Nuclear porcupine mediates XRCC6/Ku70 S-palmitoylation in the DNA damage response.

Autor: Chen Y; Department of Biochemistry and Molecular Biology, The Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Ministry of Education, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China.; Department of Radiation Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, 300060, China., Xiao M; Department of Biochemistry and Molecular Biology, The Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Ministry of Education, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China.; Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center, Shanghai, 200032, China., Mo Y; Chongqing Key Laboratory of Intelligent Oncology for Breast Cancer, Chongqing University Cancer Hospital and Chongqing University School of Medicine, Chongqing, 400030, China., Ma J; Department of Radiation Oncology, the First Affiliated Hospital, Xi'an Jiaotong University, Xi'an, 710061, China., Han Y; Department of Biochemistry and Molecular Biology, The Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Ministry of Education, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China., Li Q; Chongqing Key Laboratory of Intelligent Oncology for Breast Cancer, Chongqing University Cancer Hospital and Chongqing University School of Medicine, Chongqing, 400030, China., Zeng Q; Department of Oncology, Southern Research Institute, Birmingham, AL, 35205, USA.; Cell Biology Program, University of Alabama at Birmingham, Birmingham, AL, 35205, USA., Boohaker RJ; Department of Oncology, Southern Research Institute, Birmingham, AL, 35205, USA.; Cell Biology Program, University of Alabama at Birmingham, Birmingham, AL, 35205, USA., Fried J; Department of Oncology, Southern Research Institute, Birmingham, AL, 35205, USA.; Cell Biology Program, University of Alabama at Birmingham, Birmingham, AL, 35205, USA., Li Y; Department of Oncology, Southern Research Institute, Birmingham, AL, 35205, USA.; Cell Biology Program, University of Alabama at Birmingham, Birmingham, AL, 35205, USA., Wang H; Chongqing Key Laboratory of Intelligent Oncology for Breast Cancer, Chongqing University Cancer Hospital and Chongqing University School of Medicine, Chongqing, 400030, China., Xu B; Department of Biochemistry and Molecular Biology, The Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Ministry of Education, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China. xubo@tmu.edu.cn.; Chongqing Key Laboratory of Intelligent Oncology for Breast Cancer, Chongqing University Cancer Hospital and Chongqing University School of Medicine, Chongqing, 400030, China. xubo@tmu.edu.cn.
Jazyk: angličtina
Zdroj: Experimental hematology & oncology [Exp Hematol Oncol] 2024 Nov 04; Vol. 13 (1), pp. 109. Date of Electronic Publication: 2024 Nov 04.
DOI: 10.1186/s40164-024-00572-w
Abstrakt: Background: The activation of the DNA damage response (DDR) heavily relies on post-translational modifications (PTMs) of proteins, which play a crucial role in the prevention of genetic instability and tumorigenesis. Among these PTMs, palmitoylation is a highly conserved process that is dysregulated in numerous cancer types. However, its direct involvement in the DDR and the underlying mechanisms remain unclear.
Methods: CRISPR-Cas9 technology was used to generate the PORCN KO and PORCN NLS KO cell lines. The effects of PORCN NLS in the DDR were verified by colony formation assays, MTT assays, the DR/EJ5 homologous recombination/non-homologous end-joining reporter system, xenograft tumor growth and immunofluorescence. Mechanisms were explored by mass spectrometry, acyl-biotin exchange (ABE) palmitoylation assay, Click-iT assay, cell subcellular fractionation assay, Western blot analysis, and in vivo and in vitro co-immunoprecipitation.
Results: In this study, we introduce evidence that Porcupine (PORCN) is an integral component of and plays a critical role in the DDR. PORCN deficiency hampers nonhomologous end joining (NHEJ) and highly sensitizes cells to ionizing radiation (IR) both in vitro and in vivo. We also provide evidence that PORCN possesses a nuclear fraction (nPORCN) with S-acyltransferase activity, unlike its membrane-bound O-acyltransferase in the endoplasmic reticulum. Furthermore, we show that nPORCN is necessary for the successful activation of NHEJ. Using mass spectrometry, we reveal the existence of an nPORCN complex and show that nPORCN mediates the S-palmitoylation of XRCC6/Ku70 at five specific cysteine sites in response to IR. Mutation of these sites causes a substantial increase in radiosensitivity and delays NHEJ. Additionally, we present evidence that nPORCN-dependent Ku70 palmitoylation is required for DNA-PKcs/Ku70/Ku80 complex formation.
Conclusion: Our findings underscore the crucial role of nPORCN-dependent Ku70 S-palmitoylation in the DDR.
(© 2024. The Author(s).)
Databáze: MEDLINE
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