ERM and Rho Signal Pathways in T Cell Immune Senescence

描述（由申请人提供）： Ezrin/radixin/moesin家族的细胞骨架蛋白（ERM蛋白）有助于响应于抗原的T细胞活化，并且还有助于响应于结缔组织基质蛋白和趋化因子梯度的T细胞极化。以前的工作已经表明，来自老年小鼠的T细胞在表面大分子和底层细胞骨架框架之间形成分子连接的能力方面存在缺陷，无论是移动到突触的蛋白质还是从T细胞/APC相互作用位点排除的蛋白质。当与涂有TCR/CD 3复合物抗体的载玻片接触时，来自老年小鼠的T细胞也显示出有缺陷的细胞骨架重排和片状伪足形成。新的数据表明，老年T细胞不同于年轻的T细胞在几个方面的ERM生物化学，包括ERM磷酸化和ERM协会与结合蛋白，也显示缺陷的激活Rho家族GTP酶，控制ERM功能。提出了三个目标，以澄清，在分子水平上，在ERM和Rho为基础的信号的年龄依赖性缺陷的基础上，和他们的贡献有缺陷的T细胞极化反应。目的1将集中在T细胞细胞骨架极化响应纤连蛋白信号和刺激的趋化因子SDF-1a的梯度，测试的想法，这些T细胞从老年小鼠不能适当地响应这些信号将显示相应的缺陷，在重新定位的ERM蛋白，ERM结合蛋白，和ERM相关的表面标志物。目的2将评价与激酶、磷酸酶和表面连接蛋白相关的ERM的年龄依赖性变化，所述激酶、磷酸酶和表面连接蛋白在TCR依赖性和TCR非依赖性极化响应期间控制ERM的位置和功能。这一目标还将包括膜分馏协议，以评估年龄对ERM本地化的影响，相对于高粘度的膜微区，和研究已知的Cbp和Cbl-b蛋白调节关联的ERM与质膜。目标3的工作将调查两个假设的年龄相关的缺陷在RhoA活性，一个涉及改变Vav信号通路和其他评估的抑制RhoA再激活的GDI家族成员的RhoA解离抑制剂。提出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，确定哪些合理的控制途径实际上对改变T细胞对结缔组织和趋化因子信号的反应至关重要，并提出新的实验路线，值得在长期研究中关注。免疫系统的老化导致许多老年疾病和残疾，但对调节老年免疫系统衰退的因素知之甚少。该研究计划旨在通过研究老年小鼠细胞中T细胞活化的生物化学，探索有关年龄依赖性免疫衰竭分子基础的几个想法。

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.