Regulation of the NKG2D ligand MULT1 by E3 ubiquitin ligases

Natural killer (NK) cells are lymphocytes of the innate immune system that kill cancer cells or cells infected with certain viruses. NK cells recognize cancer cells in part by engagement of the cell surface activating receptor NKG2D with ligands expressed by cancer cells. Unlike normal cells, cancer cells often express NKG2D ligands. The binding of NKG2D with its ligands triggers NK cells to kill cancer cells. One NKG2D ligand, MULT1, is regulated by ubiquitination, which causes it to be downregulated in normal cells. The identity of the E3 ligases that mediate the degradation of MULT1 remain unclear. In order to identify the E3 ligase, we generated a construct expressing MULT1 with a TEV cleavage site and a HA tag at the C-terminus, to be used for tandem affinity purification of MULT1-interacting proteins. The MULT1-associated proteins will be identified by mass spectrometry analysis. Proteins containing either the HECT or RING domain characteristic of E3 ligases will be chosen for confirmation and further analysis. The approach may also identify other proteins that regulate MULT1 ubiquitination. Once the ligase is identified, we will focus on dissecting the underlying mechanism of MULT1 degradation and its impact on immune recognition. Deep understanding of the regulatory mechanisms may identify therapeutic targets for improving the immune response against cancer or for suppressing autoimmune disease.