Cu,Zn-superoxide dismutase increases toxicity of mutant and zinc-deficient superoxide dismutase by enhancing protein stability.
When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper- and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zinc-deficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SOD-expressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive "partially unfolded intermediate" responsible for the toxic gain of function conferred by ALS mutant SOD.
Garner
Mary Anne
MA
Burke Medical Research Institute, White Plains, New York 10605, USA.
Ricart
Karina C
KC
Roberts
Blaine R
BR
Bomben
Valerie C
VC
Basso
Manuela
M
Ye
Yaozu
Y
Sahawneh
John
J
Franco
Maria Clara
MC
Beckman
Joseph S
JS
Estévez
Alvaro G
AG
D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000737:chemistry; D007700:Kinetics; D009046:Motor Neurons / Q000378:metabolism; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D051381:Rats; D013482:Superoxide Dismutase / Q000378:metabolism*
D002614:Chelating Agents; D011506:Proteins; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D003300:Copper; D013482:Superoxide Dismutase