Fine-tuning Notch expression by lymphocytic Granzyme B production

Abstract

Abstract Notch signaling has been studied for many years and has been found to play a role in many aspects of cellular development, tissue homeostasis, and progression of disease. Upon interaction with its ligand, Delta-like-4, the Notch1 molecule undergoes two proteolytic cleavage events. This eventually leads to the translocation of the intracellular domain (NICD) into the nucleus. Once there, NICD interacts with transcription factors such as CBF1/RBPJ. Recently, Notch has been found to play a role in the Th1/Th2 imbalance found in many diseases. Using a software to determine possible cleavage target sites for Granzyme B (GzmB), two were found following the start site of NICD. One group found that cleavage of Notch1 by GzmB disables its activity, however lymphocytes were not the target tissue of research. Thus, we hypothesize that Granzyme B production plays a functional role in the translocation of intracellular Notch1 in affecting CD4+ and CD8+ T lymphocyte activation. We test our hypothesis by stimulating T lymphocytes collected from transgenic mice that are deficient in GzmB and measuring intracellular and extracellular Notch1 expression compared to wildtype following in vitro aCD3-CD28 stimulation. Flow Cytometry was used to measure Notch expression. Preliminary results show that 24hrs after stimulation, CD8+ and CD4+ T cells from GzmB-deficient mice show 12% and 6%, more NICD expression than WT counterparts and 19% and 14% more extracellular Notch1 expression, respectively. These results show that the presence of GzmB can cause an alteration in the fine-tuning expression of Notch1. This work helps us understand the role of Granzyme B in Notch activation and differentiation of lymphocytes.