3D Methodology for Interpreting Disease-Associated Genomic Variation in RAG2

项目总结 目前的建议旨在推进对RAG2中发现的基因组变异的解释机制。我们 利用先进的计算技术和现有的和新的实验数据，开发出一种新的 描述和解释个体间遗传变异和在患者中观察到的突变的方法 患有免疫缺陷综合症。我们的中心假设是，结构计算预测功能 通过捕捉特定的生化和分子力学特征来改变RAG2突变。我们的方法 以特定于域的方式调查RAG2突变，其中每个目标调查两个RAG2中的一个 结构域：1)我们询问β结构域突变对RAG异四聚体复合体的影响 2)我们从计算和功能上描述了植物中的突变 同源结构域改变稳定性和染色质结合能力。因此，在这两个域之间，我们将调查 RAG酶的变化及其通过差异靶向正确的位置进行调节的能力 基因组，通过其组蛋白阅读功能。我们的目标是独立的(使用不同的方法 结构域)，但由于每个结构域都提供了有关在 免疫缺陷患者。这两个领域都将以结构-动力学-功能范式为特征 以高分辨率阐明每个突变的细节，并识别具有相似突变的亚群 对RAG功能的影响。我们预计确定的亚群将用于对细胞的后续研究 影响以及如何潜在地解决每种类型的功能障碍。完成后，建议的研究将会 生成具有明确生物医学相关性的新数据，用于诊断免疫缺陷综合征并使 关于如何以不同方式处理调节RAG特定维度的每组突变的未来研究 复杂的函数。在更高的层面上，我们的建议解决了新的基因组学中尚未得到满足的广泛需求 计算方法来机械地解释人类变异的广泛图景。我们预料到 对于如何应用计算工具，这里使用的方法将推广到其他蛋白质 以稳健的方式确定潜在的蛋白质结构-功能关系，以解释 遗传病发病机制的结构生物学。因此，在这项试点奖励中产生的数据将为未来 通过目前的科学和多学科团队的应用，进一步扩大我们对RAG的理解 遗传变异及其对免疫系统的影响。

PROJECT SUMMARY The current proposal seeks to advance mechanisms of interpretation of genomic variation found in RAG2. We leverage advanced computational techniques with existing and new experimental data, to develop a novel approach for characterizing and interpreting inter-individual genetic variation, and mutations observed in patients with immunodeficiency syndromes. Our central hypothesis is that structural calculations predict functional changes for RAG2 mutations via capture of specific biochemical and molecular mechanic features. Our approach investigates RAG2 mutations in a domain-specific manner, where each Aim investigates one of the two RAG2 structured domains: 1) we interrogate the effects of β-domain mutations on the RAG heterotetramer complex across its enzymatic cycle; 2) we computationally and functionally characterize how mutations in the plant homeodomain alter stability and chromatin binding capacity. Thus, between the two domains, we will investigate alteration of the RAG enzyme and its ability to be regulated by differentially targeting to the correct places in the genome, via its histone reading function. Our Aims are independent (using different approaches for different domains), yet synergistic due to each providing new information about RAG2 mutations observed in immunodeficiency patients. Both domains will be characterized in structure-dynamics-function paradigm, to elucidate details for each mutation in high-resolution, and to identify subgroups of mutations that have similar effects on RAG function. The subgroups we anticipate identifying will serve for follow-up studies into cellular effects and how to potentially address each type of dysfunction. When completed, the proposed studies will generate new data with clear biomedical relevance for diagnosis of immunodeficiency syndromes and enabling future research in how to differently address each group of mutations that modulate specific dimensions of RAG complex function. At a higher level, our proposal addresses a broad unmet need in genomics for new computational approaches to mechanistically interpret the wide landscape of human variation. We anticipate that the approach used here, will be generalizable to other proteins for how the computational tools can be applied in a robust manner to determine the underlying protein structure-function relationship for interpreting the structural biology of genetic disease pathogenesis. The data generated in this pilot award will thereby seed future applications by the current scientific and multi-disciplinary team, to further expand our understanding of RAG genetic variation and its effects on the immune system.