Rbx1 Cullin-Ring ligase E3 and Ube2m neddylation E2 differentially governs the fitness of Treg cells

Abstract

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 CRL5.1-4 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.5-6 Regulatory T cells (Treg cells) are specialized immunosuppressive CD4+ T lymphocytes, that play pivotal roles in maintaining immune homeostasis in vivo.7-10 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, TNF, NF{kappa}B, 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 pevonedistat11, 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.