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

IF 9.4 1区医学 Q1 HEMATOLOGY Experimental Hematology & Oncology Pub Date : 2024-11-04 DOI:10.1186/s40164-024-00572-w

Yang Chen, Mingming Xiao, Yaqi Mo, Jinlu Ma, Yamei Han, Qing Li, Qinghua Zeng, Rebecca J Boohaker, Joshua Fried, Yonghe Li, Han Wang, Bo Xu

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Abstract

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.

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核豪猪在 DNA 损伤反应中介导 XRCC6/Ku70 S-棕榈酰化。

背景：DNA 损伤应答（DDR）的激活在很大程度上依赖于蛋白质的翻译后修饰（PTM），而蛋白质的翻译后修饰在防止遗传不稳定性和肿瘤发生方面起着至关重要的作用。在这些 PTMs 中，棕榈酰化是一个高度保守的过程，在许多癌症类型中都会出现失调。然而，其在 DDR 中的直接参与和潜在机制仍不清楚：方法：利用 CRISPR-Cas9 技术生成 PORCN KO 和 PORCN NLS KO 细胞系。方法：利用 CRISPR-Cas9 技术生成了 PORCN KO 和 PORCN NLS KO 细胞系，并通过菌落形成实验、MTT 实验、DR/EJ5 同源重组/非同源末端连接报告系统、异种移植肿瘤生长和免疫荧光等方法验证了 PORCN NLS 在 DDR 中的作用。通过质谱分析、酰基生物素交换（ABE）棕榈酰化试验、Click-iT 试验、细胞亚细胞分馏试验、Western 印迹分析以及体内和体外共免疫沉淀等方法对其机制进行了探讨：结果：在这项研究中，我们提出了证据，证明Porcupine (PORCN)是DDR不可或缺的组成部分，并在DDR中发挥着关键作用。缺乏 PORCN 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。我们还提供了证据，证明 PORCN 具有具有 S-酰基转移酶活性的核部分（nPORCN），这与其在内质网中与膜结合的 O-酰基转移酶不同。此外，我们还发现 nPORCN 是成功激活 NHEJ 的必要条件。通过质谱分析，我们揭示了 nPORCN 复合物的存在，并表明 nPORCN 在五个特定的半胱氨酸位点上介导了 XRCC6/Ku70 对 IR 的 S-棕榈酰化反应。这些位点的突变会大幅提高辐射敏感性并延迟 NHEJ。此外，我们还提出证据表明，依赖于 nPORCN 的 Ku70 棕榈酰化是 DNA-PKcs/Ku70/Ku80 复合物形成所必需的：我们的发现强调了 nPORCN 依赖性 Ku70 S-棕榈酰化在 DDR 中的关键作用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Experimental Hematology & Oncology Medicine-Oncology

CiteScore

12.60

自引率

7.30%

发文量

审稿时长

6 weeks

期刊介绍： Experimental Hematology & Oncology is an open access journal that encompasses all aspects of hematology and oncology with an emphasis on preclinical, basic, patient-oriented and translational research. The journal acts as an international platform for sharing laboratory findings in these areas and makes a deliberate effort to publish clinical trials with 'negative' results and basic science studies with provocative findings. Experimental Hematology & Oncology publishes original work, hypothesis, commentaries and timely reviews. With open access and rapid turnaround time from submission to publication, the journal strives to be a hub for disseminating new knowledge and discussing controversial topics for both basic scientists and busy clinicians in the closely related fields of hematology and oncology.