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The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro

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Cell Stress and Chaperones Aims and scope

Abstract

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease. The mechanism that underlies amyotrophic lateral sclerosis (ALS) pathology remains unclear, but protein inclusions are associated with all forms of the disease. Apart from pathogenic proteins, such as TDP-43 and SOD1, other proteins are associated with ALS inclusions including small heat shock proteins. However, whether small heat shock proteins have a direct effect on SOD1 aggregation remains unknown. In this study, we have examined the ability of small heat shock proteins αB-crystallin and Hsp27 to inhibit the aggregation of SOD1 in vitro. We show that these chaperone proteins suppress the increase in thioflavin T fluorescence associated with SOD1 aggregation, primarily through inhibiting aggregate growth, not the lag phase in which nuclei are formed. αB-crystallin forms high molecular mass complexes with SOD1 and binds directly to SOD1 aggregates. Our data are consistent with an overload of proteostasis systems being associated with pathology in ALS.

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Acknowledgments

This work was supported by the Illawarra Retirement Trust (IRT) Research Foundation and the Illawarra Health and Medical Research Institute. JJY was supported by the Motor Neurone Disease Research Institute of Australia in the form of a Bill Gole MND Postdoctoral Fellowship and is currently supported by the Australian Research Council in the form of a DECRA (DE120102840), KR is supported by a Rotary Health PhD Scholarship, and HE is supported by the Australian Research Council in the form of a Future Fellowship (FT110100586).

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  1. Illawarra Health and Medical Research Institute, School of Biological Sciences, University of Wollongong, Northfields Avenue, Wollongong, NSW, 2522, Australia

    Justin J. Yerbury, Dane Gower, Laura Vanags, Kate Roberts, Jodi A. Lee & Heath Ecroyd

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  1. Justin J. Yerbury
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  2. Dane Gower
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  3. Laura Vanags
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  4. Kate Roberts
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  5. Jodi A. Lee
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Correspondence to Justin J. Yerbury or Heath Ecroyd.

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Supplementary Figure 1

Negative control GST does not inhibit the in vitro aggregation of WT SOD1 or form a stable complex with its aggregated form. (a) Wild-type SOD1 was incubated at 30 μM in 10 mM potassium phosphate buffer containing 5 mM EDTA, pH 7.4 whilst shaking (120 rpm for 5 min each cycle, 15 min cycles) at 37 °C in the absence or presence of GST and the amount of ThT fluorescence (excitation at 440 nm and emission at 490nm) was monitored over time using a microplate-reader. The molar ratio (SOD1: GST) used was 1:1. Inset: The percent inhibition of the increase in ThT fluorescence afforded by GST. Results shown are mean ± SEM of three replicates. (b) Immuno-dot blot used to detect any interaction of GST with aggregated SOD1. Aggregated WT SOD1 (20 μM) was incubated in the absence or presence of GST (20 μM) for 1 h at 37 °C. Control samples consisted of buffer alone or GST alone. All samples were collected and centrifuged for 30 min at 4 °C and the soluble (S) and pellet (P) fractions separated. Pellet fractions were washed twice with PBS and then the soluble and pellet fractions were spotted onto a nitrocellulose membrane in duplicate. Membranes were blotted with antibodies specific to SOD1 or GST. The results shown are representative of two independent experiments. (JPEG 91 kb)

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Yerbury, J.J., Gower, D., Vanags, L. et al. The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro. Cell Stress and Chaperones 18, 251–257 (2013). https://doi.org/10.1007/s12192-012-0371-1

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  • DOI: https://doi.org/10.1007/s12192-012-0371-1

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