摘要
CD4+CD25+调节性T细胞(Tregs)是在机体整个免疫网络的负调节中发挥重要作用的一种CD4+T淋巴细胞亚群,在维持机体免疫自稳和防止自身免疫性疾病中发挥重要作用。叉状头转录因子Foxp3是诱导CD4+CD25+Tregs细胞发育及生物学功能发挥的主调控因子,具有下调免疫应答的功能。Foxp3基因突变或缺失会导致严重的自身免疫性疾病。鼠Foxp3突变导致Xsf/Y雄性杂合子小鼠发生进行性自身免疫缺陷病,表现为器官非特异性淋巴细胞浸润及多种细胞因子的大量分泌,从而导致Xsf/Y雄性杂合子小鼠多数在出生后16-25天内死亡。人类同源的Foxp3功能性突变可导致人类一种免疫调节紊乱的X连锁综合征( immune dysregulation polyendocrinopathy enteropathy X-linked syndrome, IPEX),表现为免疫失调、多种内分泌腺病、肠病、X染色体性联综合征。因此,Foxp3在免疫系统的负调节中发挥非常重要的作用。
Foxp3的两个亚型(Foxp3FL和Foxp3△2 )在人CD4+CD25+Tregs中均有表达,并且它们都具有赋予CD4+CD25-T细胞转化为CD4+CD25+Tregs的活性,但是目前关于Foxp3的作用机制尚未阐明。大量研究显示,Foxp3作为转录调控因子,并不直接调控下游靶基因,而是与其它一些转录因子或酶分子形成“动态超分子复合物”共同发挥转录调控功能。目前已知的Foxp3相互作用分子有NFAT、NF-кB、AML1/Runx1、AP-1、RORγt和TIP60,其中,在我们课题的进行中,陆续有文献报道Foxp3与AP-1、AML1/Runx1、RORγt和TIP60的相互作用。但是仅仅针对这些分子的研究并不能充分阐明Foxp3的转录调控机制,所以对未知的Foxp3相互作用分子的进一步探讨是Foxp3转录调控研究的热点。
本课题是以Foxp3△2亚型作为诱饵,从外周血白细胞cDNA文库筛选可以与其相互作用的分子。最终我们获得40个阳性克隆,经DNA测序和Blast核苷酸相似性分析发现这40个阳性克隆所编码的蛋白质在Gal4 DNA激活域开放读框内的共9个。在这9个Foxp3△2候选相互作用分子中,我们重点对泛表达转录子(ubiquitously-expressed transcript,UXT)与Foxp3△2的相互作用进行研究。通过酵母双杂交回交验证和免疫共沉淀证实了Foxp3△2与UXT在酵母细胞和哺乳动物细胞内均存在相互作用,并且激光共聚焦扫描结果显示Foxp3△2和UXT共定位于细胞核,进一步为二者的相互作用提供了理论基础。为了明确Foxp3△2与UXT的作用部位,我们构建了Foxp3△2分子的5个截短体,并通过酵母双杂交中的β-半乳糖苷酶实验发现Foxp3△2的脯氨酸富含域对二者的相互作用是必不可少的。
此外,我们对Foxp3和UXT相互作用的功能进行了初步的探讨。最近研究显示,Foxp3不但表达于淋巴细胞,还表达于多种肿瘤细胞,表达于肿瘤细胞的Foxp3可能参与了肿瘤的免疫逃逸。我们课题组已证实Foxp3在胃癌细胞系SGC-7901中的表达,现进一步探讨提高UXT的表达是否对Foxp3的表达有影响?结果显示提高胃癌细胞株SGC-7901中UXT的表达,Foxp3的表达量也相应增加,但是具体作用机制还需要深入的研究。
综上所述,本课题通过酵母双杂交筛选共获得了9个与Foxp3△2相互作用的候选分子,并确认了Foxp3△2与UXT的物理相互作用及作用位点,为阐明Foxp3的转录调节机制奠定基础。
CD4+CD25+T regulatory cells (Tregs) are a subpopulation of CD4+T lymphocytes and engaged in the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses, such as autoimmune disease and allergy. Transcriptional factor Foxp3 is the master gene for differentiation and function of regulatory T cells and plays an important role in the negative immune regulation. Dysfunction of Foxp3 gene results in fatal, early onset autoimmune disease in both human and mouse. Foxp3 mutation in mice results in lethality in hemizygous males (Xsf/Y) 16-25 days after birth, and is characterized by overproliferation of CD4+CD8-T lymphocytes, extensive multiorgan infiltration and elevation of numerous cytokines. Similarly, mutations of FOXP3 gene in human are responsible for the disease called immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). Therefore, Foxp3 as a transcription factors plays a critical role by controlling immune homeostasis mediated by CD4+CD25+ regulatory T cells.
In human CD4+CD25+ regulatory T cells, Foxp3 is expressed as two isoforms: the full-length isoform (Foxp3FL) and a splice variant lacking the exon 2 (Foxp3△2). Studies demonstrate that Foxp3 isoforms possess similar capacities to converts conventional CD4+CD25-T cells into bona fide Tregs; however, the underlying molecular mechanism remains almost entirely undefined. Recent studies indicate that Foxp3 may form a dynamic superamolecular complex with a variety of molecular partners including transcription factors and enzymatic proteins to regulate transcription, such as NFAT (nuclear factor of activated T cells), NF-кB (nuclear factorкB), AML1 (acute myeloid leukemia 1)/Runx1 (runt-related transcription factor 1), AP-1 (Activator protein 1), RORγt (retinoicacid-related orphan receptorγt) and TIP60 (Tat-interactive protein, 60 kDa). During our research, however, several reports were published, describing the interaction of Foxp3 with other proteins (AP-1, AML1/Runx1, RORγt and TIP60). However, it is not sufficient to elucidate the mechanism of how Foxp3 orchestrates the cellular and molecular programs involved in Tregs function. Therefore, the further study of unknown Foxp3 interacting protein has become a hotspot of Foxp3 transcription regulation research.
In this study, Foxp3△2 was used to screen a human leukocyte yeast two-hybrid library and 40 positive clones which encode 9 different protein within the same open reading frame of GAL4 activation domain were obtained by reporter genes screening. The interaction of Foxp3△2 and ubiquitously expressed transcript (UXT) was further confirmed in yeast and in mammalian cells. The confocal fluorescence microscopy result indicates that both Foxp3△2 and UXT were predominantly colocalized in the nucleus, which raises the possibility that these proteins interact to regulate gene expression. The proline-rich domain in the N terminal of Foxp3△2 was necessary for the interaction of Foxp3△2 and UXT.
In addition, we attempted to do some research for the function of Foxp3△2 and UXT interaction. Recent studies showed that Foxp3 was expressed not only in T lymphocyte, but also in tumor cells which probably took part in the immune escape of tumor. Our teams also have confirmed that Foxp3 expressed in gastric carcinoma cell line SGC-7901. However, will the overexpression of UXT influence on the expression of Foxp3? Preliminary result showed that the forced overexpression of UXT in gastric carcinoma cell line SGC-7901 could elevate the Foxp3 protein level. However, the mechanism of action was not clear.
In summary, we performed yeast two-hybrid screening using Foxp3△2 as a bait protein and obtained 9 interacting proteins. Physical interaction and interacting domain of Foxp3△2 were proved in vivo. Moreover, the finding of a novel Foxp3△2 interactant will lay a basis for the elucidation of Foxp3 action.
引文
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