title: A highly sensitive assay of IRE1 activity using the small luciferase NanoLuc: Evaluation of ALS-related genetic and pathological factors.
abstract: Activation of inositol-requiring enzyme 1 (IRE1) due to abnormal conditions of the endoplasmic reticulum (ER) is responsible for the cleavage of an unspliced form of X-box binding protein 1 (uXBP1), producing its spliced form (sXBP1). To estimate IRE1 activation, several analytical procedures using green fluorescence protein and firefly luciferase have been developed and applied to clarify the roles of IRE1-XBP1 signaling pathways during development and disease progression. In this study, we established a highly sensitive assay of IRE1 activity using a small luciferase, NanoLuc, which has approximately 100-fold higher activity than firefly luciferase. The NanoLuc reporter, which contained a portion of the spliced region of XBP1 upstream of NanoLuc, was highly sensitive and compatible with several types of cell lines. We found that NanoLuc was secreted into the extracellular space independent of the ER-Golgi pathway. The NanoLuc activity of an aliquot of culture medium from the neuroblastoma-spinal neuron hybrid cell line NSC-34 reflected the toxic stimuli-induced elevation of intracellular activity well. Using this technique, we evaluated the effects of several genetic and pathological factors associated with the onset and progression of amyotrophic lateral sclerosis (ALS) on NanoLuc reporter activity. Under our experimental conditions, inhibition of ER-Golgi transport by the overexpression of mutant Sar1 activated luciferase activity, whereas the co-expression of mutant SOD1 or the C-terminal fragment of TDP-43 (TDP-25) did not. The addition of homocysteine elevated the reporter activity; however, we did not observe any synergistic effect due to the overexpression of the mutant genes described above. Taken together, these data show that our analytical procedure is highly sensitive and convenient for screening useful compounds that modulate IRE1-XBP1 signaling pathways as well as for estimating IRE1 activation in several pathophysiological diseases.
authors.affiliation: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hikiji
authors.affiliation: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Junpei
authors.initials: J
authors.lastname: Norisada
authors.affiliation: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hirata
authors.affiliation: Laboratory of Pharmaceutical & Medicinal Chemistry, Gifu Pharmaceutical University, 1-25-4, Daigakunishi, Gifu 501-1196, Japan.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Okuda
authors.affiliation: Laboratory of Pharmaceutical & Medicinal Chemistry, Gifu Pharmaceutical University, 1-25-4, Daigakunishi, Gifu 501-1196, Japan.
authors.forename: Hideko
authors.initials: H
authors.lastname: Nagasawa
authors.affiliation: Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Ishigaki
authors.affiliation: Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Gen
authors.initials: G
authors.lastname: Sobue
authors.affiliation: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Kiuchi
authors.affiliation: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. Electronic address: oohashi@gifu-u.ac.jp.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Oh-hashi
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics* / Q000473:pathology; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D006801:Humans; D049409:Luciferases, Firefly / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D000071701:Regulatory Factor X Transcription Factors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism; D000071717:X-Box Binding Protein 1
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; D049409:Luciferases, Firefly; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases
keywords: ER stress; IRE1; NanoLuc; XBP1