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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要项目2将验证并研究在基因中产生和解决的表型的分子机制。核心 B 和核心 C。我们预计在资助期限内会发现大量具有修饰效应的突变，但是我们不会机械地研究所有这些，在此我们描述选择突变所使用的关键标准。研究并建议从我们的初步工作中确定为 T1D 修饰因子的四种突变开始（影响 Dusp10、Paqr8、Xpnpep1 和 Rapgef1）我们将继续重新定位 NOD/NckH 和/或中的候选基因。 NOD/NckL 背景使用 CRISPR/Cas9 创建种系敲除等位基因或替换等位基因（精确） ENU 诱导的等位基因的重新创建）或两者，以验证或排除因果关系。将研究其突变体和 WT 形式的蛋白质，以识别与它们相关的其他蛋白质，什么突变可能对这种关联产生影响，也会对细胞信号传导产生影响。直接在已知因突变而具有功能的细胞中进行测试（基于 Project 1). 质谱下拉将使用从我们拥有的小鼠中分离的原代细胞进行。直接和种系验证中WT和/或突变蛋白的工程化表位标记版本。间接相互作用将取决于目标蛋白的相互下拉和 HEK 中推定的相互作用子我们还将使用纯化的蛋白质和体外 GST 下拉研究来识别 293 细胞的直接相互作用。作为尺寸排阻色谱法的研究将被进行以精确地确定如何进行。每个相关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种相互作用相互作用时将检查突变对细胞蛋白质翻译后修饰的影响。在许多情况下，这些研究将揭示信号异常的分子基础。与项目 1 频繁交流我们的发现，会加速或抑制 T1D，反之亦然。两个项目的研究相互提供信息，从而了解和发展最优秀的人才我们的结论可能指出影响免疫或β细胞功能的信号通路的改变。然而，在每种情况下，都会对 T1D 的发展产生非常显着的影响。特别是在强而新颖的抑制突变的情况下，我们希望找到治疗机会为了预防或缓解人类患者的 T1D，项目 2 将进行诱导型的建设。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 T1D 发病后确定是否可以建立缓解，这将表明潜在的可能性。我们将收集所有修饰突变的数据。可通过 Mutagenetix Candidate Explorer 门户公开获取 CRISPR/Cas9 等位基因（如 ENU-）。诱导等位基因）将通过 MMRRC 获得。