Proteolytic cleavage regulates Foxp3 activity

描述（由申请人提供）：Foxp3是一个47-kDa的转录因子，是天然Tregs发育和功能所必需和充分的。Foxp3+ Tregs在控制自身反应性T细胞和预防自身免疫中的作用已得到证实。在比较静息和活化Tregs的Foxp3表达时，我们在活化Tregs的染色质部分检测到41-kDa的Foxp3物种，而在静息Tregs中没有检测到，这表明Foxp3可能通过蛋白水解裂解进行加工。对小鼠和人类Foxp3序列的潜在识别位点的搜索发现了两个RXXR蛋白转换酶（PC）基序，48RDLR51和414RKKR417，位于N端和c端附近。在初步研究中，我们发现Foxp3在n端和C端RXXR位点都被切割，n端切割导致41-kDa物种的产生。两个位点的切割取决于完整的RXXR基序的存在。表达Foxp3形式的转导CD4+细胞模仿N-， C-， N和C-cleaved Foxp3在功能上是不同的，这可以从它们对Treg标记物表达、抑制Teff细胞增殖和预防实验性结肠炎的不同作用中得到证明。我们的研究结果表明，裂解事件具有重要的功能后果，并为Foxp3实现其对基因表达的多种影响提供了一种手段。根据我们的初步数据，提出了2个具体目标。目的1：确定Foxp3加工对关键Treg标记表达的影响，研究蛋白水解裂解对Foxp3启动子结合能力的影响。通过对表达WT Foxp3和不同形式Foxp3的CD4+细胞中CTLA-4和IL-10 mRNA表达的qPCR分析，发现WT和加工形式Foxp3之间存在较大差异。该分析将扩展到其他关键Treg标记物。此外，Treg标记物的蛋白水平将通过ELISA、Luminex和Panomics阵列检测。不同形式的Foxp3在启动子上的dna结合已知与Foxp3直接相互作用，这些启动子上的组蛋白乙酰化水平将通过ChIP测定。目的2：确定负责在n端RDLR和C端RKKR位点切割Foxp3的pc。在涉及PC1和PC7共表达的初步研究中，这两种pc都能在c端加工Foxp3，而在n端不加工Foxp3。我们计划对所有已知的7台pc进行分析，以确定哪些能够处理Foxp3的n端或c端。作为第二种方法，shRNA技术将用于确定pc在Foxp3处理中的作用。与NIH和生物医学研究的相关性：这些研究将产生关于Foxp3不同加工形式的功能作用的第一批数据。设计改变pc处理Foxp3的能力的方法应该产生具有特定治疗目的的功能的treg。公共卫生相关性：Foxp3是一种dna结合蛋白，决定调节性T细胞的发育和功能，调节性T细胞是控制免疫反应的关键T细胞群。Foxp3可以通过增加或抑制基因的表达来影响许多基因。我们已经证明Foxp3在两个位点被切割，表达Foxp3切割形式的细胞功能非常不同，这表明了治疗的潜力。

DESCRIPTION (provided by applicant): Foxp3, a 47-kDa transcription factor, is necessary and sufficient for development and function of natural Tregs. The role of Foxp3+ Tregs in controlling self-reactive T cells and preventing autoimmunity is well established. During comparison of Foxp3 expression by resting vs. activated Tregs, we detected a 41-kDa Foxp3 species in the chromatin fraction of activated Tregs that was not detectable in resting Tregs, indicating Foxp3 may be processed by proteolytic cleavage. Searches of murine and human Foxp3 sequences for potential recognition sites revealed two RXXR proprotein convertase (PC) motifs, 48RDLR51 and 414RKKR417, located near the N- terminal and C-terminal ends. In Preliminary Studies, we show Foxp3 is cleaved at both the N-terminal and C- terminal RXXR sites and N-terminal cleavage results in the generation of the 41-kDa species. Cleavage at both sites depends on the presence of intact RXXR motifs. Transduced CD4+ cells expressing Foxp3 forms mimicking N-, C-, N&C-cleaved Foxp3 are functionally distinct, as evidenced by their differing effects on expression of Treg markers, suppression of Teff cell proliferation and prevention of experimental colitis. Our results indicate the cleavage events have important functional consequences and provide a means for Foxp3 to achieve its diverse effects on gene expression. Based on our preliminary data, 2 specific aims are proposed. Aim 1: To determine the effects of Foxp3 processing on expression of critical Treg markers and to study the effect of proteolytic cleavage on the promoter binding ability of Foxp3. The qPCR analysis of CTLA-4 and IL-10 mRNA expression in CD4+ cells expressing WT Foxp3 vs. different forms of Foxp3, showed major differences between WT and processed forms of Foxp3. This analysis will be extended to other critical Treg markers. In addition, protein levels of Treg markers will be determined by ELISA, Luminex and Panomics arrays. DNA-binding of the different Foxp3 forms on promoters Foxp3 is known to directly interact with and the histone acetylation levels at these promoters will be determined by ChIP assays. Aim 2: To determine the PCs responsible in cleaving the Foxp3 at the N-terminal RDLR and C- terminal RKKR sites. In preliminary studies involving co-expression of PC1 and PC7, both PCs were capable of processing Foxp3 at the C-terminal but not at the N-terminal site. We plan to analyze all of the 7 known PCs, to determine those capable of N-terminal or C-terminal processing of Foxp3. As a second approach, shRNA technology will be used to determine the role of PCs in Foxp3 processing. Relevance to NIH and biomedical research: These studies will generate the first data on the functional roles of the different processed forms of Foxp3. Devising methods that alter the ability of the PCs to process Foxp3 should generate Tregs with functions tailored for specific therapeutic purposes. PUBLIC HEALTH RELEVANCE: Foxp3 is a DNA-binding protein that determines development and function of regulatory T cells, a group of T cells that are critical in keeping immune responses under control. Foxp3 can affect many genes, by increasing or suppressing their expression. We have shown Foxp3 is cleaved at two sites and cells expressing the cleaved forms of Foxp3 function very differently, indicating potential in therapy.