Ubiquitination plays critical roles in immunology, including inflammatory signaling and MHC class II regulation. Among the enzymes responsible for the modification, E3 ligases and deubiquitinases (DUBs) control the addition and removal of ubiquitin (Ub), respectively. This project aims to uncover new functions for these enzymes in dendritic cells (DCs), key immune cells that initiate and modulate potent and specific immune responses mediated by T cells. Although there are over 600 putative E3 ligases and nearly 100 DUBs, few have been linked to DC biology.
We employed functional genomics and chemoproteomics to identify novel roles for Ub enzymes in DCs. First, an arrayed CRISPR/Cas9 screen targeting over 600 E3 ligases and scaffold proteins was combined with spectral flow cytometry to assess expression of 13 key surface proteins in the MuTu DC line. Cullin 3 (Cul3) emerged as a negative regulator of the co-stimulatory molecules CD80 and CD86, which are essential for T cell activation. This phenotype was confirmed in Cas9-expressing bone marrow-derived DCs (BMDCs). Cul3 also regulated programmed death ligand-2 (PD-L2), a suppressor of immune activity, following inflammatory stimulation.
Second, activity-based protein profiling identified active DUBs in resting and stimulated MuTu DCs using both lysate-based and novel cell-permeable probes. Several DUBs were differentially active depending on the DC activation state including Usp18, Otud1 and Josd1.
These findings offer new insights into the ubiquitin regulatory networks governing DC function and highlight opportunities to manipulate DC functions in health and disease.