Defining the partner interaction network of the tetraspanin CD53 in regulating B cell trafficking

项目概要/摘要 该项目的目标是了解 CD53 如何调节 B 细胞运输，并确定 CD53- 这一流程背后的合作伙伴网络。 CD53 是跨膜蛋白四跨膜蛋白家族的成员 在细胞表面组织多蛋白网络来调节多种细胞过程，例如 增殖、归巢和生存。 CD53 的缺失与免疫系统功能受损有关。 CD53 是 在正常和恶性 B 细胞中高表达，但其在这些细胞中的作用尚不清楚。我们之前 据报道，CD53 是骨髓中正常 B 细胞发育所必需的。在初步数据中，我们现在 发现 CD53 对于正常和恶性 B 细胞运输都是必需的，CD53 的缺失会导致 B 细胞粘附、迁移、骨髓归巢和抗体产生显着受损。至 了解了潜在的分子机制，我们确定了CD53的晶体结构；在 整个四跨膜蛋白家族，这是第一个以活性构象捕获的结构。我们揭示了构象如何 变化通过基于质谱 (MS) 的足迹影响 CD53 伙伴相互作用。我们已经用过 邻近标记可识别多个候选 CD53 伴侣，其功能将趋化因子信号传导与细胞联系起来 动力。这些数据支持我们的假设，即 CD53 协调粘附、信号传导和细胞的复合体 促进 B 细胞运输的运动蛋白。正常和恶性 B 细胞都依赖于准确的运输 它们在骨髓和次级淋巴器官中的生态位，以优化它们的成熟和功能。因此， 了解指导 B 细胞运输的相互作用不仅对于优化正常 B 细胞很重要 细胞功能，而且还将揭示恶性 B 细胞的潜在治疗靶点。使用体外组合 结合邻近标记、定量 MS、活细胞 MS 进行体内粘附和迁移研究 基于足迹、生化和电子显微镜分析，这个双 PI 提案提出了一个 5 年计划 目的：1) 阐明调节 B 细胞运输的 CD53-伴侣相互作用网络，以及 2) 确定功能 破坏 CD53-伴侣相互作用的后果。借助我们新开发的工具，我们将揭示 协调细胞信号传导和运动性以调节 B 细胞粘附的新型蛋白质相互作用网络， 迁移、趋化因子信号传导和生态位定位。我们将揭示这个 CD53 介导的网络是如何 B 细胞迁移过程中对趋化因子信号传导作出反应，以及跨细胞相互作用如何在 B 细胞迁移过程中建立 B细胞归巢。因此，拟议的研究将显着增进我们对 B 细胞运输如何进行的理解 得到协调和监管。鉴于四跨膜蛋白家族的保守结构和功能冗余 成员们，这将导致我们的长期目标是阐明适用于其他领域的四跨膜蛋白/合作伙伴关系 CD53 在免疫系统功能和恶性肿瘤中的作用以及其他四跨膜蛋白家族的功能 成员。

Project Summary/Abstract The goal of this project is to understand how CD53 regulates B cell trafficking, and to determine the CD53- partner network underlying this process. CD53 is a member of the tetraspanin family of transmembrane proteins that organize multi-protein networks on the cell surface to regulate a wide variety of cellular processes such as proliferation, homing and survival. Loss of CD53 is associated with impaired immune system function. CD53 is highly expressed on both normal and malignant B cells, however its role in these cells is not clear. We previously reported that CD53 is required for normal B cell development in the bone marrow. In preliminary data, we now find that CD53 is essential to both normal and malignant B cell trafficking, with the loss of CD53 causing significant impairment in B cell adhesion, migration, bone marrow homing and antibody production. To understand the underlying molecular mechanisms, we have determined the crystal structure of CD53; in the entire tetraspanin family, this the first structure captured in an active conformation. We reveal how conformational changes influence CD53 partner interactions by mass spectrometry (MS) based footprinting. We have used proximity labeling to identify several candidate CD53 partners, whose functions link chemokine signaling to cell motility. These data support our hypothesis that CD53 coordinates a complex of adhesion, signaling and cell motility proteins that facilitate B cell trafficking. Both normal and malignant B cells rely on accurate trafficking to their niches in the bone marrow and secondary lymphoid organs to optimize their maturation and function. Thus, an understanding of the interactions that guide B cell trafficking are important not only for optimizing normal B cell function, but will also reveal potential therapeutic targets of malignant B cells. Using a combination of in vitro and vivo adhesion and migration studies in combination with proximity labeling, quantitative MS, live-cell MS- based footprinting, biochemical and electron microscopic analyses, this dual-PI proposal presents a 5-year plan to: 1) elucidate the CD53-partner interaction network regulating B cell trafficking, and 2) determine the functional consequences of disrupting the CD53-partner interactions. Armed with our newly developed tools, we will reveal a novel network of protein interactions coordinating cell signaling and motility to regulate B cell adhesion, migration, chemokine signaling and niche localization. We will uncover how this CD53-mediated network responds to chemokine signaling during B cell migration and how cross-cell interactions are established during B cell homing. Thus, the proposed studies will significantly advance our understanding of how B cell trafficking is coordinated and regulated. Given the conserved structure and functional redundancy of tetraspanin family members, this will lead to our long-term goal of elucidating tetraspanin/partner relationships that apply to other roles of CD53 in immune system function and malignancy as well as the functions of other tetraspanin family members.