title: Selection of cDNA candidates that induce oligomerization of NLRP3 using a chimeric receptor approach.
abstract: Since diverse cellular events are regulated by protein oligomerization, identification of molecules that affect oligomerization of a target protein is important for understanding cellular physiology and developing therapeutics. In this study, we aimed to screen cDNA candidates that induce oligomerization of NLRP3, which is one of the important inflammatory sensor proteins, in mammalian cytoplasm. In our screening method, the chimera composed of NLRP3 and the kinase domain of c-kit, one of the receptor tyrosine kinases (RTKs) activated by oligomerization, is expressed in cytoplasm of an IL-3-dependent mammalian cell line. The cells are then transduced with a cDNA library, and cultured in the absence of IL-3. If the transduced cDNA is a NLRP3 activator, the kinase domain of the NLRP3-c-kit chimera is activated by oligomerization, which induces cell growth even in the absence of IL-3. Using this system, constitutive oligomers of two NLRP3 variants were clearly detected by cell growth. Furthermore, cDNA screening resulted in identification of three distinct cDNAs that are potential candidates of NLRP3 activators. These results demonstrate the utility of our chimeric receptor-based system for screening candidates that induce oligomerization of a target protein.
authors: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors: Shingo
authors: S
authors: Honda
authors: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors: Teruyuki
authors: T
authors: Nagamune
authors: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Electronic address: kawahara@bio.t.u-tokyo.ac.jp.
authors: Masahiro
authors: M
authors: Kawahara
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D007377:Interleukin-3; D051379:Mice; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D055503:Protein Multimerization / Q000235:genetics*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D007377:Interleukin-3; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D019009:Proto-Oncogene Proteins c-kit
keywords: Chimeric receptor; Mammalian cell; Protein oligomer; Tyrosine phosphorylation; cDNA library