Adjunct Assistant Professor Johns Hopkins University, United States
Introduction: Changes in surrounding nutrient conditions require cells to profoundly reprogram metabolic pathways as well as gene expression. As the critical effector arm of the adaptive immune system, T cells are required to adapt to a broad range of environments, and they show dramatic phenotypic changes in response to glutamine or glucose blockade. Nuclear translocation of metabolic enzymes as an adaptive response to metabolic conditions has been reported, but the role of glutamine syntheses (GLUL), a critical enzyme in glutamine metabolism, in regulating transcriptional response to metabolic challenges has not been previously explored.
Materials and
Methods: Cell culture and activation: JURKATs were cultured in normal complete RPMI media, glucose-free, or glutamine-free RPMI media at 5E5 cells/mL. Each nutrient condition contains a naive and an activated group. The activations were done in 6-well plates for 48 hours at 37C. Wells were coated with 1ug/mL a-CD3 in PBS without Calcium or Magnesium and the media contained 1ug/mL a-CD28 in culture media. For each group, another set of conditions with 1mM Methionine Sulfoximine (MSO) was included.
Western blotting 2.5E6 cells were collected in each condition and lysed sequentially with cytoplasmic and nuclear lysing media, obtaining respective protein fractions. Fractions were run on gels and transferred onto a PVDF membrane. After blocking with 5% milk, GLUL, B actin and Lamin B1 were probed with antibodies and imaged on a ChemiDox Imaging System. Band intensity was quantified on Fiji Image J and the results were normalized to its respective loading controls.
Confocal microscopy 1E5 cells in each condition were settled onto a microscope slide and fixed with 4% PFA in PBS for 15 minutes at 37C. Then the cells were permeabilized with 100% methanol for 10 minutes at -20C and blocked with 5% serum with Triton X-100 for 1 hour at room temperature. Cells were then probed for GLUL with antibodies diluted PBS containing BSA and Triton X-100 and then washed. After mounting, representative images of fluorescence showing sub-cellular localization of GLUL in nucleus were taken on Nikon SDC/TIRF(dSTROM). Fluorescence was quantified on Fiji ImageJ.
Results, Conclusions, and Discussions: Western blotting shows that nuclear glutamine synthetase is evident in the nucleus of primary mouse T cells, which has never been reported before to our knowledge. Then to establish an experimental model, we confirmed that GLUL translocates into the nucleus in the human T cell line JURKAT by Western blotting and confocal microscopy.
Next, we found that nuclear translocation of GLUL is decreased during glucose deprivation in JURKAT cells. This decrease is contrary to what we hypothesized. As glutamate, the substrate of glutamine synthase’ s metabolic activity, could be produced from glucose, we hypothesize that decreasing amounts of glucose would decrease glutamate formation. The low reactant level would result in a lowered glutamine synthase’s metabolic activity as the chemical equilibrium is shifted to the left according to Le Chatelier’s Principle. GLUL should be free to move from the cytoplasm into the nucleus, leading to an increase of such translocation for its moonlighting role. Nevertheless, this decrease still suggests that the nuclear translocation of GLUL is impacted by metabolic conditions in the environment.
We also found that treatment of JURKAT cells with GLUL inhibitor (MSO) shows distinct migration pattern in WB. MSO is a well-known inhibitor of glutamine synthase’s metabolic activity. The double band observed in the Western blots could be due to the generation of an isoform of GLUL during the direct inhibition of GLUL with MSO.
This work provides a critical experimental framework to further explore the role of GLUL in triggering metabolically-responsive transcriptional changes in T cell phenotype. In the future, it is hoped that we could continue the immunoblot and microscopy studies to confirm the initial findings of differential nuclear translocation of GLUL and catalog interventions that affect nuclear translocation of GLUL in a predictable manner. We also hope to investigate the molecular mechanism of GLUL nuclear activity in terms of transcriptional modulation, as well as to determine the association of phenotypic changes of T cell with GLUL translocation. We could perform the experiments with a truncated variant of GLUL that affects its nuclear translocation but not its metabolic function under different nutrient conditions.
