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A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1.

ABSTRACT: Since checkpoint kinase 1 (Chk1) is an essential factor for cell viability following DNA damage, the inhibition of Chk1 has been a major focus of pharmaceutical development to enhance the sensitivity of tumor cells to chemo- and radiotherapy that damage DNA. However, due to the off-target effects of conventional Chk1-targeting strategies and the toxicity of Chk1 inhibitors, alternative strategies are required to target Chk1. To facilitate such efforts, in this study, we identified a specific Chk1-binding 12-mer peptide from the screening of a phage display library and characterized the peptide in terms of cellular cytotoxicity, and in terms of its effect on Chk1 activity and sensitivity to genotoxic agents. This peptide, named N-terminal Chk1-binding peptide (Chk1?NP), bound the kinase domain of Chk1. Simulation of the binding revealed that the very N-terminus of the Chk1 kinase domain is the potential peptide binding site. Of note, the polyarginine-mediated internalization of Chk1?NP redistributed nuclear Chk1 with a prominent decrease in the nucleus in the absence of DNA damage. Treatment with Chk1?NP peptide alone decreased the viability of p53-defective HeLa cells, but not that of p53-functional NCI-H460 cells under normal conditions. The treatment of HeLa or NCI-H460 cells with the peptide significantly enhanced radiation sensitivity following ionizing radiation (IR) with a greater enhancement observed in HeLa cells. Moreover, the IR-induced destabilization of Chk1 was aggravated by treatment with Chk1?NP. Therefore, the decreased nuclear localization and protein levels of Chk1 seem to be responsible for the enhanced cancer cell killing following combined treatment with IR and Chk1?NP. The approach using the specific Chk1-binding peptide may facilitate the mechanistic understanding and potential modulation of Chk1 activities and may provide a novel rationale for the development of specific Chk1-targeting agents.

SUBMITTER: Kim KS

PROVIDER: S-EPMC5065296 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1.

Kim Kwang Seok KS Choi Kyu Jin KJ Bae Sangwoo S

International journal of molecular medicine 20160930 5

Since checkpoint kinase 1 (Chk1) is an essential factor for cell viability following DNA damage, the inhibition of Chk1 has been a major focus of pharmaceutical development to enhance the sensitivity of tumor cells to chemo- and radiotherapy that damage DNA. However, due to the off-target effects of conventional Chk1-targeting strategies and the toxicity of Chk1 inhibitors, alternative strategies are required to target Chk1. To facilitate such efforts, in this study, we identified a specific Chk ...[more]

PMID: 28025997

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Project description:BackgroundDuring the tumourigenesis and development of colorectal cancer (CRC), the inactivation of tumour suppressor genes is closely involved, although detailed molecular mechanisms remain elusive. Accumulating studies, including ours, have demonstrated that basic leucine zipper transcription factor ATF (activating transcription factor)-like 2 (BATF2) is a capable tumour suppressor that localises in the nucleus. However, its different subcellular localisation, potential functions and underlying mechanisms are unclear.MethodsThe translocation of BATF2 and its clinical relevance were detected using CRC samples, cell lines and xenograft nude mice. Candidate BATF2-binding proteins were screened using co-immunoprecipitation, quantitative label-free liquid chromatography-tandem mass spectrometry proteomic analysis, Western blotting and immunofluorescence. Recombinant plasmids, point mutations and siRNAs were applied to clarify the binding sites between BATF2 and chromosome region maintenance 1 (CRM1).ResultsThe present study found that BATF2 was mainly localised in the cytoplasm, rather than nucleus, of CRC cells in vitro and in vivo, while cytoplasmic BATF2 expression was inversely correlated with the prognosis of CRC patients. Furthermore, we identified the nuclear export and subsequent ubiquitin-mediated degradation of BATF2 in CRC cells. Mechanistically, a functional nuclear export sequence (any amino acid) was characterised in BATF2 protein, through which BATF2 bound to CRM1 and translocated out of nucleus, ultimately enhancing CRC growth via inducing activator protein 1 (AP-1)/cyclin D1/phosphorylated retinoblastoma protein (pRb) signalling pathway. Additionally, nuclear export of BATF2 can be retarded by the mutation of NES in BATF2 or the knockdown of CRM1, whereas CRM1 expression was negatively associated with nuclear BATF2 expression and the prognosis of CRC patients.ConclusionThese findings revealed the biological effects and underlying mechanisms of cytoplasmic localisation of BATF2. Furthermore, suppressing nuclear export of BATF2 via mutating its NES region or inhibiting CRM1 expression may serve as a promising therapeutic strategy against CRC.

Dataset Information

A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1.

Publications

A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1.

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