Biochemical and Biophysical Research Communications
Co-chaperone CHIP associates with mutant Cu/Zn-superoxide dismutase proteins linked to familial amyotrophic lateral sclerosis and promotes their degradation by proteasomes
Section snippets
Materials and methods
Materials. Sources of antibodies used in this study are as follows; anti-human Cu/Zn-superoxide dismutase (SOD1) (Calbiochem), anti-FLAG, and anti-FLAG M2 affinity gel (Sigma), anti-Hsc70/Hsp70 (Stressgen), anti-Hsp90 (Santa Cruz), anti-CHIP (Calbiochem), anti-S5a (Calbiochem), anti-ubiquitin (Boston Biochemical), anti-HA (Santa Cruz), and anti-Xpress (Invitrogen). All other chemicals were purchased from Sigma or Amersham–Pharmacia Biotech unless otherwise specified. Human SOD1 (wild-type and
CHIP associates with FALS-linked mutant SOD1
We initially attempted to screen cellular proteins that specifically associate with FALS-linked mutant SOD1 proteins and to study their role(s) in the degradation of mutant SOD1 proteins, which is mediated by the ubiquitin–proteasome pathway [6], [7]. For such experiments, we transiently expressed FLAG-tagged human SOD1 constructs (wild-type and G93A mutant) in BOSC 23 cells. Forty-eight hours after the transfection, we lysed cells and immunoprecipitated the lysates with anti-FLAG M2 affinity
Discussion
It seems that mutant SOD1 proteins linked to FALS do not kill neuronal cells because they compromise the cellular anti-oxidant defense system, in which normal SOD1 proteins plays a role in scavenging oxygen radicals. On the contrary, accumulating evidences have indicated that unknown toxic “gain-of-function” conferred by mutant SOD1 proteins is responsible for cell death. So far two major hypotheses have been dominating in discussion of such toxicity. The first is that mutant SOD1 proteins
Note added in proof
While this manuscript was in preparation, Takahashi and colleagues reported the similar results in their recent publication [Urushitani et al., J. Neurochem. 90 (2004) 231–244].
Acknowledgments
We thank Prof. Alfred Goldberg (Harvard Medical School) for providing FALS-linked mutant SOD1 constructs. This work was supported by grants from The Ministry of Science and Technology (MOST) of Korea and KRIBB Research Initiative Program.
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