激酶锚定蛋白AKAP8促进AID介导抗体类别转换的机制研究

Activation-induced cytidine deaminase (AID) mediates class switch recombination (CSR) and plays key roles in producing multiple diversity of antibodies in B cells. With the helps from many cofactors of AID, phosphorylated AID by Protein kinase A (PKA) is recruited to the repetitive G-rich Switch region of immunoglobulin genes and mediates double-strand breaks. However, whether A-kinase anchor proteins (AKAP) members as a cofactor involve in CSR process has yet been studied. Through data analysis and RNAi screening, we identified that AKAP8 is a novel factor which promotes CSR. Further experiments suggested that CSR promotion by AKAP8 is independent of typical PKA anchoring and probable through its association with AID. Basing of functional analysis and interaction protein clustering, AKAP8 is found to enhance histone modification, recognize G-rich sequences, and interact with RNA processing components. It is reasonable to speculate that AKAP8 may specifically promote the recruitment of AID in the Switch region, which in turn enhances CSR. In this project, we design a serial of experiments to study in depth the effects of AKAP8 on the histone modification and formation of specific structures such as R-loop in the Switch region, and dissect the molecular basis of AID recruiting to Switch regions promoted by AKAP8. Finally, we aim to reveal the functional characteristics of AKAP8 in promoting AID mediated CSR and uncover the correlated mechanism from multiple aspects. The project will also benefit the studies on diseases induced by AID off-targeting.

激活诱导胞嘧啶脱氨酶（AID）介导抗体类别转换（CSR），是B细胞产生多样性抗体的关键因子。AID被激酶磷酸化，在众多辅因子调控下，介导Ig基因Switch区双链断裂。申请人通过表型筛选，鉴定得到影响CSR的新因子激酶锚定蛋白AKAP8。进一步实验显示，AKAP8促进CSR不依赖典型的激酶锚定作用，但与AID存在互作。通过AKAP8功能分析，发现其能够增强组蛋白修饰，与RNA代谢加工组分存在互作关系，并识别G-rich序列。据此推测：AKAP8可能特异性调控Switch区AID的招募，进而影响CSR。本课题拟采用系列技术手段，深入研究AKAP8在Switch区组蛋白修饰和R-loop等特征结构形成的作用，并解析AKAP8促进Switch区招募AID的分子基础，从多层面揭示AKAP8调节AID介导CSR关键过程的功能特点，同时为AID脱靶向诱发疾病的研究提供信息。

本项目源于探索关键激酶锚定蛋白家族成员对激活诱导胞嘧啶脱氨酶AID介导抗体类别转换的影响研究课题。AID在B淋巴细胞中诱导表达，是产生多样性抗体的关键因子。AID异常激活时，可脱靶向导致Ig-非Ig基因转位、癌突变及基因组不稳定，是诱发骨髓瘤等癌症发生的重要因素之一。本项目针对筛选获得影响类别转换表型的AKAP8蛋白，采用了系列分子生物学手段，证实AKAP8通过结合R-loop区，调控免疫球蛋白基因Switch区R-loop形成机制，从抗体类别转换模型出发，解释AKAP8在调控R-loop的分子机制。.本项目证实关键蛋白AKAP8促进AID介导类别转换重组，但是不依赖于AKAP家族激酶锚定活性，而是通过促进AID表达水平实现。同时发现AKAP8在免疫球蛋白基因有富集，后续证明AKAP8能够结合R-loop区，并于R-loop结合蛋白存在互作。本项目初步揭示AKAP8通过调控R-loop区促进免疫球蛋白GLTs转录及AID表达水平，增强AID介导类别转换重组，并尝试探索AKAP8在作用于R-loop区调控机制，为深入探究AKAP8分子功能提供了实验基础。同时，利用本项目资助，验证了干细胞中AKAP8与R-loop、R-loop相关蛋白的结合，及R-loop相关蛋白在外泌体中的作用。综上，本项目从调控R-loop功能的角度，发现AKAP8新功能及调控特征，为深入研究AKAP8在抗体类型转换和R-loop结构调节提供理论和实验基础。