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.

项目摘要/摘要 项目2将验证并研究产生和遗传求解的表型的分子机制 核心B和CoreC。我们希望在资金期限内找到具有修饰效应的数十个突变，但是 我们不会机械地研究它们。本文中，我们描述了选择突变的关键标准 研究和提议首先要在我们的初步工作中被确定为T1D修饰符的四个突变（影响） DUSP10，PAQR8，XPNPEP1和RAPGEF1）。我们将通过重新定位点头/NCKH和/或重新定位候选基因 使用CRISPR/CAS9来创建种系淘汰等位基因或替换等位基因的点头/NCKL背景（确切 重新创建ENU诱导的等位基因）或两者兼而有之，以验证或排除原因。一旦验证，编码 蛋白质将在其突变体和WT形式中进行研究，以识别与之关联的其他蛋白质，什么 突变可能对此类关联产生影响，也对细胞信号传导的影响。互动将是 直接在已知由于突变而改变功能的细胞中测试（基于项目的研究 1）。将使用从我们的小鼠中分离出的原代细胞进行质谱下拉。 生殖线中的WT和/或突变蛋白的设计表位标记版。直接验证和 间接相互作用将取决于靶蛋白和HEK中推定的相互作用的相互性下拉 293个细胞。我们还将在体外使用纯化的蛋白质和GST下拉研究确定直接相互作用 作为尺寸排斥色谱。将有必要确定如何确定结构研究 每种含义的蛋白质都与其结合伴侣相互作用，以及ENU诱导的突变如何改变这一点 相互作用。当突变对细胞蛋白翻译后修饰的影响将在 怀疑信号异常。在许多情况下，这些研究将揭示 T1D的加速或抑制。我们的发现与项目1的经常沟通，反之亦然， 在这两个项目的研究中相互告知，从而理解和发展最好的 候选人。我们的结论可能指出了影响免疫或β细胞功能的信号通路的改变 以相对微妙的方式。然而，在每种情况下，都会对T1D发展产生极大的影响。 特别是在强大而新颖的抑制剂突变的情况下，我们希望确定治疗机会 用于预防或缓解人类患者的T1D。项目2将进行诱导的建设 有条件的等位基因测试这种治疗机会，立即建立基因敲除或转化 T1D发作后，确定是否可以建立缓解。这将表明潜力 T1D蜜月期治疗人类患者。我们将在所有修饰符突变上获取数据 通过诱变型候选Explorer门户网站公开获得。 CRISPR/CAS9等位基因（例如 诱导等位基因）将通过MMRRC提供。