Sunday, February 19, 2012 – 1:30 PM
Christopher Overall, University of British Columbia, Vancouver, BC, Canada
Balancing neutrophil and macrophage infiltration with their clearance is key in the pathogenesis of inflammation. We have uncovered critical roles for the control of inflammation exerted by a family of extracellular matrix metalloproteinases (MMPs) that typically have been previously viewed as being detrimental for inflammation by tissue degradation. We found that ablation of the neutrophil specific MMP8 increases inflammation by reducing neutrophil apoptosis, that in a murine model of chronic arthritis led to worsened disease. Now we have examined the role of macrophage-specific matrix metalloproteinase-12 (MMP12) in murine peritonitis, LPS lung inflammation, and arthritis by proteomics. Notably, Mmp12-/- mice display earlier and dramatic severe inflammation vs wild-type mice characterized by massive neutrophil infiltrations. Degradomics using 8-plex iTRAQ-Terminal Amino Isotopic Labelling of Substrates (TAILS1) identified 611 in vitro substrates, with many cleavage sites being confirmed in vivo by TAILS. Overall, MMP12 dampens inflammation by concerted cleavages in multiple inflammation regulatory pathways. Mechanistically, we find that 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 clotting by activation of prothrombin and inactivation of anti-thrombin-III, reducing clotting times by ~20%. Moreover, MMP12 squashes complement C3 activity at multiple levels: C3a cleavage abolishes receptor binding so halting inflammatory cell recruitment; cleavage of full-length C3 and surface-bound C3b reduces target cell lysis; whereas cleavage of C3b and iC3b increases phagocytosis of C3-opsonized particles to increase target cell elimination. This also contributes to enhanced neutrophil accumulation in these tissues. The importance of MMP12 in removing extracellular nets of actin and fibrin, identified by TAILS as new substrates, was strikingly demonstrated in vivo. Mmp12-/- mice were characterized by huge extracellular DNA/actin/fibrin nets that were absent in wild-type mice. These and multiple other inflammatory protein substrates discovered by TAILS highlights the unexpected control exerted by macrophages and MMP12 in resolving inflammation and the power of TAILS to discover new proteolytic pathways in vivo. Thus the inflammatory cell-specific MMPs dampen inflammation.
Seminar: Unlocking Biology’s Potential
Friday, February 17, 2012: 8:00 AM-9:30 AM
Christopher Mark Overall, University of British Columbia, Vancouver, BC, Canada
Among the most fundamental characteristics of a protein are the termini defining the start (amino (N) terminus) and end (carboxy (C) terminus) of the polypeptide chain. While genetically encoded, protein termini isoforms are also often generated during translation, following which, termini are highly dynamic, being frequently trimmed at their ends by a large array of exopeptidases. Neo-termini can also be generated by endopeptidases after precise and limited proteolysis, termed processing. Necessary for the maturation of many proteins, processing can also occur after synthesis and maturation, often resulting in dramatic functional consequences. For example chemokine signaling networks can be completely switched by inflammatory cell proteases. Hence, global approaches for the identification and analysis of protein terminal peptides provides key information on the stability and function of most proteins and hence, the functional state of the proteome. New proteomics methods will be presented that enrich and annotate terminomes for high throughput analyses of human tissues and cells for the Human Proteome Project, in particular for ch6 that is one of Canada’s aims for the HPP—Terminal Amino Isotopic Labelling of Substrates (TAILS, Kleifeld et al Nature Biotech 28, 281-288; Prudova et al 2010 Mol Cell Proteomics) and C-TAILS for the C-terminome (Schilling et al Nature Methods 2010). One unexpected result has been the recognition of the extensive number of “moonlighting” proteins—proteins traditionally thought to be located in one cellular compartment such as the cytosol, but which can also have bone fide and often completely different roles extracellularly, and vice versa. Our knowledgebase TopFIND (http://clipserve.clip.ubc.ca/topfind) integrates information on N and C-termini, protease cleavage sites and N-terminal modifications and reveals surprisingly widespread modification and truncation of N and C-termini in 4 model species. Notably, we find that one third of the stable chains in a proteome start distal to the expected protein maturation sites such as initiator methionine, signal peptide and pro-peptide removal points. Since the function of a protein can be entirely switched by proteolysis the implications of this are profound for understanding the functional state of a protein, its place in a network and hence the functional state of the proteome. Thus, knowledge of an organism from genomic data alone is far from complete—the proteome, as well as the termini of the constituent proteins must be recognized and the functional implications understood.