title: Screening and identification of proteins interacting with IL-24 by the yeast two-hybrid screen, Co-IP, and FRET assays.
abstract: Interleukin-24 (IL-24) is an ideal tumor-suppressor gene, but the mechanisms underlying its antitumor specificity remain to be elucidated. The best way to investigate these problems is to begin from the initiation of corresponding signaling cascades activated by IL-24 with screening and identifying those proteins that interacted with IL-24. With the aim of identifying these initial interactions, a yeast two-hybrid screening was performed by transforming AH109 cells containing PGBKT7-IL-24 with a liver cDNA plasmid library. These cells were then plated on synthetic nutrient medium (SD/-Trp/-Leu/-His) for the first screening and on quadruple dropout medium containing X-α-gal for the second screening. Positive colonies were further verified by repeating the MATE experiments, co-immunoprecipitation (Co-IP) analysis, and fluorescence resonance energy transfer (FRET) assays in vitro. Following the yeast two-hybrid screening, 15 genes were selected for sequencing, with two genes, HLA-C and NDUFA13, further verified using Co-IP assays and FRET assays. Both HLA-C and NDUFA13 were found to interact with IL-24. We found that HLA-C and NDUFA13 could interact with IL-24 and it may be involved in the signal induced by IL-24. Overall, this study contributes further insight into the cancer-specific apoptosis-inducing abilities of IL-24 to potentially enhance its therapeutic potential, and it also provides outlets for other biological functions of IL-24.
authors.affiliation: aInstitute of Aging Research, Hangzhou Normal University School of Medicine, HangzhoubInstitute of Microbiology, Jiangxi Academy of Sciences, JiangxicCollege of Chemical Engineering and Technology, Wuhan University of Science and Technology, Wuhan, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Hu
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Jiaojiao
authors.initials: J
authors.lastname: Chen
authors.forename: Fang
authors.initials: F
authors.lastname: Yu
authors.forename: Huilin
authors.initials: H
authors.lastname: Liu
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Zuo
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Yang
authors.forename: Handong
authors.initials: H
authors.lastname: Fan
mesh_terms: D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D015723:Gene Library; D006056:Golgi Apparatus / Q000378:metabolism; D015236:HLA-C Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007378:Interleukins / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D020798:Two-Hybrid System Techniques; D000099180:Interleukin-24
chemical_list: D051017:Apoptosis Regulatory Proteins; D018076:DNA, Complementary; D015236:HLA-C Antigens; D007378:Interleukins; D009247:NADH, NADPH Oxidoreductases; D000099180:Interleukin-24; C439057:NDUFA13 protein, human