E3泛素连接酶家族Rbx1和Sag/Rbx2在调节性T细胞中的作用及机制研究

Rbx1 (RING box protein-1) and Rbx2, also known as Sag (RING box protein-2 or Sensitive to Apoptosis Gene) family members are the activity-required RING components of CRLs (Cullin-RING E3 ligases) which promote ubiquitination of approximately 20% of celluar proteins for proteasome degradation. While Rbx1 is constitutively expressed, and required for proliferation; Sag/Rbx2 is stress inducible as a cellular defensive mechanism. Our previous studies using total knockout mouse models revealed that two family members are not functional redundant; While Rbx1 KO caused embryonic death at E6.5 stage due to proliferation failure, Sag KO also caused embryonic death but at E10.5 stage mainly due to defect in angiogenesis. We and others have previously showed potential involvement of Sag in regulation cellular immune functions acting at dendritic cells and macrophage cells, nothing is known as to the role of Rbx1. Furthermore, it is totally unknown whether and how these two family members regulate Treg cells, the immunosuppressive cells, which play the key role in proper immune regulation. The goal of this proposed study is to generate conditional KO mouse models (FoxP3-Cre; Rbx1fl/fl or FoxP3-Cre; Sagfl/fl) to study the role of Rbx1 and Sag/Rbx2 in immune response upon their selective KO in Treg cells, respectively, under physiological or stressed conditions. Successful execution of this proposed study would reveal, for the first time, how CRL E3 ligases regulate Treg cells and its functional significance, which may have implication in the treatment of autoimmune and infectious diseases, given a potent inhibitor of CRLs is currently in Phase I/II clinical trials for anticancer application.

E3泛素连接酶家族Cullin-RING ligases（CRLs）调控细胞内约20%蛋白质泛素化修饰和降解。Rbx1和Sag/Rbx2是CRLs的催化亚基。Rbx1组成型表达，对细胞增殖非常重要；Sag参与压力应激反应，是压力诱导表达的。我们已证实小鼠全身敲除Rbx1或Sag都会导致胚胎期死亡，且死因与存活天数不同，表明Rbx1和Sag不是功能冗余的。在免疫调控中，我们和其他课题组已经发现Sag调控多种免疫细胞，但对Rbx1的研究尚未启动。调节性T细胞（Treg 细胞）是机体免疫调控枢纽，Rbx1和Sag在Treg细胞中的作用尚无报道。本项目中，我们将特异性敲除小鼠体内Treg细胞中的Rbx1或Sag，并研究二者于静息和压力条件下对Treg细胞的作用。研究将首次阐明CRLs家族在Treg细胞中功能。CRLs抑制剂作为抗肿瘤药物已进入临床试验，本项目对自身免疫、感染等疾病治疗具有指导意义。

Cullin-RING Ligase（CRLs）连接酶家族，在许多生物过程中发挥关键调控作用。然而，CRL-neddylation如何调节Treg细胞的功能仍不清楚。Rbx1是CRLs活性所需的一个组分。我们发现，Treg特异性缺失Rbx1的小鼠，在出生后出现早发性炎症性疾病，在出生后第25天左右死亡，Treg细胞的抑制功能受损。Rbx1对于维持Treg细胞中的效应亚群至关重要，并调节多种炎症细胞信号通路。Treg细胞中Ube2m缺失的小鼠，也出现类似的炎症表型，但严重程度要小得多。Rbx1缺陷或Ube2m缺陷的Treg细胞的免疫抑制功能分别出现严重或中度的损伤。有趣的是，CRL-neddylation家族成员Rbx2/Sag或Ube2f在Treg细胞中特异性缺失，并没有引发明显表型。因此，Ube2m-Rbx1轴是维持Treg细胞内稳态和功能所必需的；同时，在Treg细胞中，Rbx1具有不依赖于Ube2m的作用，这表明了CRL-neddylation调控的复杂性。