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O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity

Abstract

A major role for the intracellular post-translational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area focused on O-GlcNAc modification of the amyloid-forming proteins themselves. Here we used synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we found that O-GlcNAc increases the ability of sHSPs to block the amyloid formation of both α-synuclein and Aβ(1–42). Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Finally, we found that, although O-GlcNAc levels are globally reduced in Alzheimer’s disease brains, the modification of relevant sHSPs is either maintained or increased, which suggests a mechanism to maintain these potentially protective O-GlcNAc modifications. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.

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Fig. 1: O-GlcNAc modification and the sHSPs.
Fig. 2: O-GlcNAcylated HSP27 is a better chaperone against α-synuclein amyloid aggregation.
Fig. 3: O-GlcNAc improves the anti-α-synuclein chaperone activity of αAC and αBC.
Fig. 4: O-GlcNAcylation is a global activator of HSP27, αAC and αBC chaperone activity against Aβ(1–42) amyloid aggregation.
Fig. 5: O-GlcNAcylation blocks the IXI–ACD HSP27 domain interaction and increases the size of the HSP27 oligomers.
Fig. 6: Global O-GlcNAc is lower in Alzheimer’s disease but is increased or maintained on HSP27 and αBC, respectively.

Data availability

Data supporting the results and conclusions are available within this paper and the Supplementary Information. Source data are provided with this paper.

References

  1. Yang, X. & Qian, K. Protein O-GlcNAcylation: emerging mechanisms and functions. Nat. Rev. Mol. Cell Biol. 18, 452–465 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wani, W. Y., Chatham, J. C., Darley-Usmar, V., McMahon, L. L. & Zhang, J. O-GlcNAcylation and neurodegeneration. Brain Res. Bull. 133, 80–87 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Wang, A. C., Jensen, E. H., Rexach, J. E., Vinters, H. V. & Hsieh-Wilson, L. C. Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration. Proc. Natl Acad. Sci. USA 113, 15120–15125 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G. & Gong, C. O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc. Natl Acad. Sci. USA 101, 10804–10809 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Liu, F. et al. Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer’s disease. Brain 132, 1820–1832 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Aguilar, A. L., Hou, X., Wen, L., Wang, P. G. & Wu, P. A chemoenzymatic histology method for O-GlcNAc detection. ChemBioChem 18, 2416–2421 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pinho, T. S., Correia, S. C., Perry, G., Ambrósio, A. F. & Moreira, P. I. Diminished O-GlcNAcylation in Alzheimer’s disease is strongly correlated with mitochondrial anomalies. Biochim. Biophys. Acta Mol. Basis Dis. 1865, 2048–2059 (2019).

    Article  CAS  PubMed  Google Scholar 

  8. Yuzwa, S. A. et al. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat. Chem. Biol. 8, 393–399 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Yuzwa, S. A., Cheung, A. H., Okon, M., McIntosh, L. P. & Vocadlo, D. J. O-GlcNAc modification of tau directly inhibits its aggregation without perturbing the conformational properties of tau monomers. J. Mol. Biol. 426, 1736–1752 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Marotta, N. P. et al. O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson’s disease. Nat. Chem. 7, 913–920 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lewis, Y. E. et al. O-GlcNAcylation of α-synuclein at serine 87 reduces aggregation without affecting membrane binding. ACS Chem. Biol. 12, 1020–1027 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Levine, P. M. et al. Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson’s disease. Proc. Natl Acad. Sci. USA 116, 1511–1519 (2019).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Haslbeck, M., Weinkauf, S. & Buchner, J. Small heat shock proteins: simplicity meets complexity. J. Biol. Chem. 294, 2121–2132 (2019).

    Article  CAS  PubMed  Google Scholar 

  15. Kappé, G. et al. The human genome encodes 10 α-crystallin-related small heat shock proteins: HspB1-10. Cell Stress Chaperon. 8, 53–61 (2003).

    Article  Google Scholar 

  16. Kriehuber, T. et al. Independent evolution of the core domain and its flanking sequences in small heat shock proteins. FASEB J. 24, 3633–3642 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Jehle, S. et al. Solid-state NMR and SAXS studies provide a structural basis for the activation of αB-crystallin oligomers. Nat. Struct. Mol. Biol. 17, 1037–1042 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Baldwin, A. J. et al. Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus. J. Mol. Biol. 413, 310–320 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. McDonald, E. T., Bortolus, M., Koteiche, H. A. & Mchaourab, H. S. Sequence, structure, and dynamic determinants of Hsp27 (HspB1) equilibrium dissociation are encoded by the N-terminal domain. Biochemistry 51, 1257–1268 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Baldwin, A. J. et al. Probing dynamic conformations of the high-molecular-weight αB-crystallin heat shock protein ensemble by NMR spectroscopy. J. Am. Chem. Soc. 134, 15343–15350 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Hochberg, G. K. A. 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).

    Article  CAS  PubMed  Google Scholar 

  22. Kudva, Y. C., Hiddinga, H. J., Butler, P. C., Mueske, C. S. & Eberhardt, N. L. Small heat shock proteins inhibit in vitro Aβ1–42 amyloidogenesis. FEBS Lett. 416, 117–121 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Rekas, A. et al. Interaction of the molecular chaperone αB-crystallin with α-synuclein: effects on amyloid fibril formation and chaperone activity. J. Mol. Biol. 340, 1167–1183 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Raman, B. et al. αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid β-peptide and β2-microglobulin. Biochem. J. 392, 573–581 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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).

    Article  CAS  PubMed  Google Scholar 

  26. Cox, D., Selig, E., Griffin, M. D. W., Carver, J. A. & Ecroyd, H. Small heat-shock proteins prevent α-synuclein aggregation via transient interactions and their efficacy is affected by the rate of aggregation. J. Biol. Chem. 291, 22618–22629 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  29. Delbecq, S. P., Jehle, S. & Klevit, R. Binding determinants of the small heat shock protein, αB-crystallin: recognition of the ‘IxI’ motif. EMBO J. 31, 4587–4594 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  31. Hilton, G. R. et al. C-terminal interactions mediate the quaternary dynamics of αB-crystallin. Philos. Trans. R. Soc. B 368, 20110405 (2013).

    Article  Google Scholar 

  32. Nappi, L. et al. Ivermectin inhibits HSP27 and potentiates efficacy of oncogene targeting in tumor models. J. Clin. Invest. 130, 699–714 (2020).

    Article  CAS  PubMed  Google Scholar 

  33. Clark, A. R. et al. Terminal regions confer plasticity to the tetrameric assembly of human HspB2 and HspB3. J. Mol. Biol. 430, 3297–3310 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Rauch, J. N. et al. BAG3 Is a modular, scaffolding protein that physically links heat shock protein 70 (Hsp70) to the small heat shock proteins. J. Mol. Biol. 429, 128–141 (2017).

    Article  CAS  PubMed  Google Scholar 

  35. Roquemore, E. P. et al. Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 267, 555–563 (1992).

    Article  CAS  PubMed  Google Scholar 

  36. Guo, K. et al. Translocation of HSP27 into liver cancer cell nucleus may be associated with phosphorylation and O-GlcNAc glycosylation. Oncol. Rep. 28, 494–500 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Rambaruth, N. D., Greenwell, P. & Dwek, M. V. The lectin Helix pomatia agglutinin recognises O-GlcNAc containing glycoproteins in human breast cancer. Glycobiology 22, 839–848 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. Wang, S. et al. Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer’s disease. J. Pathol. 243, 78–88 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Deracinois, B. et al. O-GlcNAcylation site mapping by (azide–alkyne) click chemistry and mass spectrometry following intensive fractionation of skeletal muscle cells proteins. J. Proteomics 186, 83–97 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Li, J. et al. An isotope-coded photocleavable probe for quantitative profiling of protein O-GlcNAcylation. ACS Chem. Biol. 14, 4–10 (2019).

    Article  CAS  PubMed  Google Scholar 

  41. Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl Acad. Sci. USA 95, 6705–6710 (1998).

    Article  CAS  PubMed  Google Scholar 

  42. Matveenko, M., Cichero, E., Fossa, P. & Becker, C. F. W. Impaired chaperone activity of human heat shock protein Hsp27 site-specifically modified with argpyrimidine. Angew. Chem. Int. Ed. 55, 11397–11402 (2016).

    Article  CAS  Google Scholar 

  43. Alderson, T. R. et al. Local unfolding of the HSP27 monomer regulates chaperone activity. Nat. Commun. 10, 1068 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Luk, K. C. et al. Molecular and biological compatibility with host alpha-synuclein influences fibril pathogenicity. Cell Rep. 16, 3373–3387 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Selig, E. E. et al. N- and C-terminal regions of αB-crystallin and Hsp27 mediate inhibition of amyloid nucleation, fibril binding, and fibril disaggregation. J. Biol. Chem. 295, 9838–9854 (2020).

    Article  CAS  PubMed  Google Scholar 

  46. Blanco-Canosa, J. B. & Dawson, P. E. An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew. Chem. Int. Ed. 47, 6851–6855 (2008).

    Article  CAS  Google Scholar 

  47. Metanis, N., Keinan, E. & Dawson, P. E. Traceless ligation of cysteine peptides using selective deselenization. Angew. Chem. Int. Ed. 49, 7049–7053 (2010).

    Article  CAS  Google Scholar 

  48. Shang, S., Tan, Z., Dong, S. & Danishefsky, S. J. An advance in proline ligation. J. Am. Chem. Soc. 133, 10784–10786 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ovchinnikov, S. et al. Protein structure determination using metagenome sequence data. Science 355, 294–298 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Simons, K. T., Bonneau, R., Ruczinski, I. & Baker, D. Ab initio protein structure prediction of CASP III targets using ROSETTA. Proteins 37, 171–176 (1999).

    Article  Google Scholar 

  51. De Leon, C. A., Lang, G., Saavedra, M. I. & Pratt, M. R. Simple and efficient preparation of O- and S-GlcNAcylated amino acids through InBr3-catalysed synthesis of β-N-acetylglycosides from commercially available reagents. Org. Lett. 20, 5032–5035 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Shah, N. H., Dann, G. P., Vila-Perelló, M., Liu, Z. & Muir, T. W. Ultrafast protein splicing is common among cyanobacterial split inteins: implications for protein engineering. J. Am. Chem. Soc. 134, 11338–11341 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. De Leon, C. A., Levine, P. M., Craven, T. W. & Pratt, M. R. The sulfur-linked analogue of O-GlcNAc (S-GlcNAc) is an enzymatically stable and reasonable structural surrogate for O-GlcNAc at the peptide and protein levels. Biochemistry 56, 3507–3517 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Clark, P. M. et al. Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J. Am. Chem. Soc. 130, 11576–11577 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Mymrikov, E. V., Daake, M., Richter, B., Haslbeck, M. & Buchner, J. The chaperone activity and substrate spectrum of human small heat shock proteins. J. Biol. Chem. 292, 672–684 (2017).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

M.R.P. acknowledges support from the National Institutes of Health (R01GM114537) and the Anton Burg Foundation and C.F.W.B. acknowledges support from the University of Vienna. T.W.C. thanks the Washington Research Fund for the Innovation postdoctoral fellowship. N.J.P. and S.P.M. were supported by NIGMS T32GM118289, and A.T.B. was supported as a Dornsife Chemistry–Biology Interface Trainee. SPR, ITC and SEC–MALS were performed at the USC Nanobiophysics Core Facility. TEM images were collected at the USC Core Center of Excellence in Nano Imaging. ThT measurements were performed at the USC Bridge Institute. Human tissue was obtained from the NIH NeuroBioBank. We thank K. Moremen for the generous gift of GalT(Y289L) who is supported by the National Institutes of Health (P41GM103390 and R01GM130915).

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Authors and Affiliations

Authors

Contributions

A.T.B., P.M.L., T.W.C., S.M., T.T.T., C.F.W.B., D.B. and M.R.P. designed the experiments and interpreted the data. A.T.B. and P.M.L. synthesized and purified the proteins. A.T.B. and P.M.L. performed the amyloid aggregation reactions and associated analyses. A.T.B. and T.T.T. performed the SPR analysis. A.T.B. performed the ITC, SEC–MALS and blots. T.W.C. performed the computational modelling. S.M. performed the amorphous aggregation reactions. N.J.P. and S.P.M. assisted in preparing fragments for protein synthesis. A.T.B., P.M.L., T.W.C., D.B. and M.R.P. prepared the manuscript.

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Correspondence to Matthew R. Pratt.

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The authors declare no competing interests.

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Peer review information Nature Chemistry thanks Richard Payne and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Synthesis and characterization of O-GlcNAc modified HSP27.

Unmodified and differentially O-GlcNAc modified versions of HSP27 were retrosynthetically deconstructed into a recombinant protein thioester and peptides prepared by solid phase peptide synthesis. Analytical RP-HPLC traces and MALDI-TOF-MS of the indicated synthetic proteins.

Extended Data Fig. 2 Additional TEM images of α-synuclein/HSP27 aggregation.

Larger format and additional TEM images corresponding to Figure 2c. The images are consistent between all three experimental replicates.

Extended Data Fig. 3 O-GlcNAc neither improves nor diminishes the activity of HSP27 against seeded α-synuclein aggregation.

α-Synuclein monomers (50 μM) and the indicated ratios of HSP27 or HSP27(gT184) were mixed with α-synuclein preformed fibres (2.5 μM, 5%). The reactions were placed in a plate reader and aggregation was detected by ThT fluorescence (λex = 450 nm, λem = 482 nm).

Source data

Extended Data Fig. 4 Synthesis and characterization of O-GlcNAc modified αAC.

O-GlcNAc modified αAC was retrosynthetically deconstructed into a recombinant protein thioester and two peptides prepared by solid phase peptide synthesis. Analytical RP-HPLC traces and MALDI-TOF-MS of the indicated recombinant or synthetic proteins.

Extended Data Fig. 5 Synthesis and characterization of O-GlcNAc modified αBC.

O-GlcNAc modified αBC was retrosynthetically deconstructed into a recombinant protein thioester and a peptide prepared by solid phase peptide synthesis. Analytical RP-HPLC traces and MALDI-TOF-MS of the indicated recombinant or synthetic proteins.

Extended Data Fig. 6 Additional TEM images of α-synuclein/αAC/αBC aggregation.

Larger format and additional TEM images corresponding to Figure 3. The images are consistent between all three experimental replicates.

Extended Data Fig. 7 TEM images of Aβ aggregation.

The aggregation reactions were analysed by TEM after 800 min. The images are consistent between all three experimental replicates.

Extended Data Fig. 8 Synthesis and characterization of quadruply O-GlcNAc modified HSP27.

Unmodified and differentially O-GlcNAcylated versions of HSP27 were retrosynthetically deconstructed into a recombinant protein thioester and peptides prepared by solid phase peptide synthesis. Analytical RP-HPLC traces and MALDI-TOF-MS of the indicated synthetic proteins.

Extended Data Fig. 9 HSP27 expression is upregulated in Alzheimer’s disease.

HSP27 was visualized by western blotting in brain lysates (Brodmann area 7) from Alzheimer’s disease patients and age-matched controls. These data are consistent between two biological replicates.

Source data

Extended Data Fig. 10 O-GlcNAc does not improve the chaperone activity of HSP27 against amorphous aggregation proteins.

Citrate synthase (2 μM) in the presence or absence of the indicated HSP27 proteins (0.45 μM) were incubated at 45 °C while measuring the absorbance at 400 nm. Onset-times were obtained by measuring the time required for fluorescence to reach 3-times the initial reading. Onset-time results are mean ±SEM of n=3 independent experiments. Statistical significance was determined using a one-way ANOVA test followed by Tukey’s test.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, and Table 1.

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Unprocessed western blots.

Source Data Extended Data Fig. 3

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Unprocessed western blots.

Source Data Extended Data Fig. 10

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Balana, A.T., Levine, P.M., Craven, T.W. et al. O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity. Nat. Chem. 13, 441–450 (2021). https://doi.org/10.1038/s41557-021-00648-8

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