A20促进Nrdp1聚泛素化致MyD88降解抑制炎症反应的新机制

A20 can inhibit the inflammation response by regulating the RING family E3 ubiquitin ligase through its ubiquitination or deubiquitin effect. Research showed that A20 could inhibit the RING family of E3 ubiquitin ligase TRAF6 ubiquitination, thereby reducing inflammatory reaction. However, the mechanism by which A20 negatively regulates inflammation is still not fully understood. The underlying mechanism by which A20 inhibits inflammation is still not fully understood. Research showed that Nrdp1 ubiquitinated and degradated protein MyD88 and then reduce the inflammatory response. Nrdp1 and TRAF6 belong to the RING family E3 ubiquitin ligase, which suggests that A20 may interacts with Nrdp1 to promote its ubiquitination and negative regulate the inflammatory reaction. Therefore, our previous bioinformatic predictions suggest that A20 interacted with Nrdp1, and our preliminary experiment indicated that A20 and Nrdp1 interacted with each other, which strongly suggests that A20 may promote the Nrdp1 ubiquitination, degradation of MyD88, and reduce inflammation.This project intends to further clarify the interaction between A20 and Nrdp1, and analyze the exact site of the interaction between the two, so as to clarify the exact mechanism of the interaction.This project not only can discover the new anti-inflammatory mechanism of A20, but also provide a new strategy for the treatment of inflammatory related diseases.

A20可通过泛素剪切或泛素连接作用调节RING家族E3泛素连接酶进而负性调控炎症反应。研究显示A20与RING家族E3泛素连接酶TRAF6相互作用抑制其聚泛素化，从而减轻炎症反应。但A20负性调控炎症反应的机制仍不完全清楚。研究显示Nrdp1聚泛素化后可降解MyD88进而降低炎症反应。鉴于Nrdp1与TRAF6同属于RING家族E3泛素连接酶，推测A20与Nrdp1相互作用促进其聚泛素化进而负性调控炎症反应。为此，我们前期生物信息学预测提示A20与Nrdp1存在相互作用，进一步免疫共沉淀检测发现A20与Nrdp1确有相互作用，强烈提示A20可能通过促进Nrdp1聚泛素化，降解MyD88，继而减轻炎症反应。本项目拟在进一步明确A20与Nrdp1相互作用基础上，解析二者相互作用的精确位点，从而阐明其相互作用的精确调控机制。该项目不仅能发现A20抗炎新机理，还可望为炎性相关疾病的治疗提供新策略。

A20是机体重要的内源性抗炎因子，主要通过其泛素编辑作用抑制炎症反应，从而维持机体内环境稳态。 既往，主要是通过鉴定单个靶点或沿着连接泛素编辑活性和非酶活性的生化途径的小亚群来研究A20的抗炎机制。 然而，A20的底物分布仍不甚清楚，许多参与A20抑制炎症反应的A20结合蛋白不明确。 本研究中，为了进一步阐明A20抗炎的分子机制，我们首先构建了脂多糖(LPS)刺激RAW264.7细胞的炎症模型，采用免疫共沉淀(co-IP)与质谱分析技术联合来筛选与A20相互作用的蛋白质。结果表明，与A20相互作用的蛋白主要富集于泛素连接酶活性和锌指结构域。在这些互作蛋白质中，Nrdp1的表达量高，且其与A20的相互作用既往没有报道过。因此，进一步co-IP实验证实，在LPS刺激过程中，RAW264.7细胞中A20与Nrdp1具有相互作用。 我们进一步明确了A20调控Nrdp1聚泛素化的机制。研究发现，A20促进了Nrdp1与MyD88的相互作用，导致MyD88的降解，从而抑制NF-κB的激活，进而抑制炎症反应。研究还表明，A20抑制了Nrdp1介导的TBK1激活，从而减少IFN-β的分泌。我们进一步通过构建A20锌指结构的突变体，采用基因转染技术，明确了A20调控Nrdp1聚泛素化需要其锌指结构4(ZnF4)。 我们的研究表明，Nrdp1是一种新的A20结合蛋白，参与了A20介导的抗炎活性，从而揭示了A20抑制炎症反应和IFNβ分泌的新机制。