Phase Separation Ability of ZFP91 and Its Regulation on T Cell Metabolic Pathways

Background: There is increasing evidence that protein ubiquitination plays crucial roles in adaptive immune response by regulating the function of T cells. Zinc finger protein 91 (ZFP91), as an E3 ubiquitin (Ub) ligase, has been shown to be associated with the proliferation and activation of mouse derived tumor infiltrating lymphocytes. However, whether it functions in human derived T cells and the underlying mechanism still need further exploration. Methods: We used lentivirus infection with H9 cell lines and human primary T cells to reduce ZFP91 expression. Flow cytometry and cck-8 experiments were used to compare the changes of T cell proliferation capacity before and after ZFP91 expression decreased. The expression of granzyme B and IFN-γ was detected by flow cytometry to compare the changes of T cell function. By purifying the recombinant ZFP91 protein, observe whether phase separation will occur in ZFP91, and the effects of protein concentration, salt concentration and temperature changes on phase separation phenomenon. By over expression ZFP91 in Hela cells, observe whether phase separation of ZFP91 occurs intracellularly. The interaction protein G6PD was identified by mass spectrometry analysis, and the regulatory relationship between them was verified by WB. Comparison of NADPH production before and after ZFP91 expression reduction was determined by glutathione peroxidase assay kit. The glycolytic ability of T cells before and after the reduction of ZFP91 expression was compared by lactic acid detection kit and Seahorse experiment. Results: We have verified the function of ZFP91 in H9 and human primary T cells. Compare with the control group, the T cell proliferation ability and the granzyme B, IFN- γ secretion ability of ZFP91 knockdown group enhanced significantly. The purified recombinant ZFP91 protein can separate into dynamic droplets under near-physiological conditions. After overexpressing ZFP91 in Hela cells, phase separation can also be observed in intact cells. In mass spectrometry analysis, we found that ZFP91 interacts with the key rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD) of the pentose phosphate pathway (PPP). Therefore, we continued to detect the metabolite of the PPP pathway, nicotinamide adenine dinucleotide phosphate (NADPH), and found that the level of NADPH in the ZFP91 knockdown group increased. In addition, we measured the lactic acid level of T cells and found that compared to the control group, the ZFP91 knockdown group had a decreased intracellular lactic acid level, basal glycolytic capacity and maximum glycolytic capacity. Conclusions: Our results show that ZFP91, as a functional inhibitory molecule of T cells, is able to undergo phase separation and influence T cell function by regulating the G6PD-PPP pathway, which suggest it may serve as a potential target to regulate T cell’s proliferation and function. This study was supported by NSFC 82272102.