Project 2 - Verification and Molecular Mechanisms of T1D Modifier Mutations

项目摘要/摘要 项目2将验证并研究创造和遗传解决的表型的分子机制 核心B和核心C我们预计会在资助期限内发现数十个具有修饰性效果的突变，但 我们不会机械地研究所有这些问题。在这里，我们描述了用于选择突变的关键标准 研究并建议从我们的初步工作中确定的四个T1D修饰性突变开始(影响 Dusp10、Paqr8、XpnPep1和Rapgef1)。我们将通过重新定位NOD/Nck H和/或候选基因来继续 使用CRISPR/Cas9创建生殖系基因敲除等位基因或替换等位基因的NOD/NockL背景(精确 ENU诱导的等位基因的重新创建)或两者兼而有之，以验证或排除因果关系。一旦验证，编码后的 将研究突变和WT形式的蛋白质，以确定与它们相关的其他蛋白质， 突变可能会对这种联系产生影响，也会对细胞信号产生影响。互动将是 直接在已知由于突变而改变功能的细胞中进行测试(基于Project的研究 1)。将使用从小鼠中分离出的原代细胞进行质谱下拉 生殖系中WT和/或突变蛋白的工程表位标记版本。DIRECT和DIRECT 间接相互作用将取决于目标蛋白和HEK中可能的相互作用因子的相互作用 293个细胞。我们还将使用纯化的蛋白质和体外GST下拉研究来确定直接相互作用 作为尺寸排除层析。将根据需要进行结构研究，以准确确定 每个牵连的蛋白质都与其结合伙伴相互作用，以及ENU诱导的突变如何改变这一点 互动。突变对细胞蛋白质翻译后修饰的影响将在以下情况下进行检测 怀疑信号异常。这些研究将在许多情况下揭示 加速或抑制T1D。经常将我们的发现与项目1进行沟通，反之亦然 在两个项目中相互通报研究，结果是了解和发展最好的 候选人。我们的结论可能指向影响免疫或β细胞功能的信号通路的改变 以一种相对微妙的方式。然而，在每一种情况下，都会对T1D的发展产生非常显著的影响。 特别是在强而新颖的抑制子突变的情况下，我们希望找出治疗的机会 用于预防或缓解人类患者的T1D。项目2将承建诱导式 测试这种治疗机会的条件等位基因，立即建立基因敲除或转换 在T1D发作后，以确定是否可以建立缓解。这将指向潜在的 人类患者在T1D蜜月期的治疗我们将提供所有修饰基因突变的数据 可通过Mutagenetix候选资源管理器门户网站公开获取。CRISPR/Cas9等位基因(如ENU- 诱导等位基因)将通过MMRRC获得。

PROJECT SUMMARY/ABSTRACT Project 2 will verify and then study the molecular mechanisms of phenotypes created and genetically solved in Core B and Core C. We expect to find scores of mutations with modifier effects over the term of funding, but we will not study all of them mechanistically. Herein we describe the key criteria used in selecting mutations to study and propose to begin with four mutations identified as T1D modifiers in our preliminary work (affecting Dusp10, Paqr8, Xpnpep1, and Rapgef1). We will proceed by re-targeting candidate genes in NOD/NckH and/or NOD/NckL backgrounds using CRISPR/Cas9 to create germline knockout alleles or replacement alleles (exact re-creations of the ENU-induced alleles) or both, to verify or exclude causation. Once verified, the encoded proteins will be studied in their mutant and WT forms to identify other proteins with which they associate, what effects the mutation might have on such associations, and also effects on cell signaling. Interactions will be directly tested in cells known to have altered function as a result of the mutation (based on studies from Project 1). Mass spectrometry pulldown will be performed using primary cells isolated from mice in which we have engineered epitope-tagged versions of the WT and/or mutant proteins in the germline. Verification of direct and indirect interactions will depend upon reciprocal pulldown of the target protein and putative interactors in HEK 293 cells. We will identify direct interactions using purified proteins and GST pulldown studies in vitro, as well as size-exclusion chromatography. Structural studies will be performed as warranted to determine precisely how each implicated protein interacts with its binding partners, and how the ENU-induced mutation alters this interaction. The effects of mutations on post-translational modification of cellular proteins will be examined when signaling abnormalities are suspected. These studies will, in many cases, reveal the molecular basis of acceleration or suppression of T1D. Frequent communication of our findings with Project 1, and vice versa, will mutually inform studies in both Projects, resulting in the understanding and development of the very best candidates. Our conclusions may point to alterations of signaling pathways affecting immune or β-cell function in a relatively subtle way. Yet in each case, there will be a highly significant effect on T1D development. Particularly in the case of strong and novel suppressor mutations, we hope to identify therapeutic opportunities for prevention or mitigation of T1D in human patients. Project 2 will undertake construction of inducible conditional alleles to test such therapeutic opportunities, instituting gene knockout or conversion immediately after the onset of T1D to determine whether remission may be established. This would point to the potential treatment of human patients during the honeymoon phase of T1D. We will make data on all modifier mutations publicly available via the Mutagenetix Candidate Explorer portal. The CRISPR/Cas9 alleles (like the ENU- induced alleles) will be available via the MMRRC.