Role for gluconeogenic enzyme FBP1 in T cell activation

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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.