A novel method to identify interacting partners of insoluble proteins

描述（由申请人提供）：通过类别转换重组（CSR）和体细胞超突变（SHM）形成二抗库，类别转换重组（CSR）用其他同种型替代IgM，体细胞超突变（SHM）允许产生高亲和力抗体。CSR和SHM均由免疫球蛋白基因座内胞苷残基的脱氨基作用（产生尿嘧啶）触发。脱氨作用由胞苷脱氨酶AID催化，其被认为结合并脱氨暴露在转录的免疫球蛋白基因上的ssDNA，产生U：G错配，其通过尿嘧啶-N-糖基化酶（UNG）的作用解决以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，其特征在于缺乏转换同种型产生（高IgM综合征）或与异常体细胞超突变（常见可变免疫缺陷或CVID和其他特发性体液免疫缺陷）相关。引起高IgM疾病的分子缺陷的子集已经被表征。突出地，hyperIgM与由aicda基因的隐性突变引起的AID缺陷以及与UNG功能缺陷有关。它还与信号传导途径中的缺陷有关，这些缺陷最终导致AID的产生（如CD 40和CD 40配体缺陷）。然而，在文献中已经描述了一些高IgM患者，这些位点没有缺陷。显然，未知基因的突变可导致高IgM综合征。对AID突变体的遗传实验已经导致了该领域目前的理解，即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屏幕，其目的是确定艾滋病的辅因子，然后我们建议在体内表征。我们的实验有可能直接揭示原因不明的高IgM综合征的分子基础，因此不仅提供了更好地定义这种原发性免疫缺陷的临床谱的机会，而且在某些情况下，提示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有益的突变过程是重要的，它依赖于产生针对外来物质的抗体特异性。这种突变过程是免疫系统的核心组成部分，因此它的缺失直接涉及免疫缺陷。它的失调也是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，了解参与调节这一过程的组分不仅将揭示免疫缺陷（如高IgM综合征）的分子缺陷，而且还将帮助我们更好地了解自身免疫性疾病以及B细胞淋巴瘤的遗传和环境原因。

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.