类泛素化修饰Neddylation在DNA损伤应答中的调控作用及分子机制

DNA damage induced by numerous environmental and internal hazards triggers genomic instability, and ultimately promotes tumorigenesis. To cope with these damages, cells have developed a DNA damage response （DDR）system to sense and repair DNA lesions. Various protein modifications play important roles during DNA damage response. Both Ubiquitin and members of the SUMO family have been widely studied and shown to participate in DNA damage response. However, the physiological functions of Neddylation in DNA damage remains unclear. The neddylation conjugation pathway has a pivotal role in regulation of numerous biological processes. But very limited Neddylation substrates in DNA damage response have been identified so far, and further investigations are necessary. Our previous work has found that H2A and PCNA that invovled in DNA damage response could be modified by NEDD8. In this study, we will carry out a series of experiments to investigate the regulatory functions of Neddylation in DNA damage response and elucidate the underlying mechanism. We will identify novel substrates that could be covalently conjugated by NEDD8 during DNA damage response; we will identify the E3 ligases of these NEDD8 sustrates, and elucidate the underlying molecular mechanism of Neddylation of these DDR proteins. We will further investigate the effect of Neddylation on the recruitment of DNA damage repair proteins and clarify the physiological roles of protein neddylation in DNA damage response. As ubiquitination plays important role in the DNA damage response process, the overall structure of NEDD8 is very similar to ubiquitin and these two types of modification occur on the same lysine residue in some proteins, we will also study the relationship between protein ubiquitination and neddylation, and assess the spatio-temporal variability of these two modifications during DNA damage response. These studies will help us to further understand the molecular mecahnisms of diseases such as tumorigenesis, and provide insight for drug design in the future.

DNA损伤会造成基因组的不稳定，导致肿瘤等恶性疾病的发生。蛋白质翻译后修饰在DNA损伤应答调节中发挥重要作用，目前研究较多的是泛素化和SUMO化修饰，但有关类泛素化修饰Neddylation的报道非常少，在DNA损伤应答中已鉴定的Neddylation底物非常有限。我们的前期工作发现DNA损伤应答蛋白H2A和PCNA能够发生Neddylation。本课题将围绕Neddylation在DNA损伤应答中的调控作用及分子机制这个核心科学问题开展研究：鉴定DNA损伤应答中新的Neddylation底物蛋白，寻找并鉴定其E3连接酶，揭示其发生Neddylation的分子机制；研究Neddylation与泛素化修饰在DNA损伤应答中的相互作用关系；阐明Neddylation在DNA损伤应答中的调控作用。该研究将有助于深入理解肿瘤等疾病发生的分子机理，为寻找新的治疗靶点提供理论基础。

NEDD化修饰在很多生理过程中发挥重要作用。但是已鉴定的NEDD化修饰的底物较少，尤其在DNA损伤应答中NEDD化修饰的生理功能报道很少。本课题鉴定了在DDR中能够发生NEDD化修饰的新底物，并对其在DDR中作用的分子机制开展了系统的研究。首次鉴定了PCNA能够发生NEDD化修饰。证明NEDP1是PCNA的去NEDD化酶， RAD18是PCNA NEDD化修饰的E3连接酶。在双氧水引起的氧化损伤修复过程中，NEDP1从染色质上解离，与PCNA的相互作用减弱， PCNA的NEDD化修饰增强，从而抑制泛素化PCNA的过度积累和跨损伤修复中polη在DNA损伤位点的聚集，防止保真性较低的DNA跨损伤合成途径过度进行。其次，证明E3连接酶RAD18也是NEDD化修饰的底物。在应答双氧水等刺激时，RAD18的NEDD化修饰水平显著增加，而其泛素化水平降低。NEDD8的过表达以及去NEDD化酶NEDP1的缺失会进一步抑制RAD18的泛素化修饰。 利用NEDD化修饰E1的抑制剂MLN4924处理细胞会减弱PCNA与其E3连接酶RAD18的相互作用，并进一步阻止RAD18在损伤位点形成foci，进而阻止聚合酶polη在损伤位点的招募，最终抑制跨损伤修复的效率。我们还证实MyD88能够发生NEDD化修饰。MyD88的NEDD化通过拮抗其泛素化抑制NF-κB通路，揭示了固有免疫中一个关键接头分子的活性的精细调控机制。此外，我们研究了HSV-1病毒UL36USP蛋白调控PCNA泛素化的作用、分子机制以及生理效应，为揭示病毒如何利用宿主细胞DNA损伤修复蛋白促进自身扩增提供有用信息。明确了A20通过调节组蛋白H2A的泛素化参与DNA损伤修复反应的分子机制，并探讨了其对基因组稳定性和肿瘤细胞对于DNA损伤治疗敏感性的影响。上述结果有助于我们深入了解NEDD化等翻译后修饰的生理作用,明确不同修饰发挥作用的时空关系及调控DNA损伤应答的分子机制，具有重要的生物学意义。