A novel method to identify interacting partners of insoluble proteins

描述(由申请人提供)：二级抗体谱系的形成是通过类别切换重组(CSR)和体细胞高突变(SHM)产生的，CSR用其他同种类型取代IgM，体细胞高突变允许产生高亲和力抗体。CSR和SHM都是由免疫球蛋白基因内胞苷残基的脱氨基(以产生尿嘧啶)触发的。脱氨是由胞苷脱氨酶AID催化的，它被认为与转录的免疫球蛋白基因上暴露的单链DNA结合并脱氨，产生U：G错配，这些错配通过尿嘧啶-N-糖基酶(UNG)的作用而解决，从而产生点突变或开关重组。CSR或SHM的缺陷与原发免疫缺陷直接相关，其特征是缺乏转换的同型产物(高IgM综合征)或与异常的躯体超突变相关(常见变量免疫缺陷或CVID，以及其他特发性体液免疫缺陷)。导致高IgM病的分子缺陷的一个子集已经被表征。值得注意的是，高IgM与AICDA基因隐性突变引起的AID缺乏症以及UNG功能缺陷有关。它还与信号通路中的缺陷有关，最终导致AID的产生(如CD40和CD40配体缺陷)。然而，文献中已经描述了一些高IgM患者，这些基因没有缺陷。显然，未知基因的突变可能导致高IgM综合征。对AID突变体进行的遗传实验导致了该领域目前的一种理解，即一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，其目的是识别AID的辅助因素，然后我们建议在活体中对其进行表征。我们的实验有可能直接发现病因不明的高IgM综合征的分子基础，从而不仅提供了更好地定义这种初级免疫缺陷的临床谱系的机会，而且在某些情况下，还提供了更好的诊断和治疗方法。与公共健康相关：这里提出的实验对于确定我们的免疫系统如何控制有益的突变过程非常重要，它依赖于这个过程来产生针对外来物质的抗体特异性。这一突变过程是免疫系统的中心组成部分，因此它的缺失直接导致了免疫缺陷。它的放松调控也是自身免疫性疾病的一个原因，而且，它与B细胞淋巴瘤的发生有直接联系。因此，了解参与调节这一过程的成分不仅将揭示免疫缺陷(如高IgM综合征)的分子缺陷，还将有助于我们更好地了解自身免疫性疾病以及B细胞淋巴肿大的遗传和环境原因。

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.