Journal
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
Volume 190, Issue 2, Pages 674-685Publisher
SPRINGER
DOI: 10.1007/s12010-019-03114-x
Keywords
ALS; Chaperone; NanoBiT; Selenocompound; SOD1
Funding
- Japan Society for the Promotion of Science (JSPS, Japan, KAKENHI) [17K19901, 19H04030]
- Grants-in-Aid for Scientific Research [19H04030, 17K19901] Funding Source: KAKEN
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Previously, we evaluated human SOD1 (hSOD1) dimerization in living cells using the NanoLuc complementation reporter system and found that homodimerization of G85R and G93A mutant SOD1 was lower than that of wild-type hSOD1. Since these assays were performed only using N-terminal NanoBiT-tagged hSOD1 constructs in our previous study, we constructed additional hSOD1 genes with NanoBiT-tags at the C-terminus and evaluated the NanoBiT luciferase activities. Among the tested combinations, the luciferase activity in cells expressing NanoBiT-tagged wild-type hSOD1 was higher than that in cells expressing G85R or G93A mutant hSOD1. The NanoBiT luciferase activities were detected both inside and outside of cells; however, the extracellular luciferase activities were minimally dampened by treatment with brefeldin A, which inhibits canonical ER-Golgi transport. In addition to studies on the homodimerization of SOD1, we investigated the interaction between hSOD1 and three chaperone proteins, copper chaperone for SOD1 (CCS), FKBP, and GRP78. The NanoBiT luciferase activities in cells expressing NanoBiT-tagged SOD1 and CCS were relatively high, but weak signals were also observed in cells expressing SOD1 together with FKBP or GRP78. These luciferase activities were different between wild-type and mutant hSOD1. Finally, we investigated the effects of two selenocompounds, ebselen and Se-methylselenocysteine, on SOD1 dimerization and found that ebselen increased the NanoBiT luciferase activity in cells expressing wild-type and mutant hSOD1. In conclusion, we show the differential molecular characteristics of wild-type and mutant hSOD1 in live cells by transfection with NanoBiT-tagged hSOD1 and chaperone genes and demonstrate that this assay might be useful for the development and re-evaluation of chemical compounds modulating the SOD1 conformation.
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