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

Aiting Wang,Yichao Han,Bin Li,Rui Liang,Xiaoxia Wang,Mengdi Yang,Hecheng Li,Ruirong Lin,Xinnan Liu,Xiaojun Yuan,Hui Zhao,Fangming Zhu,Dan Li,Fuxiang Zhu

doi:10.3389/fimmu.2020.01087

Abstract

Regulatory T cells (Treg cells) are essential for maintaining immune tolerance, and the dysfunction of Treg cells may cause autoimmune diseases and tumors. Forkhead box P3 (FOXP3) is the key transcription factor controlling Treg cell development and suppressive function. Mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase, has been identified as an oncoprotein that mediates the ubiquitination and degradation of tumor suppressor p53; however, whether it has functions in Treg cells remains unknown. Here, we demonstrate that MDM2 positively regulates human Treg cell suppressive function via its mediated ubiquitination and stabilization of FOXP3. Knockdown of MDM2 with shRNA in human primary Treg cells leads to the impaired ability of FOXP3 to regulate the expression levels of downstream genes and the attenuated suppressive capacity of Treg cells, due to FOXP3 instability. Consistently, MDM2 overexpression in human Treg cells enhances FOXP3 stability and Treg cell suppressive capacity. Mechanistically, MDM2 interacts with FOXP3, and mainly mediates monoubiquitination and polyubiquitination of FOXP3, thus stabilizing the protein level of FOXP3. We have also found lysine residues in FOXP3 required for MDM2-mediated ubiquitination. In addition, TCR/CD28 signaling upregulates the expression level of MDM2 and its mediated FOXP3 ubiquitination in human Treg cells. Therefore, our findings reveal that MDM2 in Treg cells could be a potential therapeutic target for treating autoimmune diseases and tumors.

Highlights

CD4+CD25+Forkhead box P3 (FOXP3)+ regulatory T cells (Treg cells) are crucial for maintaining immune tolerance and immune homeostasis [1]
Compared with Tconv cells, the expression level of Mouse double minute 2 homolog (MDM2) was higher in Treg cells, as indicated by mean fluorescence intensity (MFI) (Figure 1A), suggesting that MDM2 may be required for Treg cells
Treg cells with MDM2 knockdown displayed markedly impaired capacity of suppressing Teff cell proliferation (Figures 1G,H). These findings implicate that MDM2 knockdown in human Treg cells leads to impaired Treg cell function and acquisition of Teff cell phenotypes; MDM2 is crucial for human Treg cell suppressive function

Summary

Introduction

CD4+CD25+FOXP3+ regulatory T cells (Treg cells) are crucial for maintaining immune tolerance and immune homeostasis [1]. 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]. It has been reported that several E3 ubiquitin ligases or deubiquitinases (DUBs) modulate the K48-linked polyubiquitination of FOXP3, impacting FOXP3 stability and Treg cell function [17,18,19]. Due to the distinct sites and types, ubiquitination causes protein degradation, and stabilizes the function of substrates [21, 22]; other enzymes that regulate different types of FOXP3 ubiquitination remain to be identified

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Conclusion