ELUCIDATION OF IMMUNOGLOBULIN CLASS SWITCH RECOMBINATION AND SOMATIC HYPERMUTATIO

描述（由申请人提供）：为了获得最佳的免疫应答，成熟的B淋巴细胞经历两种遗传改变，即类开关重组（CSR）和体细胞超突变（SHM）。CSR导致产生各种同型抗体（IgG, IgE, IgA），而SHM导致产生对抗原具有更高亲和力的抗体分子。B细胞特异性蛋白AID（活化诱导脱氨酶）在这两个过程中都是必不可少的。AID通过破坏免疫球蛋白位点转录区域内的胞苷来启动CSR和SHM。这些病变随后在SHM期间转化为点突变或在CSR期间作为强制性中间产物的DNA双链断裂。艾滋病病毒的失活导致人类免疫缺陷综合征（Hyper-IgM2），患者对细菌感染非常敏感。另一方面，解除对AID的管制将其转化为一般突变体，并导致与成熟B细胞淋巴瘤发生有关的癌基因的突变和易位。因此，了解调节艾滋病活动的过程是极为重要的。本研究计划的总体目标是阐明在CSR和SHM过程中AID活性受到调节的机制。最近的研究表明，蛋白激酶A （PKA）在体外磷酸化AID，以激活其结合其辅助因子、单位点DNA结合蛋白复制蛋白A的能力，并介导转录DNA底物的脱胺作用。为了阐明AID磷酸化在体内的作用，我们将生成AID磷酸化位点突变的小鼠，并对CSR和SHM进行分析。突变小鼠也将被用于细胞和生化分析，以描述磷酸化的AID在CSR和SHM中的功能。最后，我们将对现有的PKA突变体进行分析，以验证PKA对AID的磷酸化本身是一个高度调控的事件，并可能有助于AID靶向特异性的假设。本提案中概述的项目的成功完成将提供对免疫中心反应的机制见解，以及这些生理反应的异常如何导致成熟B细胞肿瘤。公共卫生相关性：B细胞淋巴瘤是最常见的人类恶性肿瘤。现在很清楚，绝大多数B细胞肿瘤是由于靶向错误的AID活性引起的。本文提出的实验将阐明AID在免疫和癌症中的作用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: B cell lymphomas are the most common human malignancies. It is now clear that a large majority of B cell tumors arise due to mistargeted AID activity. Experiments proposed here will elucidate the role of AID in both immunity and cancer.