SOD1 mutantsSystematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associatedMikael J. Lindberg, Roberth Byström, Niklas Boknäs, Peter M. Andersen, and Mikael Oliveberg doi:10.1073/pnas.0501957102 2005;102;9754-9759; originally published online Jun 29, 2005; PNAS This information is current as of November 2006. & ServicesOnline Information www.pnas.org/cgi/content/full/102/28/9754etc., can be found at: High-resolution figures, a citation map, links to PubMed and Google Scholar, Supplementary Material www.pnas.org/cgi/content/full/0501957102/DC1Supplementary material can be found at: References www.pnas.org/cgi/content/full/102/28/9754#BIBLThis article cites 50 articles, 22 of which you can access for free at: www.pnas.org/cgi/content/full/102/28/9754#otherarticlesThis article has been cited by other articles: E-mail Alerts. click hereat the top right corner of the article orReceive free email alerts when new articles cite this article - sign up in the box Rights & Permissions www.pnas.org/misc/rightperm.shtmlTo reproduce this article in part (figures, tables) or in entirety, see: Reprints www.pnas.org/misc/reprints.shtmlTo order reprints, see: Notes:Systematically perturbed folding patterns ofamyotrophic lateral sclerosis (ALS)-associatedSOD1 mutantsMikael J. Lindberg†, Roberth Bystro¨m†, Niklas Bokna¨s†, Peter M. Andersen§, and Mikael Oliveberg†¶Departments of†Biochemistry and§Clinical Neuroscience, Umeå University, S-901 87 Umeå, SwedenEdited by Alan R. Fersht, University of Cambridge, Cambridge, United Kingdom, and approved May 18, 2005 (received for review March 9, 2005)Amyotrophic lateral sclerosis is a neurodegenerative syndromeassociated with 114 mutations in the gene encoding the cytosolichomodimeric enzyme Cu兾Zn superoxide dismutase (SOD). In thisarticle, we report that amyotrophic lateral sclerosis-associated SODmutations with distinctly different disease progression can berationalized in terms of their folding patterns. The mutations arefound to perturb the protein in multiple ways; they destabilize theprecursor monomers (class 1), weaken the dimer interface (class 2),or both at the same time (class 1 ⴙ 2). A shared feature of themutational perturbations is a shift of the folding equilibriumtoward poorly structured SOD monomers. We observed a link,coupled to the altered folding patterns, between protein stability,net charge, and survival time for the patients carrying themutations.neurodegenerative disease 兩 protein stabilityAfamilial form of the fatal neurodegenerative disease amyo-trophic lateral sclerosis (ALS) has been linked to a large set ofmutations in the ubiquitous enzyme Cu兾Zn superoxide dismutase(SOD) (1, 2). Although the mechanism by which mutant SODcauses neural death remains elusive (3), several lines of evidencesuggest that ALS is a protein-folding disease (4–13), in analogy withother neurodegenerative disorders like Parkinson’s disease, Lund–Huntington’s disease, and Creutzfeldt–Jakob’s disease (14). Thecytotoxic component of these diseases seems to be ordered oligo-meric states, or aggregates of unfolded or misfolded proteins thatescape the cellular quality-control and housekeeping mechanisms(15). More detailed elucidation of the molecular events underlyingcytotoxic gain of function has been hampered by generally complexfolding and aggregation behaviors of the disease-provoking pro-teins (15); it is not yet clear from which conformational species thecytotoxic pathway originates or whether the toxicity mechanism iscommon to all protein-folding disorders or disease-specific. In thecase of SOD, thermodynamic analysis suggests that the WT proteinis a so called three-state dimer (13, 16, 17) in which the individualmonomers adopt their correct folded structures also in the absenceof the intermolecular interface (18) and the stabilizing Zn and Cuions (16, 19). Recently, several findings have implicated suchimmature monomeric SOD species as precursors in the ALSmechanism (11–13, 20, 21).To shed more light on the origin of neurotoxicity in ALS, we havecompared the folding behavior of 15 ALS-associated SOD mutantswith survival times ranging from 1 to 17 years after the onset of thefirst symptom (Fig. 1). In support of the notion that ALS is aprotein-folding disorder, the folding patterns of the ALS-associatedmutants show different and characteristic features that can belinked to their respective disease-progression rates. Common to allmutations is a shift of the folding equilibrium toward the denaturedmonomer. However, the magnitudes and the mechanistic origins ofthis shift are found to vary; the ALS-associated mutations reducethe stability of the monomer, the strength of the dimer interface, orboth. The observation implies that therapeutic strategies (22) toprevent the noxious effects of mutant SOD at a primary proteinlevel (23) must restore at the same time perturbations of both themonomer and interface stability; restoring only one of these com-ponents may not be sufficient to suppress the disease.MaterialsAll experiments were done under oxidizing conditions, with theintramolecular disulphide linkage between C57 and C146 keptintact. To avoid artifacts from nonnative disulphide crosslinksbetween the remaining free cysteines C6 and C111, we used thedouble mutant C6A兾C111A (SODpwt) as pseudo-WT for furthermutant analysis (24). The substitutions C6A兾C111A cause only aslight decrease of protein stability but show no influence on theSOD folding behavior (25).Mutant Selection. For most of the 114 known SOD1 mutations, littleor no clinical data have been published. The 15 ALS-associatedmutants included in this study are the missense mutations fromwhich reliable and sufficient clinical data are available (see sup-porting information, which is published on the PNAS web site). Thepatient survival times from the onset of first symptom to death ortracheotomy are given in Table 2.Protein Preparation. The mutants were constructed on a back-ground of C6A兾C111A and the monomeric variant F50E兾G51E兾C6A兾C111A, and they were coexpressed with copper chaperone(yCCS) as described in ref. 26. Apo protein was prepared asdescribed in ref. 25, and the standard buffer was 10 mM Mes (pH6.3) with 10 mM EDTA to maintain the proteins metal free in allsteps of the analysis.Gel Filtration. Dimerization was analyzed by gel chromatography ona Sephadex S-75 column (Amersham Pharmacia), as described inref. 25.