SOD1 dimerization monitoring using a novel split NanoLuc, NanoBit.
In the present study, we applied a highly sensitive NanoLuc-based technology to understand the status of superoxide dismutase 1 (SOD1) within mammalian cells. Two fragments of NanoLuc (NanoBit), large N-terminal and small C-terminal regions, were fused with wild-type (wt) and mutant human SOD1 (hSOD1) genes and transfected into cells. Luciferase activity through NanoBit assembly was only detected in NanoBit-tagged wtSOD1-expressing cells. Furthermore, the developed NanoLuc system was used to investigate the role of protein-protein interactions in the pathogenesis of amyotrophic lateral sclerosis (ALS). In addition to SOD1, we also applied this NanoBit system for detecting the dimerization of wild-type, M337V-mutated human TAR-binding protein 43 kDa (hTDP43) and its cleaved C-terminal fragment (TDP25M337V) as well as their interactions with SOD1. Luciferase activities of NanoBit-tagged mutant SOD1, TDP43, or TDP25 were negligible. Finally, we found that a zinc chelator partially reduced the luciferase activity of NanoBit-wtSOD1. Collectively, these results show that the present assay is sensitive and convenient to appreciate ALS and to develop useful agents for the modulation of SOD1 conformation.
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan. oohashi@gifu-u.ac.jp.|Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan. oohashi@gifu-u.ac.jp.
Kentaro
K
Oh-Hashi
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan.|Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Yoko
Y
Hirata
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan.|Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Kazutoshi
K
Kiuchi
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry*; D055503:Protein Multimerization / Q000187:drug effects; D000072105:Superoxide Dismutase-1 / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005029:Ethylenediamines; D050505:Mutant Proteins; C000598624:TARDBP protein, human; D008156:Luciferases; D000072105:Superoxide Dismutase-1; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine
ALS; NanoBit; SOD1