Proteomics of central tolerance in NOD vs B6 mice

描述（由申请人提供）： 在这两个阶段（R21/R33）项目中，我们将开发方法来阐明NOD小鼠（人类1型糖尿病模型）中枢耐受缺陷的细胞信号传导机制。我们将比较在BDC2.5/NOD和BDC2.5/H-2g 7遗传背景下FTOC衍生的胸腺细胞中对TCP刺激的信号响应。我们的研究将集中在负选择中的几个信号转导途径。然而，我们也将监测蛋白质组范围内的蛋白磷酸化事件，因为有缺陷的克隆缺失的潜在机制可能存在于替代途径中。 在该项目的R21阶段，我们将开发方法，使从FTOC（胎儿胸腺器官培养物）中提取的胸腺细胞中的特定信号转导途径和整体蛋白磷酸化的分析成为可能，FTOC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，酶促消化免疫沉淀的蛋白质，并在LC/MS/MS分析鉴定特异性磷酸化位点之前用Fe 3 +-带电的固定化金属离子亲和色谱（IMAC）柱富集磷酸化肽。类似的策略将用于酪氨酸磷酸化蛋白质，尽管泛特异性抗磷酸酪氨酸抗体将用于免疫沉淀。在该项目的R21阶段，我们还将开发一种方法，通过从胸腺细胞中提取蛋白质，在蛋白质或肽水平上对样品进行预分级，并在LC/MS/MS分析之前在IMAC柱上富集肽，从而在全球范围内鉴定蛋白质磷酸化。 在R33阶段，我们将应用这些方法中的每一种来分析在TCR的肽刺激后从FTOC提取的胸腺细胞中的信号转导。我们将在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定量。除了Akt激酶和磷酸酪氨酸介导的信号传导外，在R33阶段将研究14-3-3结合蛋白的磷酸化状态。负选择的几种调节剂（即HDAC 7和努尔77）在磷酸化和结合14-3-3后被隔离到胞质溶胶中。 通过比较信号转导途径响应肽刺激的胸腺细胞来源于BDC2.5/NOD和BDC2.5/H-2g 7，我们希望确定信号转导事件调节自身反应性胸腺细胞的负选择，具体目的是确定的机制，在NOD小鼠中枢耐受性缺陷。

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.