a Department of Cancer Biology , Lerner Research Institute, Cleveland Clinic , Cleveland , OH , USA.
c Department of Chemistry , Cleveland State University , Cleveland , OH , USA.
Autophagy. 2018;14(11):1976-1990. doi: 10.1080/15548627.2018.1496877. Epub 2018 Aug 10.
Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair.
ATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.
最近的报告揭示了一个重要发现，即自噬/细胞自噬可直接调控 DNA 损伤反应（DDR）相关蛋白和染色质泛素化（Ubn）。在这里，我们报告了自噬和 DDR 之间以前未被探索的联系，这种联系通过去泛素化酶（DUB）USP14 介导。前列腺癌细胞中自噬的缺失导致未修复的 DNA 双链断裂（DSBs），这表现为持续的电离辐射（IR）诱导焦点（IRIF）形成γH2AFX，以及 RNF168 的蛋白水平和 IRIF 形成减少，RNF168 是一种 E3 泛素连接酶，对于染色质 Ubn 以及响应 DSBs 招募关键 DDR 效应蛋白至关重要，包括 TP53BP1。一致地，自噬缺陷细胞中 RNF168 相关的 Ubn 信号和 TP53BP1 IRIF 形成减少。活性测定鉴定了几种 DUB，包括 USP14，其在自噬缺陷细胞中的活性更高。重要的是，抑制 USP14 可以克服自噬缺陷细胞中的 DDR 缺陷。自噬缺陷细胞中 USP14 的 IRIF 形成和蛋白稳定性增加。USP14 与 MAP1LC3B 和 SQSTM1 的 UBA 结构域的免疫沉淀和共定位鉴定 USP14 是自噬的底物和 SQSTM1。此外，USP14 与 RNF168 直接相互作用，这取决于 RNF168 的 MIU1 结构域。这些发现确定 USP14 是自噬的新底物，USP14 调节 RNF168 依赖性 Ubn 和 TP53BP1 募集是 DDR 和自噬之间的关键联系。鉴于 Ubn 信号在非同源末端连接（NHEJ）中的作用，NHEJ 是修复电离辐射诱导的 DNA 损伤的主要途径，这些发现为自噬、DDR 相关的 Ubn 信号和 NHEJ DNA 修复之间的联系提供了独特的见解。
