ERM and Rho Signal Pathways in T Cell Immune Senescence

描述(由申请人提供)：Ezrin/Radisin/Moesin家族的细胞骨架蛋白(ERM蛋白)有助于在抗原反应中激活T细胞，并在结缔组织基质蛋白和趋化因子梯度反应中促进T细胞极化。先前的工作表明，老年小鼠的T细胞在表面大分子和潜在细胞骨架框架之间建立分子联系的能力存在缺陷，无论是对于移动到突触的蛋白质还是那些被排除在T细胞/APC相互作用部位之外的蛋白质。当老年小鼠的T细胞与涂有TCR/CD3复合体抗体的玻片接触时，也显示出细胞骨架重排缺陷和片状脂血症的形成。新的数据表明，老年T细胞在ERM生化的几个方面与年轻T细胞不同，包括ERM磷酸化和ERM与结合蛋白的关联，以及控制ERM功能的Rho家族GTP酶的激活缺陷。从分子水平上阐明ERM和Rho信号中年龄相关缺陷的基础，以及它们对缺陷T细胞极化反应的贡献。目的1将重点放在T细胞对纤维连接蛋白信号和趋化因子SDF-1a梯度刺激的反应中的细胞骨架极化，测试那些未能对这些信号做出适当反应的老龄小鼠的T细胞将在ERM蛋白、ERM结合蛋白和ERM相关表面标记的重新定位方面显示相应的缺陷。目的2将评估在TCR依赖和TCR非依赖的极化反应中，ERM与控制ERM位置和功能的激酶、磷酸酶和表面连接蛋白相关的年龄相关性变化。这一目标还将包括评估AGE对高粘度膜微区ERM定位的影响的膜分离方案，以及已知调节ERM与质膜结合的CBP和Cbl-b蛋白的研究。Work for Aim 3将研究与年龄相关的RhoA活性缺陷的两个假说，一个涉及Vav信号通路的改变，另一个评估RhoA解离抑制剂GDI家族成员对RhoA重新激活的抑制作用。建议的工作应该确认改变的ERM和RhoA途径在T细胞免疫衰老中的重要性，确定哪些看似合理的控制途径实际上对改变的T细胞对结缔组织和趋化因子信号的反应至关重要，并提出了值得长期研究关注的新的实验路线。免疫系统的老化导致了许多老年疾病和残疾，但人们对调节老年免疫系统衰退的因素知之甚少。本研究计划旨在通过对老龄小鼠T细胞活化的生化研究，探索年龄依赖性免疫失败的分子基础。

DESCRIPTION (provided by applicant): Cytoskeletal proteins of the ezrin/radixin/moesin family (ERM proteins) contribute to T cell activation in responses to antigen, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell/APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with antibodies to the TCR/CD3 complex. New data suggest that aged T cells differ from young T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with binding proteins, and also show defects in the activation of Rho-family GTPases that control ERM function. Three aims are proposed to clarify, at a molecular level, the basis for age-dependent defects in ERM and Rho-based signals, and their contribution to defective T cell polarization responses. Aim 1 will focus on T cell cytoskeletal polarization in responses to fibronectin signals and to stimulation by a gradient of the chemokine SDF-1a, testing the idea that those T cells from aged mice that fail to respond appropriately to these signals will show corresponding defects in re-localization of ERM proteins, ERM-binding proteins, and ERM-associated surface markers. Aim 2 will evaluate age- dependent change in ERM association with kinases, phosphatases, and surface linker proteins that control ERM position and function during TCR-dependent and TCR-independent polarization responses. This aim will also include membrane fractionation protocols to evaluate age effects on ERM localization with respect to high viscosity membrane microdomains, and studies of Cbp and Cbl-b proteins known to regulate association of ERM with the plasma membrane. Work for Aim 3 will investigate two hypotheses about the age-related defect in RhoA activity, one involving altered Vav signal pathways and the other evaluating the inhibition of RhoA reactivation by members of the GDI family of RhoA dissociation inhibitors. The work proposed should confirm the importance of altered ERM and RhoA pathways in T cell immune senescence, determine which of the plausible control pathways is actually critical to altered T cell responses to connective tissue and chemokine signals, and suggest new lines of experimentation that would merit attention in long term studies. Aging of the immune system contributes to many of the diseases and disabilities of old age, but too little is known about factors that regulate the decline of this system in old age. This research plan is aimed at exploring several ideas about the molecular basis for age-dependent immune failure by studies of the biochemistry of T cell activation in cells from aged mice.