ALS-Causing SOD1 Mutations Promote Production of Copper-Deficient Misfolded Species
Campbell Family Institute for Cancer Research, Ontario Cancer Institute/University Health Network, Department of Biochemistry, University of Toronto, TMDT 4-305, 101 College Street, Toronto, Ontario, Canada M5G 1L7. Journal of Molecular Biology
(Impact Factor: 4.33).
06/2011; 409(5):839-52. DOI: 10.1016/j.jmb.2011.04.027
Point mutations scattered throughout the sequence of Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis (ALS) cases. SOD1 is a homodimer in which each subunit binds one copper atom and one zinc atom. Inclusions containing misfolded SOD1 are seen in motor neurons of SOD1-associated ALS cases. The mechanism by which these diverse mutations cause misfolding and converge on the same disease is still not well understood. Previously, we developed several time-resolved techniques to monitor structural changes in SOD1 as it unfolds in guanidine hydrochloride. By measuring the rates of Cu and Zn release using an absorbance-based assay, dimer dissociation through chemical cross-linking, and β-barrel conformation changes by tryptophan fluorescence, we established that wild-type SOD1 unfolds by a branched pathway involving a Zn-deficient monomer as the dominant intermediate of the major pathway, and with various metal-loaded and Cu-deficient dimers populated along the minor pathway. We have now compared the unfolding pathway of wild-type SOD1 with those of A4V, G37R, G85R, G93A, and I113T ALS-associated mutant SOD1. The kinetics of unfolding of the mutants were generally much faster than those of wild type. However, all of the mutants utilize the minority pathway to a greater extent than the wild-type protein, leading to greater populations of Cu-deficient intermediates and decreases in Zn-deficient intermediates relative to the wild-type protein. The greater propensity of the mutants to populate Cu-deficient states potentially implicates these species as a pathogenic form of SOD1 in SOD1-associated ALS and provides a novel target for therapeutic intervention.
Available from: Bing-Rui Zhou
- "As mentioned above, amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein , , the human PrP and its pathogenic mutants , , , , and human SOD1 pathogenic mutants , ,  have the tendency to form fibril deposits in a variety of tissues and thereby cause Alzheimer disease, prion disease, and ALS, respectively, while the rabbit PrP , ,  and hen egg white lysozyme  do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In the present study, we demonstrated that macromolecular crowding dramatically promoted fibril formation of amyloidogenic proteins, such as GSK-3β phosphorylated human Tau protein, the human PrP and its pathogenic mutants E196K and D178N, and pathological human SOD1 mutant A4V, but remarkably inhibited aggregation of some non-amyloidogenic proteins, such as the rabbit PrP and hen egg white lysozyme. "
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ABSTRACT: Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.
As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.
We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.
PLoS ONE 04/2012; 7(4):e36288. DOI:10.1371/journal.pone.0036288 · 3.23 Impact Factor
Available from: Jessica A O Rumfeldt
- "Increased unfolding rates and accessibility of either on or off-folding pathway intermediates may increase the accessibility of transient protein species that can initiate aggregation (Dobson 2003; Wang et al. 2008). Furthermore, it has been proposed that fALS-associated mutations, even those far from the metal binding sites, promote increased levels of Cu-deficient intermediates along the holoS- S SOD1 unfolding pathway (Ip et al. 2010). Cu-deficient intermediates are lower in stability and therefore may have a higher tendency to aggregate. "
Amyotrophic Lateral Sclerosis, 01/2012; , ISBN: 978-953-307-806-9
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ABSTRACT: Kinetic experiments provide much information about protein folding mechanisms. Time-resolved signals are often best described by expressions with many exponential terms, but this hinders the extraction of rate constants by nonlinear least squares (NLS) fitting. Numerical inverse Laplace transformation, which converts a time-resolved dataset into a spectrum of amplitudes as a function of rate constant, allows easy estimation of the rate constants, amplitudes, and number of processes underlying the data. Here, we present a Tikhonov regularization-based method that converts a dataset into a rate spectrum, subject to regularization constraints, without requiring an iterative search of parameter space. This allows more rapid generation of rate spectra as well as analysis of datasets too noisy to process by existing iterative search algorithms. This method's simplicity also permits highly objective, largely automatic analysis with minimal human guidance. We show that this regularization method reproduces results previously obtained by NLS fitting and that it is effective for analyzing datasets too complex for traditional fitting methods. This method's reliability and speed, as well as its potential for objective, model-free analysis, make it extremely useful as a first step in analysis of complicated noisy datasets and an excellent guide for subsequent NLS analysis.
Analytical Biochemistry 11/2011; 421(1):181-90. DOI:10.1016/j.ab.2011.10.050 · 2.22 Impact Factor
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