Introduction: After organ transplantation, effector T cells migrate to the transplanted organ where they become activated and multiply locally, leading to rejection and host versus graft reaction (HVGR). Investigating how T cells activated immediately could offer valuable insights into transplantation immunology. Zinc finger protein 91 (ZFP91), a member of the E3 ubiquitin ligase family, has large disordered regions (IDRs) within its structure according to bioinformatics analysis by our previous study. These IDRs have the ability to induce Liquid-liquid phase separation (LLPS) of ZFP91 protein. In this study we want to explore if the LLPS of ZFP91 could control T cells function and the mechanism.
Method: Lentivirus-infected human primary T lymphocytes were used to manipulate ZFP91 expression, including knockdown, overexpression, and overexpression without the intrinsically IDRs Flow cytometry assessed cytotoxic factor secretion (GZMB, IFN-γ) post-infection, indicating T cell cytotoxicity changes. Co-culture with Huh-7 hepatocellular carcinoma cells evaluated T cell killing via Real-Time Cell Analysis. Purified ZFP91 protein exhibited LLPS extracellularly, with effects of concentration, salt, and temperature examined. Laser confocal microscopy assessed ZFP91 mobility via the fluorescence recovery after photobleaching. Mass spectrometry (MS) was utilized to identify the interacting protein of ZFP91, glucose-6-phosphate dehydrogenase (G6PD), and Western blot was used to validate the regulatory relationship between them.G6PD is the rate-limiting enzyme of pentose phosphate pathway (PPP). Metabolomics was employed to detect the metabolic changes in T cells before and after ZFP91 overexpression.
Results: Knockdown of ZFP91 in human T cells enhanced proliferation and GZMB/IFN-γ secretion, thereby improving function. Overexpression of full-length ZFP91 weakened GZMB/IFN-γ secretion, while overexpression of ZFP91 (without IDRs) only slightly reduced T cell function. The purified recombinant ZFP91 protein can form droplet-like aggregates in vitro. Furthermore, through immunofluorescence labeling, it can be observed that ZFP91 can also form droplet-like aggregates in T cells. Interestingly, as T cells are activated, the aggregates transfer from the nucleus to the cytoplasm, providing a basis for its interaction with the cytoplasmic protein G6PD. CO-IP showed that ZFP91 could bind to G6PD, and ubiquitination led to its degradation.zh In summary, ZFP91 can drive LLPS to play a role, thereby regulating T cell function through the G6PD-PPP pathway.
Conclusions: Our findings suggest that ZFP91, as a functional inhibitor of T cells, can modulate the G6PD-PPP pathway through phase separation, thereby regulating T cell function. This announcement provides a new approach to modulate T cell function through LLPS and PPP, as well as new insights into transplantation immunology.