Bcl-2 protein Noxa in human T cell metabolism and differentiation

Bcl-2蛋白Noxa在人T细胞代谢和分化中的作用

• 批准号: 10064173
• 负责人: AMEETA KELEKAR
• 金额: $ 21.77万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-18 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064173
• 关键词: Adopted; Affect; Antigen Receptors; Apoptotic; Automobile Driving; BCL-2 Protein; BCL2 gene; Biochemical; Biological Assay; Biomass; CD8-Positive T-Lymphocytes; CRISPR/Cas technology; Carbon; Cell Differentiation process; Cell physiology; Cells; Cellular Metabolic Process; Citric Acid Cycle; Clonal Expansion; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Dependence; Energy-Generating Resources; Event; Exhibits; Family; Fatty Acids; Flow Cytometry; Generations; Gluconeogenesis; Glucose; Glutaminase; Glutamine; Goals; Growth; Hematologic Neoplasms; Human; Immune; Immune response; Immunotherapeutic agent; Immunotherapy; Impairment; Invaded; Knowledge; Leukemic Cell; Link; Lymphocyte; Mediating; Memory; Messenger RNA; Metabolic; Metabolic Pathway; Metabolism; MicroRNAs; Mitochondria; Mus; Organism; Oxidative Phosphorylation; Pathway interactions; Phenotype; Play; Population; Positioning Attribute; Post-Transcriptional Regulation; Proliferating; Property; Protein Biosynthesis; Protein Family; Proteins; Proteome; Regulation; Regulatory Pathway; Regulatory T-Lymphocyte; Research; Research Personnel; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Source; Supporting Cell; T cell differentiation; T memory cell; T-Cell Activation; T-Lymphocyte; Testing; Therapeutic; Tracer; Work; acute T-cell lymphoblastic leukemia cell; aerobic glycolysis; cancer cell; cellular targeting; effector T cell; fatty acid oxidation; glucose uptake; insight; interest; leukemia; member; metabolic phenotype; metabolomics; mitochondrial fitness; novel; oxidation; prevent; programs; sugar; therapy outcome; tool; uptake

SUMMARY Naïve lymphocytes, upon receiving appropriate co-stimulatory signals, become activated and go through a rapid proliferative burst before assuming effector functions directed at killing the target ‘invading’ organism or cancer cell. This rapid proliferative burst requires increased uptake and utilization of glucose in anabolic pathways that produce building blocks for biomass. To support proliferation activated T cells switch to glutamine as the carbon source for oxidative phosphorylation (OXPHOS). The shift to aerobic glycolysis and enhanced dependence on glutamine in activated T cells is well documented, but the signaling pathways that facilitate this shift are poorly understood. Recent studies from our group point to a key role for human Bcl-2 family BH3-only protein, Noxa, in controlling this activation-induced metabolic switch in T cells. BH3-only proteins are by definition pro-apoptotic, but Noxa exhibits a pro-growth and survival function as well, in leukemia cells. This non-canonical function of Noxa is also evident in primary human T lymphocytes. Noxa is significantly upregulated in T cells following TCR engagement, and remains high as the activated cells proliferate. Additionally, it requires constant availability of glutamine to remain highly expressed. Early studies suggest that Noxa plays an essential role in T cell activation and the metabolic reprogramming associated with activation. We will test a central hypothesis that states - Noxa is required for the metabolic switch to glutaminolysis in activated human T cells and for their differentiation to the effector phenotype. The first aim will investigate the role of Noxa in T cell activation and glutamine metabolism. We will determine whether glutamine mediates the induction of Noxa expression in T cells through regulation of microRNAs. This aim will also test the hypothesis that Noxa is required for glutaminolysis and for proliferation of activated T cells using novel CRISPR-generated Noxa KO primary T cells, biochemical assays, metabolic phenotyping, and metabolomics. Although loss of Noxa impairs glutaminolysis, it does not affect mitochondrial fitness suggesting stimulated T cells shift to an alternative fuel source for oxidative phosphorylation. In Aim 2 we will use multi- parameter flow cytometry, and metabolic and functional assays to test the hypothesis that Noxa enables T effector cell generation by promoting glutaminolysis, and prevents differentiation of T cells to a regulatory (Treg) or memory (TM) phenotype by suppressing fatty acid oxidation. An understanding of the metabolic needs of T cells as they undergo expansion will help to develop therapeutic strategies targeted at metabolism. This research could reveal avenues to exploit the crosstalk between Noxa and glutamine metabolism to better understand cellular events that drive the metabolic switch in T cells. The proposed work also will offer insights into Noxa’s role in driving differentiation of T cells into effector, regulatory or memory subtypes and help evaluate the protein’s potential as a therapeutic tool.

概括 初始淋巴细胞在接收到适当的共刺激信号后被激活并经历 在发挥效应器功能以杀死目标“入侵”生物体之前快速增殖爆发或 癌细胞。这种快速增殖爆发需要增加合成代谢中葡萄糖的摄取和利用 生产生物质的构建模块的途径。为了支持增殖激活的 T 细胞切换到 谷氨酰胺作为氧化磷酸化（OXPHOS）的碳源。向有氧糖酵解的转变和 活化的 T 细胞对谷氨酰胺的依赖性增强已有充分记录，但信号通路 促进这种转变的因素人们知之甚少。 我们小组最近的研究指出，人类 Bcl-2 家族 BH3 蛋白 Noxa 在控制 T 细胞中这种激活诱导的代谢转换。根据定义，仅限 BH3 的蛋白质是促凋亡的，但 Noxa 在白血病细胞中也表现出促生长和存活功能。 Noxa 的这个非规范函数是 在原代人 T 淋巴细胞中也很明显。 TCR 后 T 细胞中 Noxa 显着上调 参与度，并且随着激活的细胞增殖而保持高水平。此外，它还需要持续可用 谷氨酰胺保持高表达。早期研究表明 Noxa 在 T 细胞激活中发挥重要作用 以及与激活相关的代谢重编程。我们将检验一个中心假设： Noxa 是活化的人类 T 细胞代谢转变为谷氨酰胺分解及其分化所必需的 到效应子表型。 第一个目标是研究 Noxa 在 T 细胞激活和谷氨酰胺代谢中的作用。我们将确定 谷氨酰胺是否通过调节 microRNA 介导 T 细胞中 Noxa 表达的诱导。这 目标还将检验 Noxa 是谷氨酰胺分解和活化 T 细胞增殖所必需的假设 使用新型 CRISPR 生成的 Noxa KO 原代 T 细胞、生化测定、代谢表型分析和 代谢组学。虽然 Noxa 的缺失会损害谷氨酰胺分解，但它不会影响线粒体的健康状况，表明 受刺激的 T 细胞转向氧化磷酸化的替代燃料来源。在目标 2 中，我们将使用多 参数流式细胞术以及代谢和功能测定来检验 Noxa 能够实现 T 的假设 通过促进谷氨酰胺分解作用产生效应细胞，并防止 T 细胞分化为调节性 T 细胞 或通过抑制脂肪酸氧化来形成记忆（TM）表型。 了解 T 细胞在扩增过程中的代谢需求将有助于开发 针对新陈代谢的治疗策略。这项研究可以揭示利用串扰的途径 Noxa 和谷氨酰胺代谢之间的关系，以更好地了解驱动代谢转换的细胞事件 在T细胞中。拟议的工作还将深入了解 Noxa 在驱动 T 细胞分化为 效应子、调节子或记忆亚型，并帮助评估蛋白质作为治疗工具的潜力。