BBB seminar: Christopher M. Overall

Quantitative proteomics and systems biology analysis of proteolytic networks in vivo

Christopher M. Overall
UBC Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada

To specifically enrich for mature protein N-termini and neo-N-termini of proteins we developed 6 and 10-plex TMT (Tandem Mass Tag) TAILS (Terminal Amine Isotopic Labeling of Substrates) (Nat Biotechnol 2010, 28:281; Nat Protoc 2011, 6:1578). In analysing the N-terminome of normal human tissues we have revealed the unexpected finding that 50-75% of proteins in vivo start at N-termini that are not as annotated in UniProt. Rather, the N-termini of protein chains in vivo can commence at many points C terminal to the predicted start site and result from proteolytic processing to generate stable protein chains: Proteolytic processing generates new protein species with characteristic neo-N-termini that are frequently accompanied by altered half-lives, function, interactions and location.

Not only does proteolysis alter protein function, but cleavage alters the protein sequence and results in neo-N-termini and hence novel semi-tryptic N-terminal peptides upon tryptic digestion proteomics. We hypothesized that some semi-tryptic N-terminal peptides will exhibit beneficial m/z (mass-to-charge ratio), ionization and fragmentation properties over their fully tryptic counterparts, rendering these peptides and cognate proteins identifiable. Hence, identifying in vivo cleavage sites in the proteome by all active proteases present generates the meta-N-terminome, which identifies proteoforms and also obtains proof for the expression of ‘missing proteins’, particularly if rare tissues and cells are analysed. We have compiled large-scale data of protease network interaction and expression to investigate the regulatory potential of the “protease web”. We used graph theory to represent this network and utilised TAILS to analyse proteolytic pathways in disease.

We explored the roles of the immune-modulatory MMP2 and macrophage MMP12 proteases by quantifying global proteome, protein N-termini (the N-terminome) and the altered abundance of proteases and inhibitors in inflammation. Cleavage and inactivation of the C1 inhibitor by MMP2 increased complement activation and bradykinin generation by plasma kallikrein, leading to increased vessel permeability during inflammation and hence influx of acute response proteins. The Mmp2 knock out mice had reduced vessel permeability, reduced acute response proteins and complement activation through pathways controlled by C1 inhibitor. In exploring the role of macrophage MMP12, we found that Mmp12-/- mice were protected from viral endocarditis and display earlier and dramatic severe arthritis vs. wild-type mice characterized by massive neutrophil infiltrations. Overall, MMP12 is essential for IFN-alpha secretion and dampens inflammation by concerted cleavages in multiple inflammation regulatory pathways. MMP12 facilitates macrophage invasion, but inactivates all CXCR2 chemokines responsible for neutrophil recruitment. This terminates neutrophil infiltration so accounting for the masses of neutrophils and the joint destruction in Mmp12-/- arthritis. MMP12 also promotes coagulation and quashes complement C3 activity at multiple levels. Such examples exemplify the general renaissance MMPs are enjoying from matrix remodellers to key cell regulators of extracellular homeostasis. By developing degradomics strategies to explore the roles of proteases in vivo many new substrates and hence functions in diverse processes have been revealed in regulating inflammation and immunity.

Chairperson: Anne Døskeland, Department of Biomedicine