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In Silico Determined Properties of Designed Superoxide Dismutase-1 Mutants Predict ALS-like Phenotypes In Vitro and In Vivo

Michèle G. DuVal, View ORCID ProfileLuke McAlary, Mona Habibi, Pranav Garg, Mine Sher, View ORCID ProfileNeil R. Cashman, View ORCID ProfileW. Ted Allison, View ORCID ProfileSteven S. Plotkin
doi: https://doi.org/10.1101/474585
Michèle G. DuVal
1Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
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Luke McAlary
2Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada
3Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada
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Mona Habibi
2Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada
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Pranav Garg
2Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada
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Mine Sher
2Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada
3Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada
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Neil R. Cashman
3Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada
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W. Ted Allison
1Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
4Centre for Prions and Protein Folding Disease, University of Alberta, Edmonton, AB, Canada
5Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada
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Steven S. Plotkin
2Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada
6Genome Sciences and Technology Program, University of British Columbia, Vancouver, BC, Canada
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  • For correspondence: steve@phas.ubc.ca
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Abstract

The underlying physical causes of SOD1-related ALS are still not well-understood. We address this problem here by computationally designing two de novo mutants, A89R and K128N, which were predicted theoretically to be either significantly destabilizing or stabilizing respectively. We subjected these in silico designed mutants to a series of experimental tests, including in vitro measures of thermodynamic stability, cell-based aggregation and toxicity assays, and an in vivo developmental model of zebrafish motor neuron axonopathy. The experimental tests validated the theoretical predictions: A89R is an unstable, highly-deleterious mutant, and K128N is a stable, non-toxic mutant. Moreover, K128N is predicted computationally to form an unusually stable heterodimer with the familial ALS mutant A4V. Consistent with this prediction, co-injection of K128N and A4V into zebrafish shows profound rescue of motor neuron pathology. The demonstrated success of these first principles calculations to predict the physical properties of SOD1 mutants holds promise for rationally designed therapies to counter the progression of ALS.

Significance Mutations in the protein superoxide dismutase cause ALS, and many of these mutants have decreased folding stability. We sought to pursue this thread using a synthetic biology approach, where we designed two de novo mutations, one stabilizing and one destabilizing, as predicted using computational molecular dynamics simulations. We then tested these mutants using in vitro, cell-based, and in vivo zebrafish models. We found that the unstable mutant was toxic, and induced a severe ALS phenotype in zebrafish; the predicted stable mutant, on the other hand, behaved even better than WT. In fact, it was able to rescue the ALS phenotype caused by mutant SOD1. We propose a mechanism for this rescue, which may provide an avenue for therapeutic intervention.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted November 21, 2018.
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In Silico Determined Properties of Designed Superoxide Dismutase-1 Mutants Predict ALS-like Phenotypes In Vitro and In Vivo
Michèle G. DuVal, Luke McAlary, Mona Habibi, Pranav Garg, Mine Sher, Neil R. Cashman, W. Ted Allison, Steven S. Plotkin
bioRxiv 474585; doi: https://doi.org/10.1101/474585
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In Silico Determined Properties of Designed Superoxide Dismutase-1 Mutants Predict ALS-like Phenotypes In Vitro and In Vivo
Michèle G. DuVal, Luke McAlary, Mona Habibi, Pranav Garg, Mine Sher, Neil R. Cashman, W. Ted Allison, Steven S. Plotkin
bioRxiv 474585; doi: https://doi.org/10.1101/474585

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