Proteomics of central tolerance in NOD vs B6 mice

描述(由申请人提供)： 在这个两个阶段(R21/R33)的项目中，我们将开发方法来阐明NOD小鼠中枢耐受缺陷的细胞信号机制，NOD小鼠是人类1型糖尿病的模型。我们将在BDC2.5/NOD和BDC2.5/H-2G7的遗传背景上比较FTOCs来源的胸腺细胞对TCP刺激的信号反应。我们的研究将集中在负选择中涉及的几个信号转导通路。然而，我们也将监测蛋白质组范围的蛋白质磷酸化事件，因为有缺陷的克隆删除的机制可能存在于另一条途径中。 在项目的R21阶段，我们将开发能够分析从FTOCs(胎儿胸腺器官培养)提取的胸腺细胞中特定信号转导途径和全球蛋白质磷酸化的方法学，FTOCs是胸腺的模型系统。为了鉴定Akt底物，我们将用Akt磷酸化基序特异性抗体进行免疫沉淀，用酶消化免疫沉淀蛋白，并在LC/MS/MS分析之前用Fe3+固定的金属离子亲和层析(IMac)柱富集磷酸化肽。酪氨酸磷酸化蛋白将采用类似的策略，尽管免疫沉淀将使用泛特异的抗磷酸酪氨酸抗体。在该项目的R21阶段，我们还将开发一种能够在全球范围内鉴定蛋白质磷酸化的方法，方法是从胸腺细胞中提取蛋白质，在蛋白质或多肽水平上对样品进行预分离，并在LC/MS/MS分析之前在iMac柱上富集肽。 在R33阶段，我们将应用这些方法中的每一种来分析在TCR多肽刺激后从FTOCs中提取的胸腺细胞的信号转导。我们将在多肽刺激后的特定时间点对系统进行采样，并测量整个时间过程中蛋白质磷酸化的相对量化。除了Akt激酶和磷酸化酪氨酸介导的信号转导外，在R33期还将研究14-3-3结合蛋白的磷酸化状态。几种负选择调节子(即HDAC7和NUR 77)在磷酸化并与14-3-3结合后被隔离到胞浆中。 通过比较BDC2.5/NOD和BDC2.5/H-2G7来源的胸腺细胞中肽刺激反应的信号转导通路，我们希望找出调节自身反应性胸腺细胞负选择的信号事件，以明确NOD小鼠中枢耐受缺陷的机制。

DESCRIPTION (provided by applicant): In this two phase (R21/R33) project we will develop methods to elucidate cellular signaling mechanisms underlying a defect in central tolerance in the NOD mouse, a model for human Type 1 diabetes. We will compare signal response to TCP stimulation in thymocytes derived from FTOCs on the BDC2.5/NOD and BDC2.5/H-2g7 genetic background. Our investigation will focus on several signal trasduction pathways implucated in negative selection. However, we will also monitor proteome-wide protein phosphorylation events, as it is possible that the mechanism underlying defective clonal deletion will lie in an alternate pathway. In the R21 phase of the project we will develop methodologies which will enable analysis of specific signal transduction pathways and global protein phosphorylation in thymocytes extracted from FTOCs (fetal thymic organ cultures), a model system for the thymus. To identify Akt substrates we will immunoprecipitate with an Akt phosphorylation motifspecific antibody, enzymatically digest immunoprecipitated proteins, and enrich phosphorylated peptides with an Fe3+- charged immobilized metal ion affinity chromatography (IMAC) column prior to LC/MS/MS analysis for identification of specific phosphorylation sites. A similar strategy will be employed for tyrosine phosphorylated proteins, although a panspecific anti-phosphotyrosine antibody will be used for immunoprecipitation. In the R21 phase of the project we will also develop a method enabling identification of protein phosphorylation on a global scale, by extracting proteins from thymocytes, prefractionating the sample at the protein or peptide level, and enriching peptides on the IMAC column prior to LC/MS/MS analysis. In the R33 phase will we apply each of these methods to the analysis of signal transduction in thymocytes extracted from FTOCs following peptide stimulation of the TCR. We will sample the system at specific time points following peptide stimulation and measure relative quantification of protein phosphorylation across the time course. In addition to Akt kinase and phospho-tyrosine mediated signaling, in the R33 phase will investigate phosphorylation state of 14-3-3 binding proteins. Several regulators of negative selection (i.e. HDAC7 and Nur 77) are sequestered to the cytosol following phosphorylation and binding to 14-3-3. By comparing signal transduction pathways responding to peptide stimulation in thymocytes derived from BDC2.5/NOD and BDC2.5/H-2g7 we hope to identify signaling events regulating negative selection of self-reactive thymocytes, with the specific aim of determining the mechanism underlying defective central tolerance in the NOD mouse.