Cullin-RING Ligases (CRLs) are a family of multi-unit E3 ubiquitin ligases with two members of RING family proteins, acting as the catalytic subunit: RING-box 1 (Rbx1) couples with CRLs1-4, whereas RING-box 2 (Rbx2/Sag) couples mainly with CRL5. Activity of CRLs requires neddylation on their Cullin subunit, catalyzed by neddylation enzyme cascades E1, E2 and E3. Ube2m and Ube2f are two neddylation E2s responsible for neddylation of Cullins in CRLs1-4 or CRL5, respectively. Regulatory T cells (Treg cells) are specialized immunosuppressive CD4+ T lymphocytes, that play pivotal roles in maintaining immune homeostasis in vivo. Whether and how Rbx1-Rbx2/CRLs and Ube2m-Ube2f/neddylation regulate Treg cell homeostasis and function are currently unknown. Here we show that while mice with a Treg-specific deletion of Rbx2/Sag showed no obvious phenotype, mice with Rbx1 deletion in Treg cells developed an early-onset fetal inflammatory disorders and death at day ~25 after birth (~p25), with disrupted homeostasis and functions of Treg cells, indicating Rbx1 as a prominent regulator of Treg cells. Single cell transcriptome assay showed that Rbx1 is essential for the maintenance of the effector subpopulations in Treg cells. The whole genome transcriptome and proteomics analysis revealed that Rbx1 regulates several inflammatory pathways, such as T-cell receptor, IL-17, TNFα, NFκB, chemokine, cytokine-cytokine receptor interaction, as well as energy and purine metabolisms. Accumulation of Acly, Fto and Nfkbib proteins, upon Rbx1 depletion suggests that these are likely the novel substrates of CRLs1-4 in Treg cells. Consistently, while Ube2f deletion showed no obvious phenotype, mice with Ube2m deletion in Treg cells also suffers from inflammatory disorders, but to a much lesser severity with a 50% of death rate at ~150 days of age. Since Rbx1 is a dual E3 as a component of CRLs1-4 ligase and as a neddylation co-E3, downstream of Ube2m E2 for neddylation activation of CRLs1-45, much severe phenotypes in Foxp3cre;Rbx1fl/fl mice suggests Rbx1 may have additional function independent of neddylation activation in Treg cells. Indeed, unbiased transcriptome comparison between Rbx1-deficient and Ube2m-deficient Treg cells, revealed that the former had greater as well as unique alteration in the signaling pathways controlling the inflammatory responses. Collectively, our study shows that the Ube2m-Rbx1 axis of the neddylation-CRL is required for the maintenance of homeostasis and functional fitness of Treg cells in the fine control of immune tolerance; with implication that targeting the neddylation/CRLs, such as a small molecule inhibitor pevonedistat, currently in the Phase II clinic trial for anticancer therapy, may have novel application in the treatment of human diseases associated with overactivated Treg cells.

中文翻译：

---

Rbx1 Cullin-Ring连接酶E3和Ube2m neddylation E2差异性地控制Treg细胞的适应性

Cullin-RING Ligases（CRLs）是一个多单元E3泛素连接酶家族，具有两个RING家族蛋白成员，起催化亚基的作用：RING-box 1（Rbx1）与CRLs1-4偶联，而RING-box 2（ Rbx2 / Sag）主要与CRL5偶联。CRLs的活性需要在其Cullin亚基上进行N-化，并由N-化酶级联E1，E2和E3催化。Ube2m和Ube2f是两个分别负责CRLs1-4或CRL5中的Cullins的糊化的糊化E2。调节性T细胞（Treg细胞）是专门的免疫抑制CD4 + T淋巴细胞，在体内维持免疫稳态方面起着关键作用。Rbx1-Rbx2 / CRLs和Ube2m-Ube2f / neddylation是否调节Treg细胞稳态和功能目前尚不清楚。在这里，我们显示，尽管具有Treg特异性Rbx2 / Sag缺失的小鼠没有明显的表型，Treg细胞中具有Rbx1缺失的小鼠在出生后约25天（〜p25）出现早发性胎儿炎症，并死​​亡，其稳态和Treg细胞功能受损，表明Rbx1是Treg细胞的重要调节剂。单细胞转录组分析表明，Rbx1对于维持Treg细胞中的效应子亚群至关重要。全基因组转录组和蛋白质组学分析表明，Rbx1调节多种炎症途径，例如T细胞受体，IL-17，TNFα，NFκB，趋化因子，细胞因子与细胞因子受体的相互作用以及能量和嘌呤的代谢。Rbx1耗尽后，Acly，Fto和Nfkbib蛋白的积累表明，这些可能是Treg细胞中CRLs1-4的新型底物。一致地，虽然Ube2f缺失未显示明显的表型，Treg细胞中Ube2m缺失的小鼠也患有炎症性疾病，但严重程度要低得多，在〜150天龄时死亡率达到50％。由于Rbx1是双重E3作为CRLs1-4连接酶的一个成分和作为一个neddylation co-E3，在Ube2m E2的下游用于CRLs1-45的neddylation活化，因此Foxp3cre中存在许多严重的表型； Rbx1fl / fl小鼠提示Rbx1可能具有其他功能不依赖于Treg细胞中的neddylation激活。确实，Rbx1缺陷型和Ube2m缺陷型Treg细胞之间无偏见的转录组比较显示，前者在控制炎症反应的信号传导途径中具有更大以及独特的改变。总的来说，我们的研究表明，neddylation-CRL的Ube2m-Rbx1轴对于维持Treg细胞的动态平衡和功能适应性，以良好地控制免疫耐受性是必不可少的。这暗示着目前针对抗癌治疗的II期临床试验中靶向小分子抑制剂（如小分子抑制剂Pevonedistat）的融合作用/ CRL可能在治疗与Treg细胞过度活化有关的人类疾病方面具有新颖的应用。