A novel role for the peptidyl-prolyl cis-trans isomerase Cyclophilin A in DNA-repair following replication fork stalling via the MRE11-RAD50-NBS1 complex-猫眼课题宝

DOI：

10.1101/2023.06.27.546694

发表时间：

2023-06

期刊：

bioRxiv

影响因子：

--

通讯作者：

中科院分区：

其他

文献类型：

--

作者：

Marisa Bedir;E. Outwin;R. Colnaghi;Lydia Bassett;I. Abramowicz;M. O’Driscoll

我们之前曾报道，非同源末端连接(NHEJ)缺陷的人LIG4-/-前B淋巴细胞对环孢素A(CsA)的杀伤出人意料地敏感，环孢素A是骨髓移植条件和维持方案的常见成分。我们还发现CsA诱导LIG4综合征患者成纤维细胞DNA双链断裂(DSB)，特别是在通过S时相。到目前为止，这些CsA影响的分子基础还没有被描述。我们推测，CsA诱导的基因组不稳定可能反映了亲环素A(Cyclophin A，CyPA)在DNA修复中的直接作用，因为CyPA是CsA的主要生理靶相互作用因子。CyPA是亲环素家族多肽-脯氨酰顺反异构酶(PPI)的创始成员。CsA通过占据CyPA的酶活性部位来抑制其PPI活性。运用CRISPR/Cas9工程、siRNA、BioID、免疫共沉淀、通路特异性DNA修复研究以及蛋白质表达-相互作用分析等方法，我们描述了CyPA缺失及其PPI活性抑制对DNA修复的新影响。在我们的CyPA-BioID邻近相互作用组的研究结果的推动下，我们验证了CyPA与DNA末端切除机制的不同组件的相互作用。此外，我们还描述了CyPA与Mre11-Rad50-NBS1(MRN)复合体的NBS1成分之间的一种新的直接相互作用，这为CyPA的PPI功能在DNA末端切除水平上通过直接的蛋白质-蛋白质相互作用影响DNA修复提供了证据。因此，我们证明，在DNA复制叉停滞后，CyPA的丢失或抑制会损害同源重组修复(HRR)。此外，我们定义了一组与CyPA丢失和抑制相关的遗传脆弱性，确定DNA复制分叉保护是这里生存能力的一个重要决定因素。利用我们已经发现的对CyPA生物学的新见解；我们探索了如何利用CyPA PPI抑制来选择性地杀死具有共同特征的基因组不稳定特征的各种不同癌症的细胞的例子。这些发现为亲环素抑制剂的非免疫抑制CsA类似物提供了一种潜在的新的疾病应用或再利用策略。

We previously reported that non-homologous end-joining (NHEJ)-defective human LIG4-/- pre-B lymphocytes were unexpectedly sensitive to killing by the cyclic peptide Cyclosporin A (CsA), a common component of bone marrow transplantation conditioning and maintenance regimes. We also found that CsA induced DNA double strand breaks (DSBs) in LIG4 syndrome patient fibroblasts, specifically upon transit through S-phase. The molecular basis underlying these CsA impacts has not been described hitherto. We postulated that CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair, as CYPA is the primary physiological target interactor of CsA. CYPA is the founding member of the Cyclophilin family of peptidyl-prolyl cis-trans isomerases (PPIs). CsA inhibits the PPI activity of CYPA through occupation of the latter’s enzymatic active site. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression-interaction analysis, we describe novel impacts of CYPA loss and inhibition of its PPI activity on DNA repair. Prompted by findings from our CYPA-BioID proximity interactome, we validate CYPA interactions with different components of the DNA end resection machinery. Moreover, we characterise a novel and direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 (MRN) complex, providing evidence that the PPI function of CYPA actively influences DNA repair via direct protein-protein interaction at the level of DNA end resection. Consequently, we demonstrate that CYPA loss or inhibition impairs Homologous Recombination Repair (HRR) following DNA replication fork stalling. Additionally, we define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability herein. Leveraging the novel insights into CYPA biology we have uncovered; we explore examples of how CYPA PPI inhibition may be exploited to selectively kill cells from a variety of different cancers with a shared characteristic genomic instability profile. These findings propose a potential new disease application or repurposing strategy for the non-immunosuppressive CsA analogue class of Cyclophilin inhibitors.