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Protein folding studies of human superoxide dismutase and ALS associated mutants
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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.
Keyword [en]
Biochemistry, superoxide dismutase, ALS, protein folding, equilibrium titration, protein stability, apo protein, disulphide bridge, chevron plot
Keyword [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-305ISBN: 91-7305-702-9 (print)OAI: oai:DiVA.org:umu-305DiVA: diva2:143020
Public defence
2004-09-25, KB3A9, KBC, Umeå Universitet, 10:00
Opponent
Supervisors
Available from: 2004-09-20 Created: 2004-09-20 Last updated: 2012-06-07Bibliographically approved
List of papers
1. Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents
Open this publication in new window or tab >>Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents
2004 (English)In: Protein Expression and Purification, ISSN 1046-5928, Vol. 37, no 2, 311-9 p.Article in journal (Refereed) Published
Abstract [en]

To fully understand the function of the Cu- and Zn-containing superoxide dismutases in normal and disordered cells, it is essential to study protein variants with full metal contents. We describe the use of an Escherichia coli-based expression system for the overproduction of human intracellular wild type CuZn-superoxide dismutase (SOD), the CuZnSOD variant F50E/G51E (monomeric), two amyotrophic lateral sclerosis-related mutant CuZnSOD variants (D90A and G93A), and PseudoEC-SOD, all with high Cu contents. This system is based on coexpression of the SOD variants with the yeast copper chaperone yCCS during growth in a medium supplemented with Cu(2+) and Zn(2+). The recombinant SOD enzymes were all found in the cytosol and represented 30-50% of the total bacterial protein. The enzymes were purified to homogeneity and active enzymes were obtained in high yield. The resulting proteins were characterized through immunochemical reactivity and specific activity analyses, in conjunction with mass-, photo-, and atomic absorption-spectroscopy.

Place, publisher, year, edition, pages
Elsevier Inc, 2004
Identifiers
urn:nbn:se:umu:diva-20475 (URN)10.1016/j.pep.2004.06.006 (DOI)15358352 (PubMedID)
Available from: 2009-03-20 Created: 2009-03-20 Last updated: 2010-10-25Bibliographically approved
2. Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: Decreased stability of the apo state
Open this publication in new window or tab >>Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: Decreased stability of the apo state
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 1.15.1.1) 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.

Keyword
ALS, amyotrophic lateral sclerosis, SOD, superoxide dismutase, CCS, copper chaperone for SOD, GdmCl, guanidinium chloride, TCEP, tris(2-carboxyethyl)phosphine hydrochloride
Identifiers
urn:nbn:se:umu:diva-8789 (URN)doi:10.1073/pnas.262527099 (DOI)
Available from: 2008-02-12 Created: 2008-02-12 Last updated: 2012-06-07Bibliographically approved
3. Folding of human apoSOD: disulphide reduction prevents dimerisation and produces marginally stable monomers
Open this publication in new window or tab >>Folding of human apoSOD: disulphide reduction prevents dimerisation and produces marginally stable monomers
In: Proc. Nat. Ac. SciArticle in journal (Refereed) Submitted
Identifiers
urn:nbn:se:umu:diva-4062 (URN)
Available from: 2004-09-20 Created: 2004-09-20Bibliographically approved
4. Folding of ALS-associated SOD mutants: combined role of monomer and interface stability in noxious gain of function.
Open this publication in new window or tab >>Folding of ALS-associated SOD mutants: combined role of monomer and interface stability in noxious gain of function.
(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-4063 (URN)
Available from: 2004-09-20 Created: 2004-09-20 Last updated: 2010-01-14Bibliographically approved

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