Role for gluconeogenic enzyme FBP1 in T cell activation

摘要 在免疫反应期间，幼稚T淋巴细胞经历大量克隆性扩增和分化，并 需要对新陈代谢进行重新编程，以满足意想不到的高能和生物合成需求。这个 代谢活动产生活性氧物种(ROS)并增加细胞内氧化应激触发 如果没有适当的管理，细胞死亡。参与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，是一种关键的 满足提高的脂质合成需求的分子，有助于中和有害的ROS。虽然T细胞 有几种抗氧化剂途径，但人们对增加 NADPH通过PPP在代谢活跃的T细胞中防止过量的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(FBP1，或FBPase)，这是一种关键 糖异生酶，以一种独特的方式控制氧化应激。我们假设一个生性活跃的人 FBP1的短亚型有助于增加葡萄糖进入戊糖磷酸途径的流量，从而增加 T细胞为快速增殖做准备时产生的NADPH。Fbp1，催化水解酶 果糖-1，6-二磷酸与果糖-6-磷酸和无机磷酸发生不可逆反应，是 在糖异生中有三种协同酶，但唯一一种在刺激的T细胞中表达和活性。 FBP1具有一个羧基末端催化域和一个抑制性氨基末端调节域。初步 观察表明，这种短的异构体可能是由于利用了一种内部翻译起始剂蛋氨酸。 目的1将确定FBP1的表达和活性是如何在T淋巴细胞被刺激后被调节的 用生化和分子方法鉴定活化T细胞中的短亚型，研究 通过鉴定共刺激T细胞中FBP1基因与蛋白质的相互作用，探讨其调控机制 细胞，并确定FBP1的短异构体是否具有酶活性。目标2将调查 利用稳定葡萄糖体外激活模型研究FBP1在活化T淋巴细胞中的生理作用 同位素示踪分析和酶活性分析，同时监测激活标志物、增殖、ROS、 NADPH和细胞凋亡，在激活过程中做出反应。CRISPR/CAS9生成的FBP1 KO和 假定起始点突变的T细胞将被用来确定是否需要结构性酶活性 刺激下T细胞的扩增和活性。最后，受FBP1活性影响的信号通路将是 通过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意义。 氧化还原动态平衡，并将揭示T细胞中FBP1独特的非糖异生功能。 具有催化活性的FBP1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。

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.