Molecular Pathways of Programmed Cell Death And Viral Cytopathicity

程序性细胞死亡和病毒细胞病变的分子途径

• 批准号: 10697667
• 负责人: michael j lenardo
• 金额: $ 63.1万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697667
• 关键词: Allergic; Antigens; Apoptosis; Autoimmune; Autoimmune Diseases; Autoimmunity; Biological; Biological Process; CRISPR/Cas technology; Candidate Disease Gene; Cell Death; Cell Survival; Cells; Ceramides; Cessation of life; Coin; Complex; Contracts; Coupled; Disease; Embryonic Development; Equation; Equilibrium; Event; Exposure to; Genes; Gold; Graft Rejection; Homeostasis; Human; Immune response; Immune system; Immunity; Immunosuppressive Agents; In Vitro; Knock-out; Lead; Libraries; Life; Lipids; Liquid Chromatography; Lymphocyte; Lymphoproliferative Disorders; Malignant lymphoid neoplasm; Maps; Mass Spectrum Analysis; Medicine; Methods; Modification; Molecular; Mus; Outcome; Pathway interactions; Phase; Play; Process; Proteins; Proteome; Reagent; Receptor Signaling; Research; Resolution; Resources; Rest; Role; Sampling; Signal Transduction; Specificity; Spectrometry; Speed; System; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Therapeutic Intervention; Transcriptional Activation; Viral; base; experimental study; genome-wide; interest; ion mobility; lipidomics; liquid chromatography mass spectrometry; mass spectrometer; metabolome; metabolomics; pathogen; programs; receptor; screening; side effect; therapeutic development; therapeutically effective

Programed cell death (PCD) is an indispensable process that takes place through a variety of different mechanisms in multicellular life. Apoptosis and necroptosis, which are essential for embryonic development and many important biological processes that rely on continuous cellular turn-over, are recognized as the two major forms of PCD that contribute to the contraction phase of immune responses. In the periphery, stimulation of the T cell antigen receptor (TCR) can have several different outcomes, including T cell survival, activation, and proliferation, or PCD. However, T cell proliferation and contraction during an immune response must be balanced to allow for both protection against pathogens and avoidance of autoimmunity and the excessive accumulation of cells that might predispose to lymphoid cancer. Although primary stimulation of TCR in resting T cells and restimulation of TCR in activated cells share many early signaling events, there are significant differences that result in either life or death respectively. We coined the term TCR restimulation-induced cell death (RICD) to conceptually distinguish the TCR process resulting in apoptosis from that leading to activation and proliferation. Clonal specificity is the important feature of RICD, which makes it an appealing method for tolerance induction in autoimmune diseases. Compared with conventional immunosuppressant medicines that debilitate the whole immune system and have many side effects, the RICD-based tolerance strategy targets only undesired T cells while keeping the general immune system intact. Thus, tolerogenic induction of antigen- specific RICD may be an effective therapeutic intervention for progressive autoimmune disorders. It is well known that the T cell undergoes dramatic changes immediately after a resting or naive cell is exposed to an antigen. During the TCR signaling cascade, the related proteins are activated, along with transcriptional activation of several T cell-specific genes, causing a wide scale modification of its metabolome. Currently, few details are known about metabolomics during this process. Liquid chromatography (LC) coupled with mass spectrometry (MS) has become the gold standard in various omics fields. However, the coverage of proteomes or metabolomes in complex biological samples remains challenging due to limited speed, sensitivity, and resolution of current mass spectrometers. Adding trapped ion mobility spectrometry (TIMS) to the equation unlocks the parallel accumulation serial fragmentation acquisition method to provide extremely high MS/MS speed and sensitivity, requiring minimal sample amounts. We have acquired a start-of-the-art TimsTOF LC-MS spectrometer system for lipidomics and metabolomics research. In our first project, we are studying the role lipids play in cell death or survival decisions when T cells are sensitive to RICD. Our preliminary in vitro findings show differences in lipid ceramide levels in T cells undergoing activation versus RICD. Further studies will help to map lipid pathways that are involved with the TCR signaling complex. Additionally, we are carrying out genome-wide CRISPR-cas 9 knockout library screening to identify candidate genes encoding proteins that promote cell survival under selected death-inducing circumstances, such as death-receptor and RICD. Due to availability of resources and reagents, these experiments are being conducted using primary mouse cells, but we will confirm the experiments with human T cells once our genes of interest are identified. Blocking key parts of these pathways highly associated with autoimmune diseases could aid in therapeutic development.

编程的细胞死亡（PCD）是一个必不可少的过程，它通过多种细胞生活中的多种不同机制进行。凋亡和坏死作用对于胚胎发育以及许多依赖连续细胞转换的重要生物学过程至关重要，被认为是有助于免疫反应收缩阶段的两种主要形式的PCD。在外围，T细胞抗原受体（TCR）的刺激可以具有几种不同的结果，包括T细胞存活，激活和增殖或PCD。但是，必须平衡在免疫反应过程中进行T细胞增殖和收缩，以允许防止病原体和避免自身免疫性以及可能易于淋巴类癌的细胞的过度积累。尽管在静息T细胞中对TCR的一级刺激和活化细胞中TCR的重新刺激共有许多早期信号事件，但存在显着差异，分别导致生与死。我们创造了术语TCR恢复诱导的细胞死亡（RICD），从概念上区分了TCR过程，导致凋亡与导致激活和增殖的凋亡。克隆特异性是RICD的重要特征，它使其成为自身免疫性疾病耐受性诱导的一种吸引人的方法。与传统的免疫抑制剂药物相比，使整个免疫系统虚弱并具有许多副作用相比，基于RICD的耐受性策略仅针对不希望的T细胞，同时保持一般的免疫系统完整。因此，抗原特异性RICD的耐受性诱导可能是进行性自身免疫性疾病的有效治疗干预措施。 众所周知，在静止或天真细胞暴露于抗原后，T细胞立即发生急剧变化。在TCR信号级联过程中，相关蛋白被激活，以及几个T细胞特异性基因的转录激活，导致其代谢组的大规模修饰。当前，在此过程中，很少有有关代谢组学的细节。液相色谱（LC）与质谱法（MS）结合已成为各种Omics领域的金标准。然而，由于速度有限，灵敏度和当前质谱仪的分辨率，蛋白质组或代谢组在复杂的生物样品中的覆盖范围仍然具有挑战性。将被困的离子迁移率（TIMS）添加到方程中，可以解锁并行的累积串行片段采集方法，以提供极高的MS/MS速度和灵敏度，需要最小的样本量。我们已经获得了用于脂质组学和代谢组学研究的LC-MS光谱仪系统的开始。在我们的第一个项目中，当T细胞对RICD敏感时，我们正在研究脂质在细胞死亡或生存决策中的作用。我们的初步体外发现显示了经受激活与RICD的T细胞中脂质神经酰胺水平的差异。进一步的研究将有助于绘制与TCR信号复合物有关的脂质途径。此外，我们正在进行全基因组CRISPR-CAS 9敲除库筛选，以识别编码蛋白质的候选基因，这些基因在选定的死亡诱导情况下（例如死亡受体和RICD）促进细胞存活。由于资源和试剂的可用性，这些实验是使用原代小鼠细胞进行的，但是一旦确定了感兴趣的基因，我们将确认人类T细胞的实验。阻止与自身免疫性疾病高度相关的这些途径的关键部分可能有助于治疗性发育。