Limits...
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.

Show MeSH

Related in: MedlinePlus

Aggregation of as-purified ALS-causing SOD1 mutants is caused by their increased propensity to expose hydrophobic surfaces. (a, b) Correlation between surface hydrophobicity measured by Sypro Orange fluorescence and aggregation measured by DLS (a) and ThT fluorescence (b). Data are means and s.e.m. from 3 independent experiments. Measurements were made 20 minutes after TFE treatment. The high linear correlation coefficient R, denotes the strength of the correlation. Note that the MBR mutants are more susceptible to TFE-induced aggregation than the WTL mutants. (c) Aggregation of SOD1G85R and SOD1G37R at room temperature, revealed by DLS.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2927901&req=5

fig6: Aggregation of as-purified ALS-causing SOD1 mutants is caused by their increased propensity to expose hydrophobic surfaces. (a, b) Correlation between surface hydrophobicity measured by Sypro Orange fluorescence and aggregation measured by DLS (a) and ThT fluorescence (b). Data are means and s.e.m. from 3 independent experiments. Measurements were made 20 minutes after TFE treatment. The high linear correlation coefficient R, denotes the strength of the correlation. Note that the MBR mutants are more susceptible to TFE-induced aggregation than the WTL mutants. (c) Aggregation of SOD1G85R and SOD1G37R at room temperature, revealed by DLS.

Mentions: In all conditions examined using two specific SOD1 mutants, we observed a correlation between exposure of hydrophobic surfaces and aggregation. To determine whether increased exposure of hydrophobic surfaces correlates with aggregation of diverse SOD1 mutants, we monitored aggregation and exposure of hydrophobic surfaces of the representative set of 12 mutants. As-purified SOD1WT and mutant proteins were exposed to 20% TFE, a condition that was found to elicit aggregation of the diverse SOD1 mutants, but not the wild-type protein. This treatment converted the soluble MBR mutants into aggregates in 20 minutes and was also efficient to provoke aggregation of the WTL mutants (Supplementary Fig. 6a). Exposure of surface hydrophobicity was monitored by Sypro Orange fluorescence and aggregation monitored both by DLS and ThT fluorescence (Fig. 6a and b). ThT fluorescence indicated that aggregates have an amyloid component. Analysis of Congo Red binding confirmed this (Supplementary Fig. 6b). We found exposure of hydrophobicity and ordered assembly of SOD1 aggregates, monitored by these two independent methods, were strongly correlated. The strength of the correlation between exposure of hydrophobic surfaces and aggregation is attested by the very high linear correlation coefficient (Fig. 6a, R = 0.95 and b, R = 0.84). This revealed that exposure of hydrophobicity is a common feature that provokes aggregation of diverse SOD1 mutants.


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 ALS-causing SOD1 mutants is caused by their increased propensity to expose hydrophobic surfaces. (a, b) Correlation between surface hydrophobicity measured by Sypro Orange fluorescence and aggregation measured by DLS (a) and ThT fluorescence (b). Data are means and s.e.m. from 3 independent experiments. Measurements were made 20 minutes after TFE treatment. The high linear correlation coefficient R, denotes the strength of the correlation. Note that the MBR mutants are more susceptible to TFE-induced aggregation than the WTL mutants. (c) Aggregation of SOD1G85R and SOD1G37R at room temperature, revealed by DLS.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927901&req=5

fig6: Aggregation of as-purified ALS-causing SOD1 mutants is caused by their increased propensity to expose hydrophobic surfaces. (a, b) Correlation between surface hydrophobicity measured by Sypro Orange fluorescence and aggregation measured by DLS (a) and ThT fluorescence (b). Data are means and s.e.m. from 3 independent experiments. Measurements were made 20 minutes after TFE treatment. The high linear correlation coefficient R, denotes the strength of the correlation. Note that the MBR mutants are more susceptible to TFE-induced aggregation than the WTL mutants. (c) Aggregation of SOD1G85R and SOD1G37R at room temperature, revealed by DLS.
Mentions: In all conditions examined using two specific SOD1 mutants, we observed a correlation between exposure of hydrophobic surfaces and aggregation. To determine whether increased exposure of hydrophobic surfaces correlates with aggregation of diverse SOD1 mutants, we monitored aggregation and exposure of hydrophobic surfaces of the representative set of 12 mutants. As-purified SOD1WT and mutant proteins were exposed to 20% TFE, a condition that was found to elicit aggregation of the diverse SOD1 mutants, but not the wild-type protein. This treatment converted the soluble MBR mutants into aggregates in 20 minutes and was also efficient to provoke aggregation of the WTL mutants (Supplementary Fig. 6a). Exposure of surface hydrophobicity was monitored by Sypro Orange fluorescence and aggregation monitored both by DLS and ThT fluorescence (Fig. 6a and b). ThT fluorescence indicated that aggregates have an amyloid component. Analysis of Congo Red binding confirmed this (Supplementary Fig. 6b). We found exposure of hydrophobicity and ordered assembly of SOD1 aggregates, monitored by these two independent methods, were strongly correlated. The strength of the correlation between exposure of hydrophobic surfaces and aggregation is attested by the very high linear correlation coefficient (Fig. 6a, R = 0.95 and b, R = 0.84). This revealed that exposure of hydrophobicity is a common feature that provokes aggregation of diverse SOD1 mutants.

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