Kirby Kim, Jensen Laran T, Binnington Janet, Hilliker Arthur J, Ulloa Janella, Culotta Valeria C, Phillips John P
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G2W1, Canada.
J Biol Chem. 2008 Dec 19;283(51):35393-401. doi: 10.1074/jbc.M807131200. Epub 2008 Oct 23.
Copper,zinc superoxide dismutase (SOD1) in mammals is activated principally via a copper chaperone (CCS) and to a lesser degree by a CCS-independent pathway of unknown nature. In this study, we have characterized the requirement for CCS in activating SOD1 from Drosophila. A CCS-null mutant (Ccs(n)(29)(E)) of Drosophila was created and found to phenotypically resemble Drosophila SOD1-null mutants in terms of reduced adult life span, hypersensitivity to oxidative stress, and loss of cytosolic aconitase activity. However, the phenotypes of CCS-null flies were less severe, consistent with some CCS-independent activation of Drosophila SOD1 (dSOD1). Yet SOD1 activity was not detectable in Ccs(n)(29)(E) flies, due largely to a striking loss of SOD1 protein. In contrast, human SOD1 expressed in CCS-null flies is robustly active and rescues the deficits in adult life span and sensitivity to oxidative stress. The dependence of dSOD1 on CCS was also observed in a yeast expression system where the dSOD1 polypeptide exhibited unusual instability in CCS-null (ccs1Delta) yeast. The residual dSOD1 polypeptide in ccs1Delta yeast was nevertheless active, consistent with CCS-independent activation. Stability of dSOD1 in ccs1Delta cells was readily restored by expression of either yeast or Drosophila CCS, and this required copper insertion into the enzyme. The yeast expression system also revealed some species specificity for CCS. Yeast SOD1 exhibits preference for yeast CCS over Drosophila CCS, whereas dSOD1 is fully activated with either CCS molecule. Such variation in mechanisms of copper activation of SOD1 could reflect evolutionary responses to unique oxygen and/or copper environments faced by divergent species.
哺乳动物中的铜锌超氧化物歧化酶（SOD1）主要通过铜伴侣蛋白（CCS）激活，在较小程度上通过一条性质不明的不依赖CCS的途径激活。在本研究中，我们对果蝇中激活SOD1所需的CCS进行了表征。构建了果蝇的CCS基因缺失突变体（Ccs(n)(29)(E)），发现其在成年寿命缩短、对氧化应激超敏以及胞质乌头酸酶活性丧失方面，表型与果蝇SOD1基因缺失突变体相似。然而，CCS基因缺失果蝇的表型较轻，这与果蝇SOD1（dSOD1）存在一些不依赖CCS的激活作用一致。不过，在Ccs(n)(29)(E)果蝇中未检测到SOD1活性，这主要是由于SOD1蛋白显著缺失。相比之下，在CCS基因缺失果蝇中表达的人SOD1具有强大的活性，并能挽救成年寿命和氧化应激敏感性方面的缺陷。在酵母表达系统中也观察到dSOD1对CCS的依赖性，其中dSOD1多肽在CCS基因缺失（ccs1Delta）的酵母中表现出异常的不稳定性。然而，ccs1Delta酵母中残留的dSOD1多肽仍具有活性，这与不依赖CCS的激活作用一致。通过表达酵母或果蝇的CCS，可轻易恢复ccs1Delta细胞中dSOD1的稳定性，这需要将铜插入酶中。酵母表达系统还揭示了CCS的一些物种特异性。酵母SOD1对酵母CCS的偏好高于果蝇CCS，而dSOD1与任何一种CCS分子都能完全激活。SOD1铜激活机制的这种差异可能反映了不同物种对独特的氧气和/或铜环境的进化反应。
