Role for gluconeogenic enzyme FBP1 in T cell activation

糖异生酶 FBP1 在 T 细胞激活中的作用

基本信息

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
项目类别：
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

项目摘要

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.

概括在免疫学反应期间，我们对基本的大规模克隆扩张和分化以及需要重新编程其代谢，以满足意外的高能量和生物合成需求。代谢活性会产生活性氧（ROS），并增加细胞内氧化物应力触发除非受到适当的调节，否则细胞死亡。有助于氧化还原调节的主要生物合成途径葡萄糖代谢增殖是五磷酸五磷酸途径（PPP），它产生了NADPH，这是关键的满足脂质合成需求升高并有助于中和有害ROS的分子。虽然T细胞有几种抗氧化剂途径，对增加生产的机制知之甚少 NADPH穿过代谢活性T细胞中的PPP，以防止超越ROS积累。我们的研究表明，活化的T细胞再利用果糖1,6-双磷酸酶1（FBP1或FBPase），一种钥匙糖原性酶，以控制氧化应激的独特方式。我们假设一个组成型活动 FBP1的短亚型促进葡萄糖的通量增加到戊糖磷酸途径中，以增加 NADPH在T细胞中的生产为快速增殖做准备。 FBP1，催化水解在不可逆反应中，果糖1,6双磷酸到果糖-6-磷酸和无机磷酸盐，是一种糖异生中的三种协调酶，但唯一一种在刺激的T细胞中表达和活性。 FBP1具有羧基末端催化域和抑制性氨基末端调节域。初步的观察结果表明，简短的同工型可能是由于内部翻译引发剂方法的利用而引起的。 AIM 1将确定在刺激后，如何在T淋巴细胞中调节FBP1的表达和活性使用生化和分子方法表征活化的T细胞中短同工型，研究 FBP1调节的机理，通过鉴定共刺激的T中FBP1 mRNA-蛋白质相互作用细胞，并确定FBP1的短同工型是否具有酶活性。 AIM 2将调查使用稳定的葡萄糖，FBP1在活化的T淋巴细胞中的生理作用同位素示踪剂分析和酶活性测定，同时监测激活标记，ROS，ROS，在激活反应过程中，纳德和凋亡。 CRISPR/CAS9生成的FBP1 KO和推定的起始位点突变T细胞将用于确定是否需要构成酶活性用于T细胞扩展和刺激时的生存能力。最后，受FBP1活动影响的信号通路将是使用FBP1 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，增生新陈代谢和维持具有广泛的影响氧化还原稳态的，并将在T细胞中揭示FBP1独特的非葡萄肽功能， FBP1的催化活性同工型也有望作为免疫疗法的工具（或目标）和糖尿病治疗。