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Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions : The Roles of Disulfide Bonds and Effects of Denaturant

Khan, Ashhar LU ; Respondek, Michal LU ; Kjellström, Sven LU ; Deep, Shashank; Linse, Sara LU and Akke, Mikael LU (2017) In ACS Chemical Neuroscience 8(9). p.2019-2026
Abstract

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril... (More)

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Amyotrophic lateral sclerosis, disulfide reduction, protein aggregation, protein unfolding, ThT fluorescence, transmission electron microscopy
in
ACS Chemical Neuroscience
volume
8
issue
9
pages
8 pages
publisher
The American Chemical Society
external identifiers
  • scopus:85029620956
  • wos:000411662000025
ISSN
1948-7193
DOI
10.1021/acschemneuro.7b00162
language
English
LU publication?
yes
id
ccd8bc46-98f9-4e38-8b8c-6471e645cd68
date added to LUP
2017-09-29 09:05:30
date last changed
2018-05-13 04:35:35
@article{ccd8bc46-98f9-4e38-8b8c-6471e645cd68,
  abstract     = {<p>Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.</p>},
  author       = {Khan, Ashhar and Respondek, Michal and Kjellström, Sven and Deep, Shashank and Linse, Sara and Akke, Mikael},
  issn         = {1948-7193},
  keyword      = {Amyotrophic lateral sclerosis,disulfide reduction,protein aggregation,protein unfolding,ThT fluorescence,transmission electron microscopy},
  language     = {eng},
  month        = {09},
  number       = {9},
  pages        = {2019--2026},
  publisher    = {The American Chemical Society},
  series       = {ACS Chemical Neuroscience},
  title        = {Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions : The Roles of Disulfide Bonds and Effects of Denaturant},
  url          = {http://dx.doi.org/10.1021/acschemneuro.7b00162},
  volume       = {8},
  year         = {2017},
}