effect on FUS-LC LLPS. The test tubes contain 150 μM FUS-LC in the presence of 30 μM αBc at 25 °C and 4 °C, respectively. The DIC and fluorescence images show the liquid-like droplets formed by FUS-LC. Scale bar: 10 μm. d, Effect of Hsp27 on the LLPS of FUS-RGG. DIC images are shown with scale bars representing 10 μm. e, Turbidity measurement of 50 μM FUS-RGG in the presence of indicated ratios of Hsp27 at 4 °C. Error bars correspond to mean ± S.D., with n = 3. N. S. indicates not significant.

Extended Data Fig. 4 Sequence alignment of Hsp27 (HSPB1) and αBc (HSPB5).

The alignment was performed by software ClustalX and ESPript 3.0. The identities of NTDs and ACDs are labeled, respectively. The CTDs vary with no significant identity.

Extended Data Fig. 5 NMR spectra of FUS-LC titrated with Hsp27.

a, The backbone assignment of FUS-LC (50 μM) with resonances labeled by one amino acid letter and the residue number in FUS. b, Overlay of the 2D 1H-15N HSQC spectra of 50 μM 15N-FUS-LC alone (red) and in the presence of Hsp27 at molar ratios (FUS-LC: Hsp27) of 5:1 (blue) and 1:1 (orange), respectively. c, Residue-specific intensity changes of 50 μM FUS-LC in the presence of 50 μM BSA (red) as a negative control. The dash line indicates the line of I/I0=1.0.

Extended Data Fig. 6 Mutation of the Ser residues of FUS-LC impairs the inhibition of Hsp27 to FUS-LC LLPS.

a, Schematics show the Ser residues (green) of FUS-LC that are mutated into Ala (red). The mutation construct is named S/A. b, Turbidity measurements of FUS-LC WT (left) and S/A (right) in the presence of Hsp27. c, Comparison of the effect of Hsp27 on the LLPS of FUS LC-WT and S/A. The turbidity values are those in (b) at 4 °C. Data shown correspond to mean ± S.D., n = 3. *** p<0.001 by Student’s t-test.

Extended Data Fig. 7 Effect of Hsp27 on the long-range and transient interactions of FUS-LC probed by PRE.

a, The PRE profile of 50 μM 15N S70C-MTSL-FUS-LC is shown on the top. The PRE attenuations were recovered with increasing concentrations of 10 μM (orange) and 50 μM (red) unlabeled FUS-LC is shown in the middle, and with 10 μM (orange) and 50 μM (red) Hsp27 is shown at the bottom. b, The PRE profiles of 50 μM 15N S127C-MTSL-FUS-LC. c, The effect of BSA (50 μM, negative control) on 50 μM 15N A16C-MTSL-FUS-LC.

Extended Data Fig. 8 Sedimentation velocity analysis of NαBc-Hsp27 and NHsp27-αBc, and the influence of Hsp27 ΔCTD-3D on FUS-LC LLPS.

a, Sedimentation velocity analysis of NαBc-Hsp27 (20 μM) and NHsp27-αBc (17 μM). b, The influence of Hsp27 ΔCTD-3D on the phase transition of FUS-LC. The turbidity (OD600 nm) of 150 μM FUS-LC in the absence and presence of ΔCTD-3D at the indicated concentrations were monitored from 19 °C to 4 °C.

Extended Data Fig. 9 Characterization of FUS P525L expression and aggregation in HeLa cells.

a, Confocal microscopic images show the pFTAA staining of FUS P525L aggregates in HeLa cells. FUS P525L was overexpressed and spontaneously aggregated in cells. b, Western blot showed that the expression level of FUS P525L remains unchanged with/without the overexpression of myc-Hsp27.

Extended Data Fig. 10 Mutagenesis of the binding surface of Hsp27 with FUS-LC impaired the inhibitory activity of Hsp27 to FUS-LC amyloid aggregation.

a, ThT fluorescence assay shows that the mutated Hsp27 (Hsp27-A) exhibits decreased inhibitory activity to FUS-LC amyloid aggregation, compared to the WT Hsp27. b, Statistics of data in (a) at 60 h. Data shown are mean ± S.D., n = 3. c, Surface representation of the structure of Hsp27 ACD dimer. Binding interface of Hsp27 ACD for FUS-LC (upper) is highlighted in orange on the surface diagram of ACD (PDB code, 4MJH); that for Tau (lower) is highlighted in green.

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Liu, Z., Zhang, S., Gu, J. et al. Hsp27 chaperones FUS phase separation under the modulation of stress-induced phosphorylation. Nat Struct Mol Biol 27, 363–372 (2020). https://doi.org/10.1038/s41594-020-0399-3

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