title: Tumor suppressor RYBP harbors three nuclear localization signals and its cytoplasm-located mutant exerts more potent anti-cancer activities than corresponding wild type.
abstract: Ectopically expressed Ring1 and YY1 binding protein (RYBP) induces tumor cell apoptosis through promoting the formation of the death-inducing signaling complex (DISC) in the cytoplasm. However, transiently overexpressed as well as endogenous RYBP in tumor tissues were observed to be mainly located in the nucleus while that in adjacent non-tumor tissues distributed majorly in the cytoplasm. Currently, we do not know the nuclear localization signals and biological function of different subcellular location of RYBP. In this study, we employed bioinformatic analysis, deletion, point mutation, enhanced green fluorescence protein (EGFP) fusion and others, to investigate the elements responsible for RYBP nuclear import and to explore the anti-tumor activities of cytoplasm- and nuclear-located RYBP. Herein, we identified three functional monopartite nuclear localization signals (NLSs), all of which located at the N-terminus of RYBP. Through four basic amino acid replacements within the NLSs, we obtained a cytoplasm-located RYBP mutant (RYBPmut). Compared with wild-type counterpart, RYBPmut exhibited more potent abilities to bind to caspase 8, to prevent MDM2-mediated polyubiquitination and degradation of p53, thereby leading to its stabilization. Further investigation revealed that, in contrast to its wild type, RYBPmut showed more potentials to inhibit tumor cell proliferation and to induce apoptosis, in both p53-dependent and -independent manner. Collectively, our current study revealed the molecular mechanism responsible for RYBP nuclear translocation, and provided evidences to support that RYBPmut could be a more promising candidate agent for cancer treatment.
authors.affiliation: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.
authors.forename: Kuan
authors.initials: K
authors.lastname: Tan
authors.affiliation: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Cong
authors.affiliation: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.
authors.forename: Bingren
authors.initials: B
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China. Electronic address: hchen_222@163.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China. Electronic address: chendeng2001@hotmail.com.
authors.forename: Deng
authors.initials: D
authors.lastname: Chen
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D019913:Nuclear Localization Signals / Q000378:metabolism*; D055550:Protein Stability; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D047908:Intracellular Signaling Peptides and Proteins; D019913:Nuclear Localization Signals; C434957:RYBP protein, human; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D053181:Caspase 8
keywords: Apoptosis; MDM2; Nuclear localization signal; RYBP; Ubiquitination; p53