A novel method to identify interacting partners of insoluble proteins

一种识别不溶性蛋白质相互作用伙伴的新方法

• 批准号: 7640354
• 负责人: F. NINA Papavasiliou
• 金额: $ 25.3万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7640354
• 关键词: Antibody Affinity; Antibody Repertoire; Antibody Specificity; Autoimmune Diseases; B-Cell Lymphomas; B-Lymphocytes; Bacteria; Binding; CD40 Ligand; Clinical; Cytidine; Cytidine Deaminase; DNA Double Strand Break; DNA lesion; Deamination; Defect; Diagnostic; Disease; Failure; Generations; Genes; Genetic; Genomic Instability; Genomics; Immune; Immune system; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Immunologic Deficiency Syndromes; Knowledge; Lead; Link; Literature; Lymphoid; Lymphomagenesis; Messenger RNA; Methods; Molecular; Mutation; Oncogenes; Oncogenic; Patients; Play; Point Mutation; Process; Production; Proteins; RNA Splicing; Reaction; Role; Screening procedure; Shapes; Signal Pathway; Structure of germinal center of lymph node; Syndrome; TNFRSF5 gene; Therapeutic; Time; Uracil; base; in vivo; mutant; novel; public health relevance; repaired; research study; tumorigenesis; uracil-DNA glycosylase

DESCRIPTION (provided by applicant): Shaping of the secondary antibody repertoire is generated by means of class- switch recombination (CSR), which replaces IgM with other isotypes, and somatic hypermutation (SHM), which allows production of high-affinity antibodies. Both CSR and SHM are triggered by deamination of cytidine residues (to yield uracil) within the immunoglobulin locus. Deamination is catalyzed by the cytidine deaminase AID, which is thought to bind and deaminate ssDNA exposed on the transcribed immunoglobulin gene, generating U:G mismatches that are resolved via the action of Uracil-N- glycosylase (UNG) to generate either point mutations or switch recombination. Defects in CSR or SHM are directly associated with primary immunodeficiencies, characterized either by a lack of switched isotype production (hyperIgM syndrome) or associated with abnormal somatic hypermutation (Common Variable Immune Deficiency or CVID, and other idiopathic humoral immunodeficiencies). A subset of the molecular defects that give rise to hyperIgM disease have been characterized. Prominently, hyperIgM has been linked with AID deficiency caused by recessive mutations of the aicda gene as well as with defects in UNG function. It has also been linked with defects in the signaling pathway that culminate in, amongst other things, AID production (such as CD40 and CD40 ligand defects). However, a number of hyperIgM patients have been described in the literature, with no defects in these loci. Clearly, mutations of as yet unknown genes can result in hyperIgM syndrome. Genetic experiments with AID mutants has led to a current understanding in the field, that a number of these unknown genes will encode proteins which interact with AID. Herein we describe a novel screen which aims to identify AID co-factors, which we then propose to characterize in vivo. Our experiments have the potential to directly uncover the molecular basis of hyperIgM syndromes for which the cause is unknown, hence offering the opportunity not only to better define the clinical spectrum of this primary immunodeficiency but also, in certain cases, to prompt better diagnostic and therapeutic approaches. PUBLIC HEALTH RELEVANCE: The experiments proposed here are important for determining how our immune system controls the beneficial mutation process upon which it depends to generate antibody specificities against foreign substances. This mutational process is a central component of the immune system and as such its absence has been directly implicated in immune deficiencies. Its deregulation has also been a cause of autoimmune diseases, and furthermore, it has been directly linked to the generation of B cell lymphomas. Therefore understanding the components involved in regulating this process will not only uncover the molecular defects underlying immune deficiencies (such as hyper-IgM syndrome) but will also help us gain better knowledge of the genetic and environmental causes of autoimmune diseases as well as B cell lymphomagenesis.

描述（由申请人提供）：二抗库的形成是通过类开关重组（CSR）和体细胞超突变（SHM）产生的，CSR用其他同型替代IgM，体细胞超突变（SHM）允许产生高亲和力抗体。CSR和SHM都是由免疫球蛋白位点胞苷残基脱胺（产生尿嘧啶）引发的。胞苷脱氨酶AID催化脱氨，该酶被认为结合并脱氨暴露在转录免疫球蛋白基因上的ssDNA，产生U:G错配，通过尿嘧啶- n -糖基化酶（UNG）的作用解决，产生点突变或开关重组。CSR或SHM的缺陷与原发性免疫缺陷直接相关，其特征要么是缺乏开关同型产生（高igm综合征），要么是与异常体细胞高突变（常见可变免疫缺陷或CVID，以及其他特发性体液免疫缺陷）相关。引起高igm疾病的分子缺陷的一个子集已经被表征。值得注意的是，hyperIgM与aida基因的隐性突变引起的AID缺陷以及UNG功能缺陷有关。它还与信号通路中的缺陷有关，这些缺陷最终导致AID的产生（如CD40和CD40配体缺陷）。然而，文献中描述的一些hyperIgM患者在这些位点上没有缺陷。显然，未知基因的突变可导致高igm综合征。对AID突变体的遗传实验使该领域目前的理解是，许多这些未知基因将编码与AID相互作用的蛋白质。在这里，我们描述了一种新的筛选，旨在识别AID辅助因子，然后我们提出在体内表征。我们的实验有可能直接揭示原因未知的高igm综合征的分子基础，因此提供了机会，不仅可以更好地定义这种原发性免疫缺陷的临床谱，而且在某些情况下，可以促进更好的诊断和治疗方法。公共卫生相关性：这里提出的实验对于确定我们的免疫系统如何控制它所依赖的有益突变过程以产生针对外来物质的抗体特异性非常重要。这种突变过程是免疫系统的核心组成部分，因此它的缺失直接与免疫缺陷有关。它的放松也是自身免疫性疾病的一个原因，而且，它与B细胞淋巴瘤的产生直接相关。因此，了解参与调节这一过程的成分不仅可以揭示免疫缺陷（如高igm综合征）的分子缺陷，还可以帮助我们更好地了解自身免疫性疾病和B细胞淋巴瘤发生的遗传和环境原因。