Regulation of B Cell Function by Membrane-Cytoskeletal Remodeling

描述（由申请方提供）：B细胞抗原受体（BCR）信号传导不仅对抗体介导的病原体免疫至关重要，而且还维持对自身的耐受性。过度活跃的BCR信号传导是自身免疫性疾病如系统性红斑狼疮（SLE）的标志，但控制B细胞对抗原的过度反应的分子机制知之甚少。因此，鉴定驱动B细胞过度活化和自身抗体产生的蛋白质对于开发SLE的有效治疗策略至关重要。林恩-/-小鼠表现出B细胞过度活化以及与人SLE相关的致病症状。人类GWAS研究显示Src家族激酶林恩与SLE之间存在遗传关联，使得林恩-/-小鼠成为该疾病的临床相关模型。我们以前曾报道，膜-细胞骨架连接蛋白ezrin调节膜动力学和BCR信号在抗原刺激的B细胞通过其能力进行动态变化的磷酸化。有趣的是，林恩-/-小鼠的B细胞中ezrin的条件性缺失导致B细胞活化和分化降低，以及自身抗体产生减少。这些数据表明，ezrin介导的调节是一个关键的机制，管理超活化的B细胞的情况下，林恩。埃兹蛋白在林恩-/- B细胞中过度磷酸化，埃兹蛋白的去磷酸化导致BCR微聚集、钙信号传导和细胞因子分泌减少。我们的数据表明了一个正反馈模型，即在林恩-/- B细胞中更强的钙信号传导导致钙依赖性蛋白激酶C活化增加，这导致埃兹蛋白的过度磷酸化，这反过来又增加了BCR微聚集和信号传导。总的来说，我们的数据揭示了ezrin和林恩之间的一种新的合作界面，调节SLE中BCR信号的强度，并可能成为治疗干预的潜在靶点。我们假设ezrin通过其调节BCR空间组织和信号传导的能力来调节林恩-/-小鼠中的B细胞过度活化和自身免疫。我们的具体目标是，（1）确定ezrin缺失对林恩-/-小鼠B细胞自身免疫发展的影响，（2）研究ezrin缺失对林恩-/- B细胞BCR组织和信号传导的影响，（3）阐明过度磷酸化ezrin介导林恩-/- B细胞过度活性的机制。我们将采用遗传学、生物化学、分子生物学、免疫学和尖端的高分辨率成像技术来实现我们的目标。我们的研究结果将为了解狼疮B细胞中BCR对抗原反应的空间调控提供新的见解，并阐明ezrin介导林恩-/- B细胞过度活跃的机制。最终，我们的发现将导致 开发治疗狼疮和其他表现出异常BCR信号传导的B细胞疾病的新策略。

DESCRIPTION (provided by applicant): B cell antigen receptor (BCR) signaling is not only critical for antibody-mediated immunity to pathogens but also maintains tolerance to self. Hyperactive BCR signaling is a hallmark of autoimmune diseases such as systemic lupus erythematosus (SLE), but the molecular mechanisms controlling B cell hyper responsiveness to antigen are poorly understood. Therefore, identification of proteins that drive B cell hyperactivation and autoantibody production is crucial for development of effective therapeutic strategies in SLE. Lyn-/- mice display B cell hyperactivation as well as pathogenic symptoms associated with human SLE. Human GWAS studies have shown a genetic association between the Src family kinase Lyn and SLE, making Lyn-/- mice a clinically relevant model for this disease. We have previously reported that the membrane-cytoskeleton linker protein ezrin regulates membrane dynamics and BCR signaling in antigen-stimulated B cells through its ability to undergo dynamic changes in phosphorylation. Interestingly, conditional deletion of ezrin in B cells of Lyn-/- mice leads to decreased B cell activation and differentiation, and reduced autoantibody production. These data suggest that ezrin-mediated regulation is a critical mechanism governing the hyper activation of B cells in the absence of Lyn. Ezrin is hyperphosphorylated in Lyn-/- B cells, and dephosphorylation of ezrin leads to reduced BCR microclustering, calcium signaling and cytokine secretion. Our data suggest a positive feedback model whereby stronger calcium signaling in Lyn-/- B cells leads to increased calcium-dependent protein kinase C activation, which results in hyperphosphorylation of ezrin, which in turn increases BCR microclustering and signaling. Collectively, our data reveal a novel collaborative interface between ezrin and Lyn that regulates the strength of BCR signaling in SLE, and may be a potential target for therapeutic intervention. We hypothesize that ezrin regulates B cell hyper activation and autoimmunity in Lyn-/- mice via its ability to regulate BCR spatial organization and signaling. Our specific aims are, (1) To determine the impact of ezrin deletion on development of B cell autoimmunity in Lyn-/- mice, (2) To investigate the effect of ezrin deletion on BCR organization and signaling in Lyn-/- B cells, and (3) To elucidate the mechanism by which hyperphosphorylated ezrin mediates hyperactivity of Lyn-/- B cells. We will employ genetic, biochemical, molecular biological, immunological and cutting-edge high resolution imaging techniques to accomplish our aims. Our results will provide insights into spatial regulation of BCR response to antigen in lupus B cells, and elucidate the mechanism by which ezrin mediates the hyperactivity of Lyn-/- B cells. Ultimately, our findings will lead to the development of novel strategies for treatment of lupus and other B cell disorders that exhibit aberrant BCR signaling.