Mutant Cu/Zn-superoxide dismutase proteins have altered solubility and interact with heat shock/stress proteins in models of amyotrophic lateral sclerosis.
Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis. In humans and experimental models, death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD-1 protein. In our previous studies, heat shock protein 70 (HSP70) prolonged viability of cultured motor neurons expressing mutant human SOD-1 and reduced formation of aggregates. In this paper, we report that mutant SOD-1 proteins have altered solubility in cells relative to wild-type SOD-1 and can form a direct association with HSP70 and other stress proteins. Whereas wild-type human and endogenous mouse SOD-1 were detergent-soluble, a portion of mutant SOD-1 was detergent-insoluble in protein extracts of NIH3T3 transfected with SOD-1 gene constructs, spinal cord cultures established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and alphaB-crystallin with mutant SOD-1 (G93A or G41S), but not wild-type or endogenous mouse SOD-1, was demonstrated by coimmunoprecipitation. Mutant SOD-1.HSP70 complexes were predominantly in the detergent-insoluble fraction. However, only a small percentage of total cellular mutant SOD-1 was detergent-insoluble, suggesting that mutation-induced alteration of protein conformation may not in itself be sufficient for direct interaction with heat shock proteins.
Shinder
G A
GA
Department of Neurology/Neurosurgery and Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
Lacourse
M C
MC
Minotti
S
S
Durham
H D
HD
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D004195:Disease Models, Animal; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D016472:Motor Neuron Disease / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D006360:Heat-Shock Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D013482:Superoxide Dismutase