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

Nature Chemistry volume 13, pages 441–450 (2021)Cite this article

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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.

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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).

Author information

Authors and Affiliations

  1. Departments of Chemistry, University of Southern California, Los Angeles, CA, USA

    Aaron T. Balana, Paul M. Levine, Nichole J. Pedowitz, Stuart P. Moon, Terry T. Takahashi & Matthew R. Pratt

  2. Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, WA, USA

    Timothy W. Craven & David Baker

  3. Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria

    Somnath Mukherjee & Christian F. W. Becker

  4. Biological Sciences, University of Southern California, Los Angeles, CA, USA

    Matthew R. Pratt

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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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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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Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 3

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Source Data Extended Data Fig. 9

Unprocessed western blots.

Source Data Extended Data Fig. 10

Statistical source data.

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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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