Zhang Li, Shimoji Mika, Thomas Bobby, Moore Darren J, Yu Seong-Woon, Marupudi Neena I, Torp Reidun, Torgner Ingeborg A, Ottersen Ole P, Dawson Ted M, Dawson Valina L
Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Hum Mol Genet. 2005 Jul 15;14(14):2063-73. doi: 10.1093/hmg/ddi211. Epub 2005 Jun 8.
Both homozygous (L166P, M26I, deletion) and heterozygous mutations (D149A, A104T) in the DJ-1 gene have been identified in Parkinson's disease (PD) patients. The biochemical function and subcellular localization of DJ-1 protein have not been clarified. To date the localization of DJ-1 protein has largely been described in studies over-expressing tagged DJ-1 protein in vitro. It is not known whether the subcellular localization of over-expressed DJ-1 protein is identical to that of endogenously expressed DJ-1 protein both in vitro and in vivo. To clarify the subcellular localization and function of DJ-1, we generated three highly specific antibodies to DJ-1 protein and investigated the subcellular localization of endogenous DJ-1 protein in both mouse brain tissues and human neuroblastoma cells. We have found that DJ-1 is widely distributed and is highly expressed in the brain. By cell fractionation and immunogold electron microscopy, we have identified an endogenous pool of DJ-1 in mitochondrial matrix and inter-membrane space. To further investigate whether pathogenic mutations might prevent the distribution of DJ-1 to mitochondria, we generated human neuroblastoma cells stably transfected with wild-type (WT) or mutant (M26I, L166P, A104T, D149A) DJ-1 and performed mitochondrial fractionation and confocal co-localization imaging studies. When compared with WT and other mutants, L166P mutant exhibits largely reduced protein level. However, the pathogenic mutations do not alter the distribution of DJ-1 to mitochondria. Thus, DJ-1 is an integral mitochondrial protein that may have important functions in regulating mitochondrial physiology. Our findings of DJ-1's mitochondrial localization may have important implications for understanding the pathogenesis of PD.
在帕金森病（PD）患者中已鉴定出DJ-1基因的纯合突变（L166P、M26I、缺失）和杂合突变（D149A、A104T）。DJ-1蛋白的生化功能和亚细胞定位尚未明确。迄今为止，DJ-1蛋白的定位在很大程度上是在体外过表达带标签的DJ-1蛋白的研究中描述的。尚不清楚过表达的DJ-1蛋白的亚细胞定位在体外和体内是否与内源性表达的DJ-1蛋白相同。为了阐明DJ-1的亚细胞定位和功能，我们制备了三种针对DJ-1蛋白的高度特异性抗体，并研究了内源性DJ-1蛋白在小鼠脑组织和人神经母细胞瘤细胞中的亚细胞定位。我们发现DJ-1广泛分布且在脑中高表达。通过细胞分级分离和免疫金电子显微镜，我们在线粒体基质和膜间隙中鉴定出内源性DJ-1池。为了进一步研究致病突变是否可能阻止DJ-1向线粒体的分布，我们构建了稳定转染野生型（WT）或突变型（M26I、L166P、A104T、D149A）DJ-1的人神经母细胞瘤细胞，并进行了线粒体分级分离和共聚焦共定位成像研究。与WT和其他突变体相比，L166P突变体的蛋白水平大幅降低。然而，致病突变并未改变DJ-1向线粒体的分布。因此，DJ-1是一种完整的线粒体蛋白，可能在调节线粒体生理学方面具有重要功能。我们关于DJ-1线粒体定位的发现可能对理解PD的发病机制具有重要意义。
