Chen Yang, Xiao Mingming, Mo Yaqi, Ma Jinlu, Han Yamei, Li Qing, Zeng Qinghua, Boohaker Rebecca J, Fried Joshua, Li Yonghe, Wang Han, Xu Bo
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.
Exp Hematol Oncol. 2024 Nov 4;13(1):109. doi: 10.1186/s40164-024-00572-w.
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.
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.
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.
Our findings underscore the crucial role of nPORCN-dependent Ku70 S-palmitoylation in the DDR.
DNA损伤反应（DDR）的激活严重依赖于蛋白质的翻译后修饰（PTM），这些修饰在预防基因不稳定和肿瘤发生中起着关键作用。在这些PTM中，棕榈酰化是一个高度保守的过程，在多种癌症类型中失调。然而，其在DDR中的直接参与及潜在机制仍不清楚。
使用CRISPR-Cas9技术生成PORCN基因敲除（KO）和PORCN核定位信号（NLS）基因敲除细胞系。通过集落形成试验、MTT试验、DR/EJ5同源重组/非同源末端连接报告系统、异种移植瘤生长和免疫荧光验证PORCN NLS在DDR中的作用。通过质谱、酰基生物素交换（ABE）棕榈酰化试验、Click-iT试验、细胞亚细胞分级试验、蛋白质印迹分析以及体内和体外共免疫沉淀探索机制。
在本研究中，我们提供证据表明猬因子（PORCN）是DDR的一个组成部分并在其中起关键作用。PORCN缺陷会阻碍非同源末端连接（NHEJ），并在体外和体内使细胞对电离辐射（IR）高度敏感。我们还提供证据表明，与内质网中其膜结合的O-酰基转移酶不同，PORCN具有具有S-酰基转移酶活性的核部分（nPORCN）。此外，我们表明nPORCN是成功激活NHEJ所必需的。通过质谱，我们揭示了nPORCN复合物的存在，并表明nPORCN在响应IR时介导XRCC6/Ku70在五个特定半胱氨酸位点的S-棕榈酰化。这些位点的突变导致放射敏感性大幅增加并延迟NHEJ。此外，我们提供证据表明DNA-PKcs/Ku70/Ku80复合物形成需要nPORCN依赖性的Ku70棕榈酰化。
我们的研究结果强调了nPORCN依赖性的Ku70 S-棕榈酰化在DDR中的关键作用。
