Defining the molecular mechanisms of COPA syndrome through computational modeling and functional studies

项目总结： 我们的实验室最近发现了COPA综合征，这是一种影响肺部和关节的自身免疫性疾病 由辅酶A亚单位(COPA)基因的显性突变引起。COPA综合征通常表现为 患有炎症性关节炎或表现为肺毛细管炎或间质性肺疾病的儿童 肺部疾病。虽然基因检测是用来确认诊断的，但在测序时可能会产生不确定性 发现未知意义的COPA变种(VU)。这种情况发生得比较频繁，因为经过验证 突变存在于~50个碱基对(BP)区域，该区域跨度不到该基因完整编码序列的1%。 COPA是外壳蛋白复合体I(COPI)的一个亚单位，它介导蛋白质从 高尔基体至内质网(ER)。已建立的COPA突变改变了蛋白质的WD40 通过C-末端二赖氨酸基序标记的内质网检索标记的COPA与货运蛋白的结构域和损伤结合。在……里面 这笔赠款，我们将使用分子动力学模拟和特征向量中心性分析来解决如何 突变改变COPA的变构结合部位并预测新的突变。我们将从功能上验证 基于我们最近发现的突变体CopA通过互补的实验方法 损害从高尔基体针刺的恢复，并导致I型干扰素信号。我们已经确定了几个 在需要进一步功能分析的患者中存在意义未知的COPA变体，并已被 世界各地的临床医生与VUS联系，让我们研究。我们将寻找新奇的COPA变体 通过对在PedVas倡议注册中心登记的受试者进行完整的外显子组测序，最大的队列 世界上最大的儿童脉管炎患者。将使用以下筛选标准确定受试者的排序 在最近发表的一项国际队列中描述了常见的肺和肺外特征 COPA综合征患者。在没有COPA变异的受试者中，我们将寻找可能导致 COPA综合征基于与疾病有关的细胞和分子途径。 我们的资助将使用计算建模和功能研究来提供对基本 通过分析致病突变如何破坏COPA与C-末端的结合来研究COPA的作用机制 货物的双胞胎图案。这项工作将使我们能够准确地预测突变是否会导致COPA综合征 并有可能扩大这种疾病的分子定义。我们将进行完整的外显子组测序 在疑似患有COPA综合征的患者中，识别新的致病COPA变体或识别新的 导致炎症性肺和关节疾病的基因。

PROJECT SUMMARY: Our lab recently discovered COPA syndrome, an autoimmune disease affecting the lungs and joints caused by dominant mutations in the coatomer subunit alpha (COPA) gene. COPA syndrome typically presents in childhood with either inflammatory arthritis or lung disease that manifests as pulmonary capillaritis or interstitial lung disease. Although genetic testing is used to confirm the diagnosis, uncertainty can arise when sequencing uncovers COPA variants of unknown significance (VUS). This occurs relatively frequently because verified mutations lie within a ~50 base pair (bp) region that spans less than 1% of the full coding sequence of the gene. COPA is a subunit of coat protein complex I (COPI) that mediates retrograde movement of proteins from the Golgi apparatus to the endoplasmic reticulum (ER). Established COPA mutations alter the protein’s WD40 domain and impair binding of COPA to cargo proteins marked for ER retrieval by a C-terminal dilysine motif. In this grant, we will employ molecular dynamics simulations and eigenvector centrality analysis to resolve how mutations alter the allosteric binding site of COPA and to predict novel mutations. We will functionally validate mutants through complementary experimental approaches based on our recent discovery that mutant COPA impairs retrieval of STING from the Golgi and leads to type I interferon signaling. We have identified several COPA variants of unknown significance in patients that require further functional analysis and have been contacted by clinicians throughout the world with VUS for us to study. We will search for novel COPA variants by performing whole exome sequencing in subjects enrolled in the PedVas initiative registry, the largest cohort of pediatric vasculitis patients in the world. Subjects will be identified for sequencing using screening criteria of common pulmonary and extra-pulmonary features delineated in a recently published international cohort of COPA syndrome patients. In subjects without COPA variants, we will search for novel genes suspected to cause COPA syndrome based on the cellular and molecular pathways implicated in disease. Our grant will use computational modeling and functional studies to provide insight into the basic mechanisms of COPA function by analyzing how pathogenic mutations impair binding of COPA to the C-terminal dilysine motif of cargo. This work will allow us to accurately predict whether a mutation causes COPA syndrome and has the potential to expand the molecular definition of the disease. We will perform whole exome sequencing in patients suspected to have COPA syndrome to identify new disease-causing COPA variants or identify novel genes that lead to inflammatory lung and joint disease.