PARP1 ufmylation modulates replication stress response

PARP1 ufmylation 调节复制应激反应

• 批准号: 391593922
• 负责人: Professor Dr. Zhao-Qi Wang
• 金额: --
• 依托单位: Leibniz-Institut für Alternsforschung - Fritz-Lipmann-Institut e.V. (FLI)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/391593922?language=en
• 关键词: PARP1; ufmylation; modulates; replication; stress

High-fidelity DNA replication ensures the accurate transmitting of parental genetic information to daughter cells. Studying the molecular mechanisms of the cellular response to replication stress has important implications on aging, tissue homeostasis, and pathogenesis, including cancer and neurodegeneration. UFM1 is the most recently identified ubiquitin-like protein and conjugates to target protein(s), a process known as ufmylation, via an E1-like enzyme (UBA5), an E2-like enzyme (UFC1), and an E3 ligase (UFL1). However, the biological function of ufmylation remains largely unknown. We found that defects in UFM1 compromise the DNA replication stress response by delaying the activation of the S-phase checkpoint kinase CHK1. Moreover, PARP1 is found ufmylated, which enhances its enzymatic activity. Furthermore, a defective ufmylation compromises the recruitment of the MRN complex to DNA damage sites. We showed previously that the PARP1 product PAR binds to CHK1 to facilitate its activation in the S-phase checkpoint. Thus, we hypothesize that a PARP1 ufmylation promotes CHK1-mediated replication stress and coordinates a MRN-mediated repair of the stalled forks. This project aims to study (1) whether ufmylation plays an important role in the S-phase checkpoint; (2) its underlying molecular mechanism, and (3) its biological function in vivo. To achieve these goals, my lab at the Fritz Lipmann Institute (FLI), Germany and Prof. Dr. Xingzhi Xu from the Shenzhen University, China decided to combine our effort to carry out biochemical and cellular experiments, as well as genetic studies. Specifically, we will investigate whether the ATR-CHK1 pathway is directly modulated by ufmylation and how PARP1 ufmylation coordinates the response to replication stress with the repair of stalled replication forks. To this end, we will employ human cell lines and mouse models, in which ufmylation is genetically modified. We will perform a biochemical study to map the ufmylation site in PARP1 and test its impact on the CHK1 activation and fork stability. Finally, we will examine a possible interplay of PARylation and ufmylation by generating and using hypoPARylated or enzymatic dead PARP1 cells and mice. Our research will reveal a novel function of UFM1 signaling in handling DNA replication stress and the maintenance of genome stability. Exploring the specific function of ufmylation in the network of the ATR-CHK1 and PARP1/CHK1 pathways may provide scientific knowledge to modulate this novel post-translational modification process as new therapeutic strategies for the treatment of human malignancy and aging-related diseases.

高保真DNA复制确保了亲本遗传信息准确地传递到子细胞。研究细胞对复制应激反应的分子机制对衰老、组织稳态和发病机制（包括癌症和神经变性）具有重要意义。UFM 1是最近鉴定的泛素样蛋白，并通过E1样酶（UBA 5）、E2样酶（UFC 1）和E3连接酶（UFL 1）与靶蛋白缀合，这一过程称为ufmylation。然而，ufmylation的生物学功能在很大程度上仍然未知。我们发现UFM 1的缺陷通过延迟S期检查点激酶CHK 1的激活而损害DNA复制应激反应。此外，发现PARP 1被ufmylated，这增强了其酶活性。此外，有缺陷的ufmylation损害了MRN复合物向DNA损伤位点的募集。我们先前表明，PARP 1产物PAR与CHK 1结合，以促进其在S期检查点的激活。因此，我们假设PARP 1的修饰促进了CHK 1介导的复制应激，并协调了MRN介导的停滞叉的修复。本项目旨在研究（1）ufmylation是否在S期检查点中起重要作用;（2）其潜在的分子机制;（3）其体内生物学功能。为了实现这些目标，我在德国弗里茨·李普曼研究所（FLI）的实验室和中国深圳大学的Xingzhi Xu博士教授决定联合收割机我们一起努力进行生化和细胞实验，以及遗传研究。具体来说，我们将研究ATR-CHK 1通路是否直接通过ufmylation调节，以及PARP 1 ufmylation如何协调对复制应激的反应和对停滞复制叉的修复。为此，我们将采用人细胞系和小鼠模型，其中ufmylation是遗传修饰的。我们将进行一项生化研究，以定位PARP 1中的ufmylation位点，并测试其对CHK 1激活和分叉稳定性的影响。最后，我们将通过产生和使用低PAR化或酶促死亡的PARP 1细胞和小鼠来检查PAR化和ufmylation的可能相互作用。我们的研究将揭示UFM 1信号在处理DNA复制应激和维持基因组稳定性方面的新功能。探索Ufmylation在ATR-CHK 1和PARP 1/CHK 1通路网络中的特定功能，可能为调节这种新型翻译后修饰过程提供科学知识，作为治疗人类恶性肿瘤和衰老相关疾病的新治疗策略。