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Exposure of hydrophobic surfaces initiates aggregation of diverse ALS-causing superoxide dismutase-1 mutants.

Münch C, Bertolotti A - J. Mol. Biol. (2010)

Bottom Line: The remarkable diversity of the effects of these mutations on SOD1 properties has suggested that they promote aggregation by a variety of mechanisms.Our results uncover the biochemical nature of the misfolded aggregation-prone intermediate and reconcile the seemingly diverse effects of ALS-causing mutations into a unifying mechanism.Furthermore, the method we describe here will be useful for investigating and interfering with aggregation of various proteins and thereby provide insight into the molecular mechanisms underlying many neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

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Aggregation of as-purified SOD1 mutants follows exposure of hydrophobic surfaces. (a) Time course of measurements of exposed hydrophobicity monitored by Sypro Orange fluorescence and (b) aggregation of SOD1A4V at acidic pH monitored by DLS. Note that demetallation first provoked a decrease in the size of particles measured, indicating that metal loss provoked monomerization of the protein. (c) Time course of measurements of exposed hydrophobicity and (d) aggregation of apo-SOD1A4V exposed to 50 °C. (e, f) aggregation of SOD1A4V monitored by ThT after 3 days of incubation at acidic pH (e) or 50 °C (f). Data are means and s.d. values of replicate experiments (n = 3).
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fig5: Aggregation of as-purified SOD1 mutants follows exposure of hydrophobic surfaces. (a) Time course of measurements of exposed hydrophobicity monitored by Sypro Orange fluorescence and (b) aggregation of SOD1A4V at acidic pH monitored by DLS. Note that demetallation first provoked a decrease in the size of particles measured, indicating that metal loss provoked monomerization of the protein. (c) Time course of measurements of exposed hydrophobicity and (d) aggregation of apo-SOD1A4V exposed to 50 °C. (e, f) aggregation of SOD1A4V monitored by ThT after 3 days of incubation at acidic pH (e) or 50 °C (f). Data are means and s.d. values of replicate experiments (n = 3).

Mentions: Aggregation of SOD1H46R occurred within minutes after TFE addition, while a longer time was required to detect aggregation of SOD1A4V at low pH (Figs. 3, 4 and data not shown). We took advantage of the slow aggregation kinetics of both as-purified and apo-SOD1A4V at acidic pH to analyze the aggregation process in greater detail. As-purified and apo-SOD1A4V were adjusted to low pH (day 0) and aliquots taken every day to analyze both Sypro Orange-derived fluorescence and aggregation. Immediately after exposure to low pH, both as-purified and apo-SOD1A4V increased the fluorescence of the conformation-sensitive dye (Fig. 5a, day 0). The increase in fluorescence preceded the onset of aggregation (Fig. 5a and b, day 0). Fluorescence of Sypro Orange then reached a maximum after 1 day and gradually decreased after 2 days (Fig. 5a). Concomitantly, aggregates gradually grew (Fig. 5b and Supplementary Fig. 5a). Note that the soluble proteins were barely detectable after 2 days (Supplementary Fig. 5a). Together, these analyses reveal that low pH exposes hydrophobic regions on the surface of SOD1A4V and such regions are buried in aggregates.


Exposure of hydrophobic surfaces initiates aggregation of diverse ALS-causing superoxide dismutase-1 mutants.

Münch C, Bertolotti A - J. Mol. Biol. (2010)

Aggregation of as-purified SOD1 mutants follows exposure of hydrophobic surfaces. (a) Time course of measurements of exposed hydrophobicity monitored by Sypro Orange fluorescence and (b) aggregation of SOD1A4V at acidic pH monitored by DLS. Note that demetallation first provoked a decrease in the size of particles measured, indicating that metal loss provoked monomerization of the protein. (c) Time course of measurements of exposed hydrophobicity and (d) aggregation of apo-SOD1A4V exposed to 50 °C. (e, f) aggregation of SOD1A4V monitored by ThT after 3 days of incubation at acidic pH (e) or 50 °C (f). Data are means and s.d. values of replicate experiments (n = 3).
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fig5: Aggregation of as-purified SOD1 mutants follows exposure of hydrophobic surfaces. (a) Time course of measurements of exposed hydrophobicity monitored by Sypro Orange fluorescence and (b) aggregation of SOD1A4V at acidic pH monitored by DLS. Note that demetallation first provoked a decrease in the size of particles measured, indicating that metal loss provoked monomerization of the protein. (c) Time course of measurements of exposed hydrophobicity and (d) aggregation of apo-SOD1A4V exposed to 50 °C. (e, f) aggregation of SOD1A4V monitored by ThT after 3 days of incubation at acidic pH (e) or 50 °C (f). Data are means and s.d. values of replicate experiments (n = 3).
Mentions: Aggregation of SOD1H46R occurred within minutes after TFE addition, while a longer time was required to detect aggregation of SOD1A4V at low pH (Figs. 3, 4 and data not shown). We took advantage of the slow aggregation kinetics of both as-purified and apo-SOD1A4V at acidic pH to analyze the aggregation process in greater detail. As-purified and apo-SOD1A4V were adjusted to low pH (day 0) and aliquots taken every day to analyze both Sypro Orange-derived fluorescence and aggregation. Immediately after exposure to low pH, both as-purified and apo-SOD1A4V increased the fluorescence of the conformation-sensitive dye (Fig. 5a, day 0). The increase in fluorescence preceded the onset of aggregation (Fig. 5a and b, day 0). Fluorescence of Sypro Orange then reached a maximum after 1 day and gradually decreased after 2 days (Fig. 5a). Concomitantly, aggregates gradually grew (Fig. 5b and Supplementary Fig. 5a). Note that the soluble proteins were barely detectable after 2 days (Supplementary Fig. 5a). Together, these analyses reveal that low pH exposes hydrophobic regions on the surface of SOD1A4V and such regions are buried in aggregates.

Bottom Line: The remarkable diversity of the effects of these mutations on SOD1 properties has suggested that they promote aggregation by a variety of mechanisms.Our results uncover the biochemical nature of the misfolded aggregation-prone intermediate and reconcile the seemingly diverse effects of ALS-causing mutations into a unifying mechanism.Furthermore, the method we describe here will be useful for investigating and interfering with aggregation of various proteins and thereby provide insight into the molecular mechanisms underlying many neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

Show MeSH
Related in: MedlinePlus