Mutant SOD1 detoxification mechanisms in intact single cells
Mutant SOD1 detoxification mechanisms in intact single cells
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DOI：
10.1038/sj.cdd.4402262
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发表时间：
2008-02-01
影响因子：
12.4
通讯作者：
Weishaupt, J. H.
中科院分区：
文献类型：
--
作者：
Ganesan, S.;Rohde, G.;Weishaupt, J. H.
突变的超氧化物歧化酶1(MtSOD1)主要导致遗传性肌萎缩侧索硬化症(ALS)。MtSOD1毒性的机制尚不清楚。两个主要假说是蛋白酶体功能受损和mtSOD1错误折叠导致的伴侣蛋白缺失。在这里，我们使用FRET/FLIM和生物传感器成像来定量定位mtSOD1的泛素化和伴侣结合，并评估它们对蛋白酶体和蛋白质折叠活性的影响。我们发现，在完整的单细胞中，野生型(Wt)SOD1和mtSOD1的泛素化和伴侣相互作用水平存在很大差异。此外，蛋白酶体结构和细胞质物质的SOD1泛素化水平也不同。Wt和mtSOD1物种的HSP70结合和泛素化是高度相关的，表明这两种细胞解毒机制对mtSOD1表达的上调是耦合的。在单个细胞中的生物传感器成像显示，mtSOD1的表达改变了所研究的神经元细胞系中的细胞蛋白折叠活性，但不改变蛋白酶体功能。我们的结果首次提供了SOD1泛素化和伴侣相互作用的逐个细胞分析。此外，我们的研究为ALS的细胞生物学研究开辟了新的方法学途径。
Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quantitatively localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 versus mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examined. Our results provide the first cell-bycell- analysis of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodological avenues for cell biological research on ALS.