Ring finger protein 31–mediated atypical ubiquitination stabilizes forkhead box P3 and thereby stimulates regulatory T-cell function

Abstract

The CD4 ؉ CD25 ؉ FOXP3 ؉ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-␥, resulting in a Th1 helper cell-like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.

FoxP3 in Treg cell biology: a molecular and structural perspective

“…This interaction positively regulates both FoxP3 stability and T reg cell function. Moreover, RNF31 expression is correlated with intratumoral T reg cells in gastric cancer, implying a potential role of RNF31 in mediating tumor immunity .…”

Mouse Double Minute 2 Homolog-Mediated Ubiquitination Facilitates Forkhead Box P3 Stability and Positively Modulates Human Regulatory T Cell Function

“…To determine whether the ubiquitination modification of FOXP3 is mediated by MDM2 directly, we performed in vitro ubiquitination assay by using recombinant GST-MDM2 (purchased from R&D systems), 6×his-E1, 6×his-UbcH5b (E2), 6×his-Flag-ubiquitin, and StrepII-FOXP3a, which were purified previously (17,20). The result demonstrated that MDM2 could ubiquitinate FOXP3 directly in this cell-free system, especially through monoubiquitination ( Figure 5C).…”

“…(C) 6×his-E1 (400 ng), 6×his-UbcH5b (E2) (800 ng), 6×his-Flag-ubiquitin (12 µg), StrepII-FOXP3a (400 ng), and GST-MDM2 (1.5 µg) were incubated at 37 • C in a cell-free system for 2 h, followed by termination with SDS loading buffer. Lys63-linked polyubiquitination and monoubiquitination can stabilize target proteins at post-translational level (20,34,35). To determine which types of ubiquitination on FOXP3 are mediated by MDM2, HA-tagged FOXP3 and Flag-tagged MDM2 were co-expressed with several His-tagged ubiquitin mutants in HEK293T cells, followed by His-pull-down assay.…”

“…CD4 + CD25 − T cells that ectopically expressed FOXP3 acquired Treg cell phenotypes, because they exhibited suppressive activity, and could inhibit interleukin-2 (IL-2) expression while facilitating the expression of CD25 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) (5,6). Studies have established that FOXP3 could be regulated at the level of post-translational modifications, which include acetylation, phosphorylation, poly(ADPribosyl)ation, arginine methylation, and ubiquitination, affecting the stability and function of FOXP3 and Treg cells (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In recent years, it has been reported that several E3 ubiquitin ligases or deubiquitinases (DUBs) modulate the K48-linked polyubiquitination of FOXP3, thus impacting FOXP3 stability and Treg cell function (17)(18)(19).…”

Immune Response in H. pylori-Associated Gastritis and Gastric Cancer

“…The shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of IFN-γ, resulting in an increase in Th1 cells. In mouse Treg cells, specific removal of RNF31 causes severe Treg cell deficiency and lethal immune diseases, revealing the importance of LUBAC activity for Treg cell homeostasis [27].…”