ACTIVATION INDUCED DEAMINASE (AID) INTERACTING PROTEINS

激活诱导脱氨酶 (AID) 相互作用蛋白

基本信息

批准号：
8169094
负责人：
Michel C Nussenzweig
金额：
$ 0.23万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8169094
关键词：
Affect Alanine Antibody Formation B-Lymphocytes Computer Retrieval of Information on Scientific Projects Database DNA DNA Damage DNA Double Strand Break Deaminase Enzymes Funding Genome Stability Grant Guanine Human Immunoglobulin Class Switching Immunoglobulin Somatic Hypermutation Immunoglobulin Switch Recombination Immunoglobulin Variable Region Institution Ions Lymphocyte Manuscripts Mass Spectrum Analysis Mus Mutation Oncogenes Pathway interactions Phosphorylation Phosphorylation Site Play Point Mutation Positioning Attribute Post-Translational Protein Processing Process Proteins Publishing RNA Editing Regulation Research Research Personnel Resources Role Serine Site Somatic Mutation Source Testing Threonine United States National Institutes of Health Uracil Work activation-induced cytidine deaminase in vivo repaired

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The "Activation Induced Deaminase" (AID-protein) is potentially involved in RNA editing in human lymphocytes. In search for possible interacting proteins, that could function in the assumed recruitment of AID to specific RNAs, we have employed MALDI-ion trap MS/MS analysis to identify proteins that are co-purified with flag/HA-tagged AID. We have also identified sites of phosphorylation on AID at a site that appears to play an important role in regulating AID activity. Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching. A manuscript describing this work has been published: K.M. McBride, A. Gazumyan, E.M. Woo, T.A. Schwickert, B.T. Chait, M.C. Nussenzweig, "Regulation of class switch recombination and somatic mutation by AID phosphorylation", J Exp Med. 205 (2008) 2585-94.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目及研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是中心，不一定是研究者的机构。 “激活诱导脱氨酶”（AID 蛋白）可能参与人类淋巴细胞的 RNA 编辑。为了寻找可能的相互作用蛋白质，这些蛋白质可以在假定的 AID 招募到特定 RNA 的过程中发挥作用，我们采用 MALDI 离子阱 MS/MS 分析来鉴定与标记/HA 标记的 AID 共纯化的蛋白质。我们还鉴定了 AID 上的磷酸化位点，该位点似乎在调节 AID 活性中发挥重要作用。激活诱导胞苷脱氨酶 (AID) 是一种增变酶，通过将尿嘧啶：鸟嘌呤错配引入 DNA 来启动 B 淋巴细胞中的体细胞突变和类别转换重组。修复途径处理这些错配，在 Ig 可变区产生点突变，或在转换区 DNA 中产生双链 DNA 断裂。然而，AID 也会产生脱靶 DNA 损伤，包括癌基因突变。因此，需要对 AID 进行严格调节，以在抗体反应成熟期间维持基因组稳定性。有人提出，AID 丝氨酸 38 (S38) 的磷酸化可调节其活性，但这尚未在体内进行测试。通过结合质谱和免疫化学方法，我们发现除了 S38 之外，AID 也在苏氨酸 140 (T140) 位点被磷酸化。 S38 或 T140 突变为丙氨酸不会影响催化活性，但会干扰体内类别转换和体细胞超突变。这种效应在 AID 水平有限的单倍剂量不足的小鼠中尤其明显。尽管 S38 对这两个过程同样重要，但 T140 磷酸化优先影响体细胞突变，这表明翻译后修饰可能有助于超突变和类别转换之间的选择。描述这项工作的手稿已经出版： K.M.麦克布莱德 (A. Gazumyan)、E.M. 吴 (T.A.)施维克特，B.T.柴特，M.C. Nussenzweig，“AID 磷酸化对类别转换重组和体细胞突变的调节”，J Exp Med。 205（2008）2585-94。