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Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: Decreased stability of the apo state
Umeå University, Faculty of Science and Technology, Chemistry.
Umeå University, Faculty of Science and Technology, Chemistry.
Umeå University, Faculty of Science and Technology, Chemistry.
2002 (English)In: PNAS, Vol. 99, no 26, 16607-12 p.Article in journal (Refereed) Published
Abstract [en]

More than 100 point mutations of the superoxide scavenger Cu/Zn superoxide dismutase (SOD; EC have been associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, these mutations are scattered throughout the protein and provide no clear functional or structural clues to the underlying disease mechanism. Therefore, we undertook to look for folding-related defects by comparing the unfolding behavior of five ALS-associated mutants with distinct structural characteristics: A4V at the interface between the N and C termini, C6F in the hydrophobic core, D90A at the protein surface, and G93A and G93C, which decrease backbone flexibility. With the exception of the disruptive replacements A4V and C6F, the mutations only marginally affect the stability of the native protein, yet all mutants share a pronounced destabilization of the metal-free apo state: the higher the stability loss, the lower the mean survival time for ALS patients carrying the mutation. Thus organism-level pathology may be directly related to the properties of the immature state of a protein rather than to those of the native species.

Place, publisher, year, edition, pages
2002. Vol. 99, no 26, 16607-12 p.
Keyword [en]
ALS, amyotrophic lateral sclerosis, SOD, superoxide dismutase, CCS, copper chaperone for SOD, GdmCl, guanidinium chloride, TCEP, tris(2-carboxyethyl)phosphine hydrochloride
URN: urn:nbn:se:umu:diva-8789DOI: doi:10.1073/pnas.262527099OAI: diva2:148460
Available from: 2008-02-12 Created: 2008-02-12 Last updated: 2012-06-07Bibliographically approved
In thesis
1. Protein folding studies of human superoxide dismutase and ALS associated mutants
Open this publication in new window or tab >>Protein folding studies of human superoxide dismutase and ALS associated mutants
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are among the most abundant biological macromolecules. The cellular machinery is coupled to exact structural shape and properties of the more than 100 000 different proteins. Still, proteins can sometimes completely change their character and as a result trigger neuro degenerative disease. Exactly what happens is yet poorly understood but misfolding and aggregation leading to toxic gain of function is probable causes, i.e. the protein adopts new noxious properties. In 1993 the protein superoxide dismutase (SOD) was found to be associated with the neuro degenerative disease ALS. Up to date more than 100 mutations in SOD have been associated with ALS. However, the mutations are scattered all over the structure and no common denominator for the disease mechanism has been found.

This work has been focused on the molecular mechanism of the toxic gain-of - function of mutant SOD from the perspective of protein folding and structural stability. To facilitate the studies of SOD and its ALS associated mutations, an expression system resulting in increased copper content was developed. Coexpression with the copper chaperone for superoxide dismutase (yCCS) leads to increased expression levels, especially for the destabilised ALS mutants. Through thermodynamic studies, I show that with the exception of the most disruptive mutations the holo protein is only marginally destabilised, whereas all mutations show a pronounced destabilisation on the apo protein. Kinetic studies suggest further that the dimeric apoSOD folds via a three-state process where the dimerisation proceeds via a marginally stable monomer. The apoSOD monomer folds by a two-state process. The disulphide bond is not critical for the folding of the apoSOD monomer although it contributes significantly to its stability. Interestingly, in the absence of metals, reduction of the disulphide bond prevents the formation of the dimer. A mutation can affect the protein stability in various ways: either from destabilisation of the monomer (case 1), weakening of the dimer interface (case 2) or, in the worst case, from a combination of both (case 1+2). Thus, therapeutic strategies to prevent the noxious effects of mutant SOD must include both mechanisms. An important finding in this study is that we can see a correlation between the stability for each mutation and the mean survival time. This could be an opening in the development of therapeutic substances that counteract the defect in SOD upon mutation.

Place, publisher, year, edition, pages
Umeå: Kemi, 2004. 74 p.
Biochemistry, superoxide dismutase, ALS, protein folding, equilibrium titration, protein stability, apo protein, disulphide bridge, chevron plot, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:umu:diva-305 (URN)91-7305-702-9 (ISBN)
Public defence
2004-09-25, KB3A9, KBC, Umeå Universitet, 10:00
Available from: 2004-09-20 Created: 2004-09-20 Last updated: 2012-06-07Bibliographically approved

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