Role for gluconeogenic enzyme FBP1 in T cell activation

糖异生酶 FBP1 在 T 细胞激活中的作用

• 批准号: 10433367
• 负责人: AMEETA KELEKAR
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-24 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433367
• 关键词: Antigens; Antioxidants; Apoptosis; Binding Proteins; Biochemical; Biological Assay; CD8-Positive T-Lymphocytes; CRISPR/Cas technology; Carbon; Catalytic Domain; Cell Death; Cell division; Cells; Clonal Expansion; Cyclic AMP; Data; Diabetes Mellitus; Differentiation Antigens; Ensure; Enzymes; Fructose; Gel; Gluconeogenesis; Glucose; Goals; Homeostasis; Hydrolysis; Immune; Immune response; Immunoprecipitation; Immunotherapy; In Vitro; Investigation; Isotopes; Kinetics; Knowledge; Label; Length; Lipids; Maintenance; Mass Spectrum Analysis; Messenger RNA; Metabolic; Metabolism; Methionine; Modeling; Molecular; Monitor; Mutation; NADP; Natural regeneration; Nucleotides; Nutrient; Oxidation-Reduction; Oxidative Stress; Oxides; Pathway interactions; Pentosephosphate Pathway; Phase; Phosphoric Monoester Hydrolases; Physiological; Positioning Attribute; Post-Transcriptional Regulation; Production; Protein Isoforms; Proteins; Proteomics; Reaction; Reactive Oxygen Species; Reduced Glutathione; Regulation; Research; Resistance; Role; Signal Pathway; Site; T cell response; T-Cell Activation; T-Lymphocyte; Testing; Tracer; Transcript; Translations; base; cancer therapy; comparative; crosslink; design; enzyme activity; experience; experimental study; fructose-6-phosphate; glucose metabolism; immunoregulation; improved; inorganic phosphate; insight; interest; mutant; novel; prevent; response; tool

SUMMARY During an immune response, naïve T lymphocytes undergo massive clonal expansion and differentiation and are required to reprogram their metabolism to meet unexpected high energy and biosynthetic demands. The metabolic activity generates reactive oxygen species (ROS) and increases intracellular oxidative stress trigger cell death unless properly regulated. A major biosynthetic pathway that contributes to redox regulation in proliferative glucose metabolism is the pentose phosphate pathway (PPP) which produces NADPH, a critical molecule that meets the elevated demand for lipid synthesis and helps neutralize harmful ROS. Although T cells have several antioxidant pathways in place, less is known about mechanisms that increase the production of NADPH through the PPP in the metabolically active T cell to prevent excess ROS build up. Our studies suggest that activated T cells repurpose fructose 1,6-bisphosphatase 1 (FBP1, or FBPase), a key gluconeogenic enzyme, in a unique way to control oxidative stress. We hypothesize that a constitutively active short isoform of FBP1 facilitates an increased flux of glucose into the pentose phosphate pathway, to increase the production of NADPH in T cells as they prepare for rapid proliferation. FBP1, which catalyzes hydrolysis of fructose-1,6 bisphosphate to fructose-6-phosphate and inorganic phosphate in an irreversible reaction, is one of three coordinated enzymes in gluconeogenesis, but the only one expressed and active in stimulated T cells. FBP1 has a carboxy terminal catalytic domain and an inhibitory amino terminal regulatory domain. Preliminary observations suggest the short isoform may result from utilization of an internal translation initiator methionine. Aim 1 will determine how FBP1 expression and activity are regulated in T lymphocytes following stimulation by characterizing the short isoform in activated T cells using biochemical and molecular approaches, investigating mechanism of regulation of FBP1, through identification of FBP1 mRNA-protein interactions in co-stimulated T cells, and determining whether the short isoform of FBP1 is enzymatically active. Aim 2 will investigate the physiological role of FBP1 in activated T lymphocytes using an in vitro activation model, with stable glucose isotope tracer analysis and enzyme activity assays, while monitoring activation markers, proliferation, ROS, NADPH and apoptosis, over the course of the activation response. CRISPR/Cas9-generated FBP1 KO and putative start site mutant T cells will be utilized to determine whether constitutive enzymatic activity is required for T cell expansion and viability upon stimulation. Finally, signaling pathways impacted by FBP1 activity, will be identified using comparative proteomic analyses of FBP1 KO and control T cells. The outlined research has broad implications for immune regulation, proliferative metabolism and maintenance of redox homeostasis, and will reveal a unique non-gluconeogenic function for FBP1 in T cells The novel, catalytically active isoform of FBP1 also holds promise as a tool (or target) for immunotherapy, cancer therapy and diabetes treatment.

概括 在免疫反应期间，幼​​稚 T 淋巴细胞经历大规模克隆扩增和分化， 需要重新编程他们的新陈代谢以满足意想不到的高能量和生物合成需求。这 代谢活动产生活性氧 (ROS) 并增加细胞内氧化应激触发因素 除非适当调节，否则细胞死亡。有助于氧化还原调节的主要生物合成途径 增殖性葡萄糖代谢是磷酸戊糖途径 (PPP)，它产生 NADPH，NADPH 是一种关键的 满足脂质合成增加的需求并有助于中和有害活性氧的分子。虽然T细胞 存在多种抗氧化途径，但人们对增加产生的机制知之甚少 NADPH 通过代谢活跃的 T 细胞中的 PPP 来防止过多的 ROS 积聚。 我们的研究表明，活化的 T 细胞重新利用果糖 1,6-二磷酸酶 1（FBP1 或 FBPase），这是一种关键的酶 糖异生酶，以独特的方式控制氧化应激。我们假设本构活跃 FBP1 的短亚型有助于增加葡萄糖进入磷酸戊糖途径的流量，从而增加 T 细胞在准备快速增殖时产生 NADPH。 FBP1，催化水解 1,6-二磷酸果糖转化为6-磷酸果糖与无机磷酸盐发生不可逆反应，是其中之一 糖异生过程中存在三种协调酶，但只有一种在受刺激的 T 细胞中表达并具有活性。 FBP1 具有羧基末端催化结构域和抑制性氨基末端调节结构域。初步的 观察结果表明，短亚型可能是由于使用内部翻译起始子甲硫氨酸造成的。 目标 1 将确定 T 淋巴细胞刺激后如何调节 FBP1 表达和活性 使用生化和分子方法表征活化 T 细胞中的短亚型，研究 通过鉴定共刺激 T 中 FBP1 mRNA-蛋白质相互作用，研究 FBP1 的调节机制 细胞，并确定 FBP1 的短亚型是否具有酶活性。目标 2 将调查 使用体外激活模型，具有稳定的葡萄糖，FBP1 在激活的 T 淋巴细胞中的生理作用 同位素示踪分析和酶活性测定，同时监测激活标记、增殖、ROS、 NADPH 和细胞凋亡，在激活反应过程中。 CRISPR/Cas9 生成的 FBP1 KO 和 假定的起始位点突变 T 细胞将用于确定是否需要组成型酶活性 用于 T 细胞在刺激后的扩增和活力。最后，受 FBP1 活性影响的信号通路将被 使用 FBP1 KO 和对照 T 细胞的比较蛋白质组学分析进行鉴定。 概述的研究对免疫调节、增殖代谢和维持具有广泛的影响 氧化还原稳态，并将揭示 T 细胞中 FBP1 独特的非糖异生功能 FBP1 的催化活性异构体也有望作为免疫治疗、癌症治疗的工具（或靶标） 和糖尿病治疗。