Antigens of the RNA-induced silencing complex in autoimmunity

描述(由申请人提供)：P.I.和合作者的实验室是第一个发现被称为GW小体(GWB)的新的细胞质病灶，目前已知GW小体富含在RNA干扰(RNAi)途径的效应臂中重要的人类自身抗原。最初的鉴定是通过使用含有GW182抗体的人自身免疫血清的表达克隆实现的。GW182是一种182KD的蛋白自身抗原，具有独特的甘氨酸-色氨酸(GW)重复序列，定位于GWBS。GW182与特定的信使RNA(MRNAs)和Ago2蛋白相关，Ago2蛋白是RNAi介导的mRNA加工过程中的关键酶。我们最近的数据表明，短干扰RNAs(SiRNAs)和microRNAs(MiRNAs)定位于GWB，有效的RNAi功能需要完整的GWB。综上所述，工作假说是GWB是通过依赖siRNA/miRNA的RNAi过程通过维持稳定和/或控制mRNA的降解来调节细胞质mRNA水平的新位点。到目前为止，已知人类抗GWB自身抗体具有重叠的亚群，识别GW182、AGO2和其他自身抗原，这些自身抗原是中国人民解放军S实验室的特征。基于将抗GW182自身抗体与系统性红斑狼疮(SLE)和干燥综合征(SjS)这两种自身免疫性疾病相关联的初步数据，设计了三个特定的目标来帮助进一步表征GWBS在正常和系统性风湿性疾病中的生物学意义。随着小涎腺组织中GW182的过度表达和抗GW182阳性自身免疫血清中抗52kD SSA/Ro自身抗体的共同表达，人们对SjS的兴趣进一步加深。这项研究的目的是探索这些新的亚细胞病灶的生物学功能，这可能有助于了解它们在SLE和SjS发病机制中的作用。具体目标1将解决在GWB中发现的GW182、Ago2和其他密切相关的自身抗原的生物学问题。具体目标2将在已建立的自身免疫性疾病小鼠模型中表征抗GWB抗体，并测试对GWB的自身免疫反应与靶器官GWB表达的解除调控可能导致RNAi缺陷的假设。特指目标3将描述抗GWB抗体在人类疾病中的意义，并追踪已知的SjS和SLE的主要自身抗原GW182和SS-B/La在SjS唾液腺活检病灶中过表达的初步数据。建议的研究将有助于确定GWBS在SLE和SjS发病机制中的生物学作用。动物模型将提供测试与诱导这些针对GWB的自身抗体相关的假说的方法。最近已经描述了miRNA在癌症中的意义，这项拟议的研究将讨论miRNA放松调控是否可能在自身免疫性疾病中发挥作用。

DESCRIPTION (provided by applicant): The P.I. and collaborator's laboratories were the first to identify novel cytoplasmic foci known as GW bodies (GWBs), which are now known to be enriched with human autoantigens important in the effector arm of the RNA interference (RNAi) pathway. The initial characterization was achieved via expression cloning using a human autoimmune serum containing antibody to GW182, an 182KD protein autoantigen with unique glycine-tryptophan (GW) repeats that is specifically localized to GWBs. GW182 was shown to be associated with selected messenger RNAs (mRNAs) and the protein Ago2 which is the key enzyme in the RNAi mediated processing of mRNA. Our recent data showed that short interference RNAs (siRNAs) and microRNAs (miRNAs) are localized to GWBs and effective RNAi function requires intact GWBs. Taken together, the working hypothesis is that GWBs are novel sites regulating cytoplasmic mRNA levels via the siRNA/miRNA dependent RNAi process by maintaining stability and/or controlling degradation of mRNA. To date, human autoantibodies to GWBs are known to have overlapping subsets that recognize GW182, Ago2, and other autoantigens being characterized in the P.l.'s lab. Three Specific Aims have been designed to help further characterize the biological significance of GWBs in normal and in systemic rheumatic diseases including systemic lupus erythematosus (SLE) and Sjogren's syndrome (SjS) based on the preliminary data linking anti-GW182 autoantibody to these 2 autoimmune diseases. The interest in SjS is further developed with the observed overexpression of GW182 in certain foci of SjS minor salivary gland biopsies and the co- expression of anti-52kD SSA/Ro autoantibody in anti-GW182 positive autoimmune sera. The goal of the proposed study is to explore the biological functions of these novel subcellular foci which may shed some insight as to their involvement in the pathogenesis of SLE and SjS. Specific Aim 1 will address the biology of GW182, Ago2 and other closely associated autoantigens detected in GWBs. Specific Aim 2 will characterize anti-GWB antibodies in established mouse models of autoimmune diseases and test the hypothesis that autoimmune responses to GWBs are related to deregulation of GWB expression in target organs possibly resulting in defects in RNAi. Specific Aim 3 will characterize the significance of anti-GWB antibodies in human diseases and follow up on the preliminary data that GW182 and SS-B/La, a known major autoantigen in SjS and SLE, were overexpressed in foci of SjS salivary gland biopsies. The proposed studies will help define the biological role of GWBs in the pathogenesis of SLE and SjS. The animal models will provide the means to test hypotheses that are relevant to the induction of these autoantibodies targeting GWBs. Implication for mi RNA in cancer has been described recently and this proposed study will address whether there may be a role for miRNA deregulation in autoimmune diseases.