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: 10554289
• 负责人: AMEETA KELEKAR
• 金额: $ 18.47万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10554289
• 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; Pathway interactions; Pentosephosphate Pathway; Phase; Phosphoric Monoester Hydrolases; Physiological; Positioning Attribute; Post-Transcriptional Regulation; Production; Proliferating; 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; cancer therapy; cell preparation; comparative; crosslink; design; enzyme activity; experience; experimental study; fructose-6-phosphate; glucose metabolism; immunoregulation; improved; inorganic phosphate; insight; interest; mutant; novel; prevent; programs; 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.

总结 在免疫应答期间，初始T淋巴细胞经历大规模克隆扩增和分化， 需要重新编程它们的新陈代谢，以满足意想不到的高能量和生物合成需求。的 代谢活动产生活性氧（ROS）并增加细胞内氧化应激触发因子 细胞死亡，除非得到适当的调控。一个主要的生物合成途径，有助于氧化还原调节， 增殖性葡萄糖代谢是戊糖磷酸途径（PPP），其产生NADPH，一种关键的 分子，满足脂质合成的需求增加，并有助于中和有害的活性氧。虽然T细胞 有几种抗氧化剂途径，但对增加抗氧化剂产生的机制知之甚少。 NADPH通过PPP在代谢活跃的T细胞中阻止过量的ROS积聚。 我们的研究表明，激活的T细胞重新利用果糖1，6-二磷酸酶1（FBP 1或FBPase），这是一个关键的功能。 促氧化酶，以独特的方式控制氧化应激。我们假设一个组成性的活跃分子 FBP 1的短同种型促进葡萄糖进入磷酸戊糖途径的通量增加，以增加 当T细胞准备快速增殖时，T细胞中NADPH的产生。FBP 1，其催化 果糖-1，6-二磷酸转化为果糖-6-磷酸和无机磷酸的不可逆反应，是 三个协调酶在造血，但唯一的一个表达和激活的刺激T细胞。 FBP 1具有羧基端催化结构域和抑制性氨基端调节结构域。初步 观察表明短同种型可能是由于利用了内部翻译起始剂甲硫氨酸而产生的。 目的1将确定FBP 1的表达和活性是如何调节T淋巴细胞刺激后， 使用生物化学和分子方法表征活化T细胞中的短同种型，研究 通过鉴定共刺激T细胞中FBP 1 mRNA-蛋白质相互作用， 细胞，并确定FBP 1的短同种型是否具有酶活性。目标2将调查 FBP 1在体外活化T淋巴细胞中的生理作用，使用稳定葡萄糖 同位素示踪分析和酶活性测定，同时监测活化标记物、增殖、ROS NADPH和细胞凋亡，在激活反应的过程中。CRISPR/Cas9产生的FBP 1 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 用于刺激后的T细胞扩增和活力。最后，受FBP 1活性影响的信号通路将被 使用FBP 1 KO和对照T细胞的比较蛋白质组学分析鉴定。 概述的研究对免疫调节、增殖代谢和维持具有广泛的意义 氧化还原稳态，并将揭示T细胞中FBP 1的独特非致突变功能。 FBP 1的催化活性亚型也有望成为免疫治疗、癌症治疗的工具（或靶点） 和糖尿病治疗。