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Direct observation of prion-like propagation of protein misfolding templated by pathogenic mutants

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Abstract

Many neurodegenerative diseases feature misfolded proteins that propagate via templated conversion of natively folded molecules. However, crucial questions about how such prion-like conversion occurs and what drives it remain unsolved, partly because technical challenges have prevented direct observation of conversion for any protein. We observed prion-like conversion in single molecules of superoxide dismutase-1 (SOD1), whose misfolding is linked to amyotrophic lateral sclerosis. Tethering pathogenic misfolded SOD1 mutants to wild-type molecules held in optical tweezers, we found that the mutants vastly increased misfolding of the wild-type molecule, inducing multiple misfolded isoforms. Crucially, the pattern of misfolding was the same in the mutant and converted wild-type domains and varied when the misfolded mutant was changed, reflecting the templating effect expected for prion-like conversion. Ensemble measurements showed decreased enzymatic activity in tethered heterodimers as conversion progressed, mirroring the single-molecule results. Antibodies sensitive to disease-specific epitopes bound to the converted protein, implying that conversion produced disease-relevant misfolded conformers.

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Fig. 1: Force spectroscopy of propagated misfolding in SOD1.
Fig. 2: Observing prion-like conversion directly in individual refolding–unfolding cycles.
Fig. 3: Conversion is templated by the misfolded mutant.
Fig. 4: Different mutants generate different misfolding patterns.
Fig. 5: Conversion in tethered heterodimers monitored by enzymatic activity.
Fig. 6: Prion-like conversion of immature SOD1.

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Data availability

Data supporting this work have been deposited on Figshare (https://doi.org/10.6084/m9.figshare.25751289; ref. 51). Source data are provided with this paper.

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Acknowledgements

We thank N. Cashman (University of British Columbia, Canada) for helpful discussions and for providing antibody samples. This work was supported by the Alberta Prion Research Institute (grant 201600020), Canadian Institutes of Health Research (grant PJT-185931), Natural Sciences and Engineering Research Council Canada (grant RGPIN-2018-04673) and National Research Council Canada. A.N. acknowledges support from a Banting Postdoctoral Fellowship and an Alberta Innovates Postdoctoral Fellowship. G.A. acknowledges support from an Alberta Innovates studentship.

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Author notes

  1. Supratik Sen Mojumdar

    Present address: Department of Chemistry, Indian Institute of Technology Palakkad, Palakkad, India

  2. These authors contributed equally: Krishna Neupane, Abhishek Narayan.

Authors and Affiliations

  1. Department of Physics, University of Alberta, Edmonton, Alberta, Canada

    Krishna Neupane, Abhishek Narayan, Supratik Sen Mojumdar, Gaurav Adhikari, Craig R. Garen & Michael T. Woodside

  2. Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada

    Michael T. Woodside

  3. Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada

    Michael T. Woodside

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  1. Krishna Neupane
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Contributions

K.N., A.N., S.S.M. and M.T.W. designed the research. K.N., A.N. and C.R.G. provided reagents. K.N., A.N., S.S.M. and G.A. performed experiments. K.N. and A.N. analyzed data. A.N. and M.T.W. wrote the manuscript. All authors edited the manuscript.

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Nature Chemical Biology thanks Gregory Merz, Tae-Young Yoon and Yongli Zhang for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Unfolding force distributions for native and misfolded states.

The distribution of unfolding forces for the native state (black) is similar to the distribution of unfolding forces seen for all misfolded states (red).

Source data

Extended Data Fig. 2 Sample unfolding FECs for G127X mutant.

Examples of unfolding curves for the misfolded G127X mutant showing total length changes of different amounts, reflecting different misfolded states.

Source data

Extended Data Fig. 3 Effect of chemical denaturant and reducing agent on conversion.

WST-1 formazan production by superoxide ions is normally inhibited by SOD1 activity; the drop in inhibition by SOD1 indicates that the enzyme becomes less active owing to conversion. (A) As the amount of chemical denaturant (GdnHCl) used to destabilize SOD1 is reduced from 1 M, the lag phase for conversion increases, until at 0 M no conversion is detected after 10 days. In each measurement, 5 mM TCEP was also present. (B) In the absence of any TCEP, but with 1 M GdnHCl, no conversion is detected after 10 days. All measurements were done using wt-G85R heterodimer. Error bars represent s.e.m. from 3 replicates.

Source data

Extended Data Fig. 4 Detection of disease-specific misfolded epitopes in converted wt SOD1.

Mass spectra from single-molecule mass photometry of (A) antibody 10C12 (sensitive specifically to disease-related misfolded C-terminal residues) and (B) 10C12 with wt-G127X heterodimer before conversion show distinct peaks for unbound antibody (dotted yellow line) and heterodimer (dotted gray line). (C) After 48 h conversion in the ensemble assay, the heterodimer peak is smaller and the antibody peak shifts. (D) Difference spectrum before and after conversion shows a decrease in unbound antibody and heterodimer and formation of a new peak at the mass of the bound complex.

Source data

Supplementary information

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Supplementary Tables 1 and 2 and Supplementary Figs. 1–3.

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Supporting data for Supplementary Figs. 1–3.

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Neupane, K., Narayan, A., Sen Mojumdar, S. et al. Direct observation of prion-like propagation of protein misfolding templated by pathogenic mutants. Nat Chem Biol 20, 1220–1226 (2024). https://doi.org/10.1038/s41589-024-01672-8

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