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

项目摘要/摘要 本项目的目标是了解CD53如何调节B细胞的贩运，并确定CD53- 作为这一流程基础的合作伙伴网络。CD53是Tetraspanin跨膜蛋白家族的成员 在细胞表面组织多蛋白质网络来调节各种各样的细胞过程，如 扩散、归宿和生存。CD53的缺失与免疫系统功能受损有关。CD53是 在正常和恶性B细胞上均高表达，但其在这些细胞中的作用尚不清楚。我们之前 报道称，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对正常和恶性B细胞的运输都是必不可少的，CD53的丢失导致 B细胞黏附、迁移、骨髓归巢和抗体产生明显受损。至 了解潜在的分子机制，我们已经确定了CD53的晶体结构；在 整个Tetraspanin家族，这是第一个以活性构象捕捉到的结构。我们揭示了如何构象 通过基于质谱仪(MS)的足迹分析，变化会影响CD53合作伙伴的相互作用。我们已经使用了 邻近标记以确定几个候选的CD53伙伴，其功能将趋化因子信号与细胞联系起来 能动性。这些数据支持我们的假设，即CD53协调了黏附、信号和细胞的复合体 促进B细胞运输的运动性蛋白质。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利基，以优化其成熟和功能。因此， 了解引导B细胞转运的相互作用不仅对优化正常B细胞非常重要 但也将揭示恶性B细胞的潜在治疗靶点。使用体外试验的组合 体内黏附和迁移研究结合邻近标记、定量MS、活细胞MS- 基于足迹、生化和电子显微镜分析，这项双PI提案提出了一个五年计划 目的：1)阐明CD53-伴侣相互作用网络调节B细胞转运；2)确定CD53-伙伴相互作用网络的功能 中断CD53与合作伙伴互动的后果。用我们新开发的工具武装起来，我们将揭示 一种新的蛋白质相互作用网络，协调细胞信号和运动来调节B细胞黏附， 迁移、趋化因子信号和生态位定位。我们将揭开CD53介导的网络是如何 在B细胞迁移过程中对趋化因子信号的反应，以及在B细胞迁移过程中如何建立跨细胞相互作用 B细胞归巢。因此，拟议的研究将极大地促进我们对B细胞如何贩运的理解 是协调的和受监管的。鉴于Tetraspanin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，即阐明Tetraspanin/合作伙伴关系适用于 CD53在免疫系统功能和恶性肿瘤中的作用以及其他Tetraspanin家族的功能 会员。

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.