Use of integrated proteomic approaches to identify novel functions of iRhom2.

iRhom2 is a catalytical “inactive” member of rhomboid proteases which is required for tumor necrosis factor signalling by promoting trafficking and stabilization of A disintegrin and metalloprotease 17 (ADAM17). iRhom1 and iRhom2 are unique in the regulation of ADAM17 but iRhom2 is the only iRhom family member expressed in the immune cells. As such, iRhom2 is a pivotal player in controlling inflammation and a valuable drug target for several inflammatory diseases. Herein, I used different unbiased proteomics approaches to characterize novel functions of iRhom2. First, I investigated the role of iRhom2 in immune cells, including identification of substrates shed by the iRhom2/ADAM17 complex and new ADAM17-independent functions of iRhom2. I used a number of proteomics and biochemical approaches to identify MHC class I molecules as ADAM17 substrates in murine and human primary immune cells. Then, I found that iRhom2 plays a role in MHC class I trafficking to the cell surface, in an ADAM17 independent mechanism. Thus, iRhom2 expression supports CD8+ T cells immune response in a chronic lymphocytic leukaemia in vitro model by increasing the levels of MHC class I molecules on the cell surface. Second, I investigated the role of iRhom1 and iRhom2 in giving guidance to ADAM17 substrate’s selectivity. I used shotgun proteomics on cells expressing iRhom1 and/or iRhom2 and by using bioinformatic tools to analyse high throughput data, I evaluated ADAM17 substrates released in an iRhom1-depedent manner and in an iRhom2-dependent manner, by suggesting a predisposition of the sheddase to cleave substrates in presence of both iRhoms. Finally, I developed a novel proteomic workflow, called HEP-SEC, to analyse secretomes that encompass the problem of identifying low-abundant proteins that tend to bind to the extracellular matrix. By using heparin, which enhances solubility of extracellular matrix binding proteins, I was able to identify several heparin binding proteins that have not been identified by standard secretome analysis. Finally, I used HEP-SEC to identify novel ADAM17 substrates, demonstrating that HEP-SEC performed better than standard secretome analysis in identifying known and novel ADAM17 substrates.