Article

Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase

Department of Chemistry and Biochemistry, California State University Long Beach, Long Beach, CA, USA.
PLoS ONE (Impact Factor: 3.23). 02/2009; 4(3):e5004. DOI: 10.1371/journal.pone.0005004
Source: PubMed

ABSTRACT Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

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    • "These conditions cause release of Ca 2+ , leading to reduction of thermal stability and formation of partially folded conformations (Goers et al., 2002; Veprintsev et al., 1997; Ebrahim-Habibi et al., 2010a). Similarly, loss of metal ions, disulfide reduction, and pathologic mutations may drive superoxide dismutase toward amyloid fibrillation (Oztug Durer et al., 2009; Fee and Phillips, 1975; Mei et al., 1992). Fibril formation is also observed in apo-carbonic anhydrase, which may take up a pre-molten globular structure after removal of its zinc ion (Es-haghi et al., 2012). "
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    ABSTRACT: A number of ligands with affinities for the heme binding site of apomyoglobin were tested to control amorphous and fibrillar aggregation in the protein. Several techniques, including fluorescence, dynamic light scattering, transmission electron microscopy, dot blot analysis combined with viability studies were employed for structural characterization and cytotoxicity assessment of the intermediate and final protein structures formed during the aggregation process. Of the small molecules investigated, chrysin and Nile red with high structural similarities to heme were chosen for further studies. Only fibril formation was found to be prevented by Nile red, while chrysin, with a greater structural flexibility, was able to prevent both types of aggregate formation. The two ligands were found to influence aggregation at different stages of intermediate structure formation, an ability determined by their degrees of similarities with heme. Based on structural characterization and toxicity studies, it is concluded that ligands similar in structure to heme may be effective in influencing various stages of aggregate formation and toxicity potencies of the protein structures. Since metalloproteins constitute more than thirty percent of all known proteins, it is concluded that the present strategy may be of general significance.
    The international journal of biochemistry & cell biology 10/2012; 45(2). DOI:10.1016/j.biocel.2012.10.004 · 4.24 Impact Factor
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    • "One of the conditions for triggering aggregation of non-reduced SOD1 is the addition of a chaotropic agent, guanidine hydrochloride (GdnHCl). Valentine and colleagues have performed agitation of 50 M apo-SOD1 S-S with Teflon balls in a buffer (10 mM K-Pi, pH 7.4) containing 1 M GdnHCl and found the formation of Thioflavin-T (ThT)-positive aggregates after approximately 40 hours (Chattopadhyay et al., 2008; Oztug Durer et al., 2009). These apo-SOD1 S-S aggregates possess fibrillar morphologies, and its formation is accelerated when the solution pH becomes acidic (pH 3.0, 4.0, 5.0). "
    Amyotrophic Lateral Sclerosis, 01/2012; , ISBN: 978-953-307-806-9
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    • "Importantly, the aggregates formed from holoS-S SOD1 in this study exhibited similar structural, dye-binding, and immunological characteristics as the aggregates found in fALS patients (Hwang et al. 2010). In contrast, other studies have reported that SOD1 does not aggregate from the holoS-S form (Chattopadhyay et al. 2008), or requires extremely destabilizing conditions with agitation to promote fibrilization (Oztug Durer et al. 2009). The differences between these findings may be related to the different experimental conditions for studying SOD1 aggregation, such as length of incubation, frequency of sampling, and methods for monitoring aggregation. "
    Amyotrophic Lateral Sclerosis, 01/2012; , ISBN: 978-953-307-806-9
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