ELUCIDATION OF IMMUNOGLOBULIN CLASS SWITCH RECOMBINATION AND SOMATIC HYPERMUTATIO

项目总结 为了获得最佳的免疫反应，成熟的B淋巴细胞在体内经历了两次基因改变 类切换重组(CSR)和体细胞超突变(SHM)的形式。企业社会责任导致生产 不同同种类型的抗体(Ig G、Ig E、Ig A)，而SHM导致产生具有 对抗原的亲和力要高得多。B细胞特异性蛋白AID(激活诱导脱氨酶)是 这两个过程。AID通过在转录区域内脱氨胞苷来启动CSR和SHM 免疫球蛋白基因座。这些损伤在SHM过程中被转化为点突变或DNA加倍 在企业社会责任过程中作为强制性中间体的断链。艾滋病灭活导致人类 免疫缺陷综合征(Hyper-IgM2)患者对细菌高度敏感 感染。另一方面，解除对艾滋病的管制会将其转化为通用的突变子，并导致突变和 与成熟B细胞淋巴瘤有关的癌基因易位。一种理解 因此，控制艾滋病活动的进程是至关重要的。这项研究的总体目标是 建议阐明在CSR和SHM过程中AID活性的调节机制。近期 研究表明，蛋白激酶A(PKA)在体外磷酸化AID，激活其与其结合的能力 辅因子、单链DNA结合蛋白复制蛋白A及其介导的转录产物脱氨基 DNA底物。为了阐明AID磷酸化在体内的作用，将AID基因突变的小鼠 将生成并分析CSR和SHM的磷酸化位点。突变的小鼠也将用于 细胞和生化分析以阐明磷酸化AID在CSR和SHM中的作用。最后， 将对现有的PKA突变体进行分析，以检验PKA使AID磷酸化本身是一种高度 受调控的事件，并可能潜在地有助于AID靶标的特异性。项目圆满完成 这项提案中概述的将提供对免疫核心反应的机械性洞察以及如何 这种生理反应中的异常可导致成熟的B细胞肿瘤。

PROJECT SUMMARY To mount an optimum immune response, mature B lymphocytes undergo two genetic alterations in the forms of class switch recombination (CSR) and somatic hypermutation (SHM). CSR leads to the production of antibodies of various isotypes (IgG, IgE, IgA) while SHM results in the generation of antibody molecules with a much higher affinity for antigens. The B cell specific protein AID (activation induced deaminase) is essential to both processes. AID initiates CSR and SHM by deaminating cytidines within transcribed regions of the immunoglobulin locus. These lesions are then converted into point mutations during SHM or into DNA double strand breaks that serve as obligatory intermediates during CSR. Inactivation of AID leads to human immunodeficiency syndromes (Hyper-IgM2) where patients suffer from profound susceptibility to bacterial infections. On the other hand, deregulation of AID converts it into a general mutator and leads to mutations and translocations of oncogenes that have been implicated in mature B cell lymphomagenesis. An understanding of the processes that regulate AID activity is thus of utmost importance. The overall objective of this research proposal is to elucidate the mechanism by which AID activity is regulated during CSR and SHM. Recent studies have shown that protein kinase A (PKA) phosphorylates AID in vitro to activate its ability to bind its cofactor, the single-stand DNA binding protein Replication Protein A and mediate deamination of transcribed DNA substrates. To elucidate the role of AID phosphorylation in vivo, mice with a mutation in the AID phosphorylation site will be generated and analyzed for CSR and SHM. The mutant mouse will also be used in cellular and biochemical assays to delineate the function of phosphorylated AID in CSR and SHM. Finally, existing PKA mutants will be analyzed to test the hypothesis that AID phosphorylation by PKA is itself a highly regulated event and could potentially contribute to AID target specificity. Successful completion of the projects outlined in this proposal will provide mechanistic insights into reactions central to immunity and how aberrations in such physiological reactions can cause mature B cell tumors.