ELUCIDATION OF IMMUNOGLOBULIN CLASS SWITCH RECOMBINATION AND SOMATIC HYPERMUTATIO

描述（由申请方提供）：为了产生最佳免疫应答，成熟B淋巴细胞经历类别转换重组（CSR）和体细胞超突变（SHM）形式的两种遗传改变。CSR导致产生各种同种型（IgG、IgE、伊加）的抗体，而SHM导致产生对抗原具有高得多的亲和力的抗体分子。B细胞特异性蛋白AID（活化诱导脱氨酶）对这两个过程都是必不可少的。AID通过使免疫球蛋白基因座的转录区域内的胞苷脱氨基来启动CSR和SHM。然后这些病变在SHM期间转化为点突变，或转化为DNA双链断裂，其在CSR期间充当强制性中间体。AID的失活导致人类免疫缺陷综合征（Hyper-IgM 2），其中患者遭受对细菌感染的高度易感性。另一方面，AID的失调将其转化为一般的增变因子，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，了解规范援助活动的程序至关重要。本研究的总体目标是阐明在CSR和SHM期间调节AID活性的机制。最近的研究表明，蛋白激酶A（PKA）在体外磷酸化AID以激活其结合其辅因子（单链DNA结合蛋白复制蛋白A）的能力，并介导转录的DNA底物的脱氨基作用。为了阐明AID磷酸化在体内的作用，将产生在AID磷酸化位点中具有突变的小鼠并分析CSR和SHM。突变小鼠也将用于细胞和生物化学测定，以描述磷酸化AID在CSR和SHM中的功能。最后，现有的PKA突变体将进行分析，以测试这一假设，即由PKA的AID磷酸化本身是一个高度调节的事件，并可能有助于AID靶特异性。本提案中概述的项目的成功完成将提供对免疫反应的机制性见解，以及这些生理反应中的畸变如何导致成熟的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，大多数B细胞肿瘤是由于AID活性的错误设定而产生的。这里提出的实验将阐明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.