Identification and molecular characterization of binding partners for the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor (MIF) is an immune modulator which is synthesized by various cell types and originally has been named by its ability to inhibit the random migration of human monocytes. More recently, evidence has accumulated that MIF may function in the inhibition of the deneddylase activity of the COP9 signalosome (CSN) by interacting with its subunit Jab1/CSN5. The target of the CSN deneddylase activity is the cullin component of SCF E3 ubiquitin ligases that are significantly more active when neddylated. These ligases are responsible for the selective ubiquitinylation of substrates which are hydrolysed in the 26S proteasome. Thus, ubiquitinylating enzymes and the proteasome form the two main components of the ubiquitin proteasome system (UPS). To better understand the biological role of MIF in the UPS, a systematic interactome screen was performed in NIH3T3 cells that are constitutively expressing biotinylated MIF.Biotinylated MIF and its associated proteins were purified by streptavidin binding. Subsequent identification of MIF interacting proteins by mass spectrometry detected a number of already known MIF interacting proteins including MIF itself, peroxiredoxin 1 and RPS19, thus emphasizing the validity of the approach. Besides other new candidates, endoplasmic reticulum (ER) associated chaperones and the AAA ATPase valosin-containing protein (VCP), which are all involved in ER-associated degradation were identified. As many new MIF interacting partners are involved in the UPS, further investigations were concentrated on the association of MIF with Jab1/CSN5 and VCP. While the binding of MIF to VCP is mediated by Jab1/CSN5, the interaction of VCP with Jab1/CSN5 is direct as verified independently by co-immunoprecipitation (co-IP), in vitro pull-down assays and fluorescence resonance energy transfer experiments.The interaction interface between VCP and Jab1/CSN5 was investigated by transient transfection of wild type and mutant proteins followed by co-IP. It emerged that Jab1/CSN5 associates with VCP via the core of its MPN domain that is contacting the ND1 domain of VCP. Its binding is independent of the JAMM motif harboring the NEDD8 isopeptidase activity. Using gel filtration chromatography, VCP co-migrated with the CSN and co-IP experiments confirmed that the interaction of VCP is not restricted to Jab1/CSN5, but involves the whole CSN complex in the associationRNA interference mediated knockdowns demonstrated that the JAMM motif of CSN5, a functional CSN complex and the CSN-associated deubiquitinase USP15 are all required for deubiquitinylation of substrates bound to VCP.Taken together, these results suggest that the CSN together with VCP forms a complex. Therefore, the CSN together with VCP could have the function to extract ubiquitinylated and abnormal folded proteins from larger protein complexes or membranes and to determine their fate.