Upon DNA damage, numerous proteins are targeted for ubiquitin-dependent proteasomal degradation, which is an integral part of the DNA repair program. Although details of the ubiquitination processes have been intensively studied, little is known about whether and how the 26S proteasome is regulated in the DNA damage response (DDR). Here, we show that human Rpn10/PSMD4, one of the three ubiquitin receptors of the 26S proteasome, is rapidly phosphorylated in response to different types of DNA damage. The phosphorylation occurs at Rpn10-Ser266 within a conserved SQ motif recognized by ATM/ATR/DNA-PK. Blockade of S266 phosphorylation attenuates homologous recombination-mediated DNA repair and sensitizes cells to genotoxic insults. In vitro and in cellulo experiments indicate that phosphorylation of S266, located in the flexible linker between the two ubiquitin-interacting motifs (UIMs) of Rpn10, alters the configuration of UIMs, and actually reduces ubiquitin chain (substrate) binding. As a result, essential DDR proteins such as BRCA1 are spared from premature degradation and allowed sufficient time to engage in DNA repair, a scenario supported by proximity labeling and quantitative proteomic studies. These findings reveal an inherent self-limiting mechanism of the proteasome that, by controlling substrate recognition through Rpn10 phosphorylation, fine-tunes protein degradation for optimal responses under stress.

中文翻译：

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DNA 损伤诱导的蛋白酶体磷酸化控制底物识别并促进 DNA 修复


DNA 损伤后，许多蛋白质被靶向进行泛素依赖性蛋白酶体降解，这是 DNA 修复程序的一个组成部分。尽管泛素化过程的细节已被深入研究，但对于 26S 蛋白酶体是否以及如何在 DNA 损伤反应 (DDR) 中受到调节仍知之甚少。在这里，我们发现人类 Rpn10/PSMD4（26S 蛋白酶体的三个泛素受体之一）会因不同类型的 DNA 损伤而快速磷酸化。磷酸化发生在 ATM/ATR/DNA-PK 识别的保守 SQ 基序内的 Rpn10-Ser266 处。阻断 S266 磷酸化会减弱同源重组介导的 DNA 修复，并使细胞对基因毒性损伤敏感。体外和细胞实验表明，位于 Rpn10 的两个泛素相互作用基序 (UIM) 之间的柔性接头中的 S266 的磷酸化会改变 UIM 的构型，并实际上减少泛素链（底物）结合。因此，BRCA1 等必需的 DDR 蛋白不会过早降解，并有足够的时间进行 DNA 修复，这一情况得到了邻近标记和定量蛋白质组学研究的支持。这些发现揭示了蛋白酶体固有的自我限制机制，通过 Rpn10 磷酸化控制底物识别，微调蛋白质降解以获得应激下的最佳反应。