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）：通过类开关重组（CSR）生成二级抗体库的形状，该抗体重组（CSR）用其他同种型代替IgM，以及允许产生高亲和力抗体的体细胞超松（SHM）。 CSR和SHM均由胞苷残基（在免疫球蛋白基因座中产生尿嘧啶）脱氨基触发。脱氨酸是由胞苷脱氨酶辅助催化的，被认为是在转录的免疫球蛋白基因上暴露于接触的和脱氨酸的ssDNA，产生了通过尿素 - n-糖基酶（UNG）的作用解决的U：G失配，以产生点突变或开关重新组合。 CSR或SHM中的缺陷与原发性免疫缺陷直接相关，其特征是缺乏开关的同型产生（Hyperigm综合征）或与异常的体细胞过度儿模（常见的可变免疫缺陷或CVID以及其他特发性体液疗法的体液性免疫免疫缺陷）有关）。已经表征了引起过度疾病的分子缺陷的一个子集。显着地，Hyperigm与AICDA基因隐性突变以及UNG功能的缺陷引起的辅助缺陷有关。它也与信号通路中的缺陷有关，这些缺陷在其他方面达到了帮助生产（例如CD40和CD40配体缺陷）。然而，文献中已经描述了许多Hyperigm患者，在这些基因座中没有缺陷。显然，AS基因的突变可能导致高成像综合征。用辅助突变体进行的基因实验导致了当前在田间的理解，许多这些未知基因将编码与AID相互作用的蛋白质。在本文中，我们描述了一个新颖的屏幕，该屏幕旨在识别辅助辅助因素，然后我们建议它以体内表征。我们的实验有可能直接发现原因未知的高素质综合征的分子基础，因此，不仅为更好地定义了这种原发性免疫缺陷的临床光谱，而且在某些情况下，还提供了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其依赖于产生针对异物物质的抗体特异性的有益突变过程很重要。该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。它的放松管制也已成为自身免疫性疾病的原因，此外，它与B细胞淋巴瘤的产生直接相关。因此，了解调节这一过程所涉及的成分不仅会发现免疫缺陷的分子缺陷（例如Hyper-IGM综合征），而且还将有助于我们更好地了解自身免疫性疾病的遗传和环境原因以及B细胞淋巴瘤的遗传和环境原因。