title: NEIL1 p.Gln282Stop variant is predominantly localized in the cytoplasm and exhibits reduced activity in suppressing mutations.
abstract: Human NEIL1 protein is a DNA glycosylase known to be involved in the repair of oxidized DNA lesions. A c.C844T germline variant of the NEIL1 gene has recently been identified in the Japanese population, however, the p.Q282Stop-type protein produced from this variant gene has not yet been characterized. In this study to determine whether the NEIL1 c.C844T variant might be a defective allele, we investigated the subcellular localization of the p.Q282Stop-type protein and its ability to suppress the development of mutations in mammalian cells. In contrast to the nuclear localization of wild-type (WT) NEIL1, the p.Q282Stop-type protein tagged with GFP or FLAG was localized predominantly in the cytoplasm of human H1299 cells. Mutant forms of the putative nuclear localization signal (NLS, amino acid sequences 359 to 378) of NEIL1-GFP resulted in predominant cytoplasmic localization of the mutants, suggesting that the abnormal localization of p.Q282Stop-type NEIL1 may also be caused by a loss of the putative NLS in the protein. Next, V79 mammalian cell lines inducibly expressing WT NEIL1 or p.Q282Stop-type NEIL1 were established using the piggyBac transposon vector system, and the mutation frequency was compared between the cell lines by HPRT assay. The frequency of mutations induced by glucose oxidase, an oxidative stress inducer, was higher in the p.Q282Stop-type NEIL1-transposed cells than that in the WT NEIL1-transposed cells. Finally, the Cancer Genome Atlas (TCGA) data showed an increased number of somatic mutations in primary carcinomas containing a truncating NEIL1 mutation. These results suggest that p.Q282Stop-type NEIL1 is predominantly localized in the cytoplasm, possibly due to a loss of the NLS, and possesses a reduced ability to suppress the onset of mutations, both findings suggesting that NEIL1 c.C844T is a defective allele.
authors.affiliation: Department of Tumor Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan. Electronic address: kzshinmu@hama-med.ac.jp.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Shinmura
authors.affiliation: Department of Tumor Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors.forename: Hisami
authors.initials: H
authors.lastname: Kato
authors.affiliation: Research Equipment Center, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Kawanishi
authors.affiliation: Division of Carcinogenesis and Prevention, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Goto
authors.affiliation: Department of Tumor Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors.forename: Hong
authors.initials: H
authors.lastname: Tao
authors.affiliation: Department of Tumor Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Inoue
authors.affiliation: Department of Tumor Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors.forename: Satoki
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Department of Tumor Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sugimura
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology; D018389:Codon, Nonsense / Q000235:genetics*; D003593:Cytoplasm / Q000201:enzymology*; D045647:DNA Glycosylases / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017354:Point Mutation
chemical_list: D018389:Codon, Nonsense; D049452:Green Fluorescent Proteins; D045647:DNA Glycosylases; C473634:NEIL1 protein, human
keywords: DNA glycosylase; HPRT assay; NEIL1; Nuclear localization signal (NLS); TCGA; p.Q282Stop-type NEIL1