去類小泛素特異性蛋白酶2在Th17細胞分化中所扮演的角色

Abstract

近日越來越多的研究顯示轉譯後修飾（Posttranslational modification）具有調節細胞功能的作用及影響疾病發展的進程。有一些研究報告指出，SUMOylation這種轉譯後的修飾會透過調節細胞訊息傳遞及影響細胞生理功能進而導致疾病發生。在本篇研究中，我們將探討SUMOylation在免疫細胞發育中的分子機制以及SUMOylation在疾病發展過程中所扮演的角色。 SUMO是一種類小泛素的修飾蛋白，可以修飾在特定蛋白質的氨基酸羧基上。被SUMO修飾的蛋白以可透過去類小泛素特異性蛋白酶 (SUMO-specific protease, SENP) 去除SUMO使其能再循環被重複使用。SUMOylation是對於調節細胞內訊息傳遞、核質脂蛋白運輸和調節基因表有重要功能的轉譯後修飾。文獻研究結果顯示轉錄調節因子Smad4可以被SUMO1修飾，並被Senp2去除SUMO的修飾。此外，Smad4在Th17分化中扮演重要的角色。 Smad4也可以透過與負向調控因子SKI的相互作用進而抑制Th17分化中RORγt的表現。然而，目前仍不清楚Senp2是否通過Smad4調節T細胞的穩定平衡或Th17分化。在本篇論文中，我們將探討SUMOylation在免疫細胞發育中的所扮演的調控角色與其中的分子機制，以及其作用與疾病發展的關係。 先前的研究觀察到小鼠的飲水中若含有帶負電荷硫酸葡聚醣 (Dextran sulfate sodium, DSS) 會誘發的結腸炎的發生，且在小鼠的結腸炎模式中可以觀察到健康部位和病變部位之間調控SUMOylation的 E2連接酶 (UBC9) 的表現量有顯著差異，但SUMOylation在免疫細胞發育中的調節機制及其在結腸炎致病過程中的角色仍不清楚。在本篇論文中，我們使用了帶有T細胞專一性Senp2剔除的小鼠進行研究，以探討Senp2在調控T細胞發育和細胞功能中所扮演的角色。結果顯示Senp2具有負向調控致病性Th17細胞的分化的功能，進一步導致結腸炎發展的更嚴重並影響小鼠的腸道微生物群的菌相平衡。此外，與過繼轉移野生型CD4+ T細胞的小鼠相比，轉移了缺乏Senp2 CD4+ T細胞的Rag2剔除小鼠會導致更為嚴重的結腸炎的發展。使用誘發性剔除Senp2於T細胞的實驗，顯示T細胞內源之Senp2具有調控Th1和Th17細胞分化的作用。在剔除Senp2之Th17細胞中表現缺乏酵素活性的Senp2，我們發現Senp2的酵素活性是抑制Th17分化所必需的。在分子機制上，我們發現被SUMO修飾的Smad4的表現量在缺乏Senp2的 Th17中較對照組的 Th17有增加，並且SUMO修飾在Smad4的氨基酸K159的位點對於T細胞中Smad4的入核非常重要，並調控RORγt在Th17細胞中的表現。 總結，此論文發現在致病性Th17細胞中的Senp2可藉由調控Smad4的SUMOylation與影響其入核表現基因，進而避免過度產生IL-17A 和GM-CSF，並抑制結腸炎的發展。

Recently, accumulating evidence suggested that posttranslational modifications (PTMs) play a critical role in the regulation of cellular functions and disease progression. Several reports indicated that SUMOylation, a type of PTMs, contributed to diseases through modulation of cell signaling and functions. Here, we aim to study the insights of the molecular mechanisms of SUMOylation in immune cell development and the association of SUMOylation with diseases. SUMO is a small ubiquitin-like modifier protein that can be attached to target proteins on specific lysine residues. SUMOylation is a type of post-translational modifications (PTMs) which is crucial for signaling regulation, nucleocytoplasmic transportation and the regulation of gene expression. SUMOylation can be regulated by sentrin-specific protease (SENP) family, The process of SUMOylation is dynamic and reversibly removed by the SUMO proteases. Some studies indicated that the transcription factor Smad4 can be modified by SUMO1, and deSUMOylated by Senp2. In addition, Smad4 plays a role in Th17 differentiation, and that Smad4 can suppress the RORt expression through the interaction with the repressor SKI. However, whether Senp2 regulates T-cell homeostasis or Th17 differentiation through Smad4 is unclear. Here, we sought to study the molecular actions of SUMOylation in immune cell development and its association with diseases. Previous studies observed that the expression level of SUMO E2 enzyme UBC9 was different between the healthy and lesion sites of dextran sulfate sodium (DSS)-induced colitis model, but the underlying regulatory mechanisms of SUMOylation in immune cell development and its association with diseases remain elusive. In this study, we generated T-cell specific Senp2 knockout mice to investigate the role of Senp2 in regulation of T-cell development and functions. We found Senp2 restricts the differentiation of pathogenic Th17 cells, which results in the more severe development of colitis and affects gut microbiota in mice. Besides, adoptive transfer of naïve CD4+ T cells lacking Senp2 also showed more severe development of colitis in Rag2 knockout recipient mice as compared with mice received WT naïve CD4+ T cells. Using an inducible culture in which Senp2 in T cells can be deleted upon the addition of 4-hydroxytomaxifan, we demonstrated that Senp2 plays a role in Th1 and Th17 cell differentiation. Re-introduction of Senp2 enzymatic mutant into Senp2 deficient Th17 cells revealed that the enzymatic activity of Senp2 is required for the suppression of Th17 differentiation. Mechanistically, we demonstrated that Smad4 is an important SUMO targeted substrate in Th17 cells, and the levels of Smad4 SUMOylation is different between control and Senp2 deficient Th17 cells and SUMOylation at K159 site of Smad4 is important for the nuclear translocation of Smad4 in T cells, and RORγt expression in Th17 differentiation. Taken together, in this study, we found that Senp2 negatively regulates the differentiation of pathogenic Th17 cells by modulating the SUMOylation of Smad4 and its nuclear translocation and gene expression, thereby preventing the excessive production of IL-17A and GM-CSF in the progression of DSS-induced colitis.