Project 2 - Verification and Molecular Mechanisms of T1D Modifier Mutations

项目2-T1D修饰突变的验证和分子机制

• 批准号: 10642554
• 负责人: BRUCE A BEUTLER
• 金额: $ 34.03万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-13 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642554
• 关键词: Acceleration; Actins; Affect; Alleles; Animals; Apoproteins; Apoptotic; Attention; B-Lymphocytes; Beta Cell; Binding; Biological Assay; CRISPR/Cas technology; CTLA4 gene; Candidate Disease Gene; Cell Separation; Cell Survival; Cell physiology; Cells; Communication; Communities; Complex; Crystallization; DNase-I Footprinting; Data; Development; Diabetes Mellitus; Disease; Disease remission; Educational process of instructing; Engineering; Epitopes; Ethylnitrosourea; Etiology; Exclusion; Funding; Genes; Genetic; Human; Immune; In Vitro; Induced Mutation; Insulin-Dependent Diabetes Mellitus; Investigation; Islets of Langerhans; Knock-out; Knowledge; Lead; Location; Maps; Mass Spectrum Analysis; Measures; Meiosis; Methodology; Methods; Microfilaments; Missense Mutation; Molecular; Molecular Sieve Chromatography; Mus; Mutagens; Mutant Strains Mice; Mutation; N-terminal; Pathogenesis; Patients; Peptides; Phase; Phenotype; Post-Translational Protein Processing; Prevention; Protein Analysis; Protein Isoforms; Protein Overexpression; Proteins; Proteomics; Recovery; Recreation; Signal Pathway; Signal Transduction; Splice-Site Mutation; Suppressor Mutations; T cell receptor repertoire sequencing; T cell response; T-Cell Depletion; T-Lymphocyte; T-cell receptor repertoire; Testing; Therapeutic; Thymus Gland; Time; Tissues; Tumor Necrosis Factor Receptor; Untranslated RNA; Work; autoreactivity; cell motility; cell type; conditional knockout; epigenetic regulation; genetic pedigree; interest; knockout gene; mutant; novel; phosphoproteomics; protein complex; protein function; protein protein interaction; protein purification; response; synergism; transcriptomics

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.

项目总结/摘要 项目2将验证，然后研究的表型创造和遗传解决的分子机制， 核心B和核心C。我们希望在资助期限内发现大量具有修饰效应的突变， 我们不会机械地全部研究。在此，我们描述了选择突变的关键标准， 研究并建议开始于我们初步工作中鉴定为T1 D修饰物的四种突变（影响 Dusp 10、Paqr 8、Xpnpep 1和Rapgef 1）。我们将通过重新靶向NOD/NckH和/或NckH中的候选基因来进行。 使用CRISPR/Cas9的NOD/NckL背景来创建种系敲除等位基因或替换等位基因（精确的 ENU诱导的等位基因的重建）或两者，以验证或排除因果关系。一旦验证，编码的 蛋白质将以其突变体和WT形式进行研究，以确定与它们相关的其他蛋白质， 突变可能对这种关联产生的影响，以及对细胞信号传导的影响。互动将是 直接在已知因突变而改变功能的细胞中进行测试（基于Project 1）。将使用从小鼠分离的原代细胞进行质谱下拉，其中我们已经 在一些实施方案中，本发明的方法包括在种系中的WT和/或突变蛋白的工程化表位标记形式。核查直接和 间接相互作用将取决于靶蛋白和HEK中推定的相互作用物的相互下拉 293细胞我们将确定直接相互作用，使用纯化的蛋白质和GST下拉研究在体外，以及 如尺寸排阻色谱法。将根据需要进行结构研究，以准确确定 每一个相关的蛋白质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查： 怀疑信号异常。在许多情况下，这些研究将揭示 加速或抑制T1 D。经常与项目1交流我们的发现，反之亦然， 相互通报两个项目的研究，从而了解和发展最好的 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 以相对微妙的方式。然而，在每一种情况下，都会对T1 D的发展产生非常重要的影响。 特别是在强和新的抑制突变的情况下，我们希望确定治疗的机会 用于预防或减轻人类患者的T1 D。项目2将建设可诱导的 条件等位基因来测试这种治疗机会，立即进行基因敲除或转换 在T1 D发作后，以确定是否可以建立缓解。这表明 在T1 D的蜜月期治疗人类患者。我们将收集所有修饰突变的数据 可通过Mutagenetix Candidate Explorer门户网站公开获取。CRISPR/Cas9等位基因（如ENU- 诱导的等位基因）将通过MMRRC获得。