Co-chaperone CHIP associates with mutant Cu/Zn-superoxide dismutase proteins linked to familial amyotrophic lateral sclerosis and promotes their degradation by proteasomes

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Abstract

Although the ubiquitin–proteasome system and the molecular chaperones are implicated to play an important role in pathogenesis of familial amyotrophic lateral sclerosis (FALS) caused by mutations in Cu/Zn-superoxide dismutase (SOD1), the mechanism underlying the causes of this fatal disease is still poorly understood. Here we found that co-chaperone CHIP (carboxyl terminus of Hsc70-interacting protein), together with molecular chaperones Hsc70/Hsp70 and Hsp90, associates with FALS-linked mutant SOD1 proteins in cultured human cells. S5a subunit of 26S proteasomes, which recognizes polyubiquitylated proteins, also interacts with mutant SOD1 proteins. Over-expression of CHIP leads to the reduction in cellular levels of mutant SOD1 as well as the suppression of cytotoxicity induced by mutant SOD1. Unusually, rather than increasing the level of poly-ubiquitylated SOD1, over-expressed CHIP alters the ubiquitylation pattern of mutant SOD1 proteins. Both down-regulation and ubiquitylation of mutant SOD1 are greatly reduced by a mutant CHIP protein lacking U-box domain. Taken together, these results suggest that co-chaperone CHIP, possibly with another E3 ligase(s), modulates the ubiquitylation of mutant SOD1 and renders them more susceptible for proteasomal degradation.

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.

References (27)

  • L. Neckers

    Hsp90 inhibitors as novel cancer chemotherapeutic agents

    Trends Mol. Med.

    (2002)
  • S. Hatakeyama et al.

    U box proteins as a new family of ubiquitin–protein ligases

    J. Biol. Chem.

    (2001)
  • J.S. Valentine et al.

    Misfolded CuZnSOD and amyotrophic lateral sclerosis

    Proc. Natl. Acad. Sci. USA

    (2003)
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