Background: The Gipie protein, a member of the hook-related protein family, plays an important role in forming the immune synapse—a critical interface between immune cells and their targets. Published studies have identified Gipie as a master regulator of T-cell maturation and effector functions, and as a key player in the movement of Natural Killer (NK) cell granules during activation. This research aims to elucidate how Gipie regulates the clustering of lytic granules and the repositioning of the microtubule-organizing centre (MTOC) during immune cell-mediated killing. Furthermore, clinical data shows that ectopic expression of Gipie in ER/PR breast cancer tissue microarrays (TMAs) correlates with metastasis, lymph node involvement, and recurrence, suggesting a potential role for Gipie in cancer progression / spread.
Methods and Results: In immune cells we have identified potential binding and regulatory partners of Gipie. The immune marker CD16 was identified, and its co-localization and interaction with Gipie were demonstrated through immunofluorescence (IF) and immunoprecipitation (IP) assays. Mass spectrometry analysis of hook-related protein family immunoprecipitants from PBMCs identified novel binding partners, including BICD2, Coroin7, DNM1L, and DNAM-1. These interactions were further confirmed through IF and IP. Colocalization studies revealed distinct trafficking pathways for Gipie in cancer versus immune cells, and differential localization patterns between resting and active states of Gipie. The partners of Gipie also differ from its innate expression in immune cells compared with ectopic expression in cancer. To investigate Gipie's functional role, we established Gipie overexpressing A431 cancer cell lines and Jurkat T-cell lines. ADCC assays comparing Gipie overexpressing A431 cells to wild-type (WT) cells showed a 10% decrease in killing rate, suggesting a role for Gipie in modulating cancer survival under immune cell attack.
Conclusions: In the immune cell setting Gipie is involved in immune synapse formation and the regulation of immune cell-mediated killing. These findings advance our understanding of immune cell activation and highlight Gipie as a potential target for modulating immune responses in therapeutic settings. Gipie is ectopically expressed in some cancers. The reduced killing rate in Gipie overexpressing cancer cells suggests a mechanism by which cancer cells may evade immune detection and destruction.