Canada Research Chair in Metalloproteinase Proteomics & Systems Biology.

Research Summary

The Overall Lab founded the field of ‘degradomics’. Degradomics is the application of genomic and proteomic techniques to determine protease and protease-substrate repertoires—or ‘degradomes’—on an cell, tissue and organism-wide scale. The Overall Lab has developed world-leading innovative quantitative proteomic techniques to enrich and identify proteolysis products in vivo. This awareness and understanding derived from application of “omics” technologies to complex biological systems has revolutionized the understanding of the roles of proteases and matrix metalloproteinases (MMP) in particular, in physiological and pathological processes in vivo.

For technical reasons protein cleavage is opaque to conventional proteomics, which limits functional insight. Therefore, to specifically enrich for mature protein N-termini and neo-N-termini of proteins we utilize 4-8plex iTRAQ and 10plex TMT TAILS (Terminal Amine Isotopic Labeling of Substrates) (Nature Biotech 28, 281-288 (2010); Nature Protocols 6, 1578-1611 (2011). Using quantitative proteomics information determined through TAILS, our lab’s signature approach, the protein substrate and the exact cleavage site are determined in the same analysis. By analyzing all N-termini, the original mature N terminus of a protein together with protease generated neo-N termini, the N-terminome of the tissue is elucidated. By then analyzing all quantifiable peptides that change in abundance significantly between samples, protein expression and new protease substrates are identified. On a global scale this generates the proteolytic signature of the tissue. The proteolytic signature can be used to identify protease networks in vivo that are operative in the system under study and to identify new disease biomarkers, with the advantage of being mechanistically informative. Resultant new drug targets and new clinical tests for early, accurate patient diagnosis can thereby by translated.

We now aim to develop clinically relevant system-wide knowledge of inflammation in humans in vivo e.g., in response to bacterial infection in placental chorioamnionitis and periodontal diseases, in viral infection, in chronic inflammatory diseases, e.g., arthritis and periodontitis, and in tumor-associated inflammation.

In analyzing 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.

To identify and understand the function of all proteins in the human body and to map the human proteome on a disease-centric and chromosome-centric basis, the international HUPO consortium was formed. By knowing all the proteins and their interactions and roles in pathways, knowledge will be gained about the fundamental ways cells and tissues function. Thus, our studies of human disease have the added value of contributing highly desired and unique knowledge of human protein N-termini to the Human Proteome Project (HPP) of HUPO.

Proteolysis 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 N terminal semi-tryptic peptides will exhibit beneficial m/z, 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 in the tissue identifies protein speciation and also obtains proof for the expression of ‘missing proteins’, particularly if rare tissues and cells are analyzed. In our HPP project, “Termini Orientated Proteomics-Human Amino Terminome” (TOP-HAT), we have identified >80 missing proteins in less commonly studied cells e.g. erythrocytes, platelets, placenta, gingiva, dental pulp and lymphocytes.

Recently we found that an extracellular protease (MMP12) secreted from macrophages targets virus-infected target cells where it segues to the nucleus (Nature Medicine 20, 493-502 2014). Here MMP12 acts as a transcription factor and cleaves intracellular substrates that are essential for IFN-alpha secretion and antiviral immunity. Hence, by proteomically identifying proteolytic networks by TAILS and their actions on signaling and cytokine pathways in cancer, inflammation, and infection we have uncovered remarkable roles for proteases inside, outside and outside-to-inside the cell.


Dr. Overall is a Professor and Tier 1 Canada Research Chair in Proteinase Proteomics and Systems Biology at the University of British Columbia, Centre for Blood Research. He completed his undergraduate, Honors Science and Masters degrees at the University of Adelaide, South Australia; his Ph.D. in Biochemistry at the University of Toronto; and was a MRC Centennial Fellow in his post-doctoral work with Dr. Michael Smith, Nobel Laureate, Biotechnology Laboratory, University of British Columbia (UBC). On Sabbatical in 1997-1998 he was a Visiting Senior Scientist at British Biotech Pharmaceuticals, Oxford, UK and in 2004/2008 he was a Visiting Senior Scientist at the Expert Protease Platform, Novartis Pharmaceuticals, Basel, Switzerland. At the Freiburg Institute for Advanced Studies, Albert-Ludwigs Universität Freiburg, Germany he was an External Senior Fellow, and in November 2014 was appointed as an Honorary Professor, Albert-Ludwigs Universität Freiburg. Dr. Overall was awarded the Institute of Musculoskeletal Health and Arthritis CIHR Award as 2002 CIHR Scientist of the Year, the UBC Killam Senior Researcher Award (Science) 2005, and was the Chair of the 2003 Matrix Metalloproteinase Gordon Research Conference and the 2010 Protease Gordon Research Conference. He was elected to the HUPO Council and to the Executive Committee of the Chromosome–Centric Human proteome Project (CHPP) in 2014. With over 12,626 citations for his 210 papers (since 2000 his papers have been cited >8,813 times) and with an h factor of 62 he is a highly influential scientist in the field. Professor Overall is also the pioneer of degradomics, a term he coined. With 22 Nature Review, Nature Journal, Cell Journal, Science and Science Signaling papers he is a leader in the field, which was recently recognized by the International Society of Proteolysis with a Lifetime Achievement Award in 2011; by the Matrix Biology Society of Australia and New Zealand with the 2012 Barry Preston Award; in 2013 by the IADR Distinguished Scientist Award for Research in Oral Biology; and in 2014 by the Tony Pawson Canadian National Proteomics Network Award for Outstanding Contribution and Leadership to the Canadian Proteomics Community. He is an Associate Editor of the Journal of Proteomics and on the Editorial Boards of the Journal of Molecular Cellular Proteomics and Biological Chemistry.


Selected Honors and Awards:

1984-1989 Postdoctoral Fellowship, MRC; 1987 Edward H. Hatton Award, 1st Place Postdoctoral, International Association for Dental Research (IADR); 1988 Young Investigator Award, 3rd Int. Conference Chemistry & Biology Mineralized Tissues; 1989 William J. Gies Award, AADR for the best publication in 1988; 1989 Young Investigator Award, MMP Conference; 1989-1992 Centennial Fellowship (1st place), MRC; 1991 Young Investigator Award, IADR (for best scientist under 35 years); 1999 Scientist Award, MRC; 2001 Tier 1 Canada Research Chair in Metalloproteinase Proteomics & Systems Biology; 2002 CIHR Researcher of the Year; 2003 CIHR Award for Research Excellence in Oral Health; 2003 Chairman, MMP Gordon Research Conference; 2004 Listed in the Contemporary Who’s Who; 2005 Fellow, Canadian Academy of Health Sciences; 2005 International Proteolysis Society Council; 2006 Killam Award Research Prize, Senior Science Category, UBC; 2010 Chairman, Protease Gordon Research Conference; 2011 Lifetime Achievement Award, International Proteolysis Society; 2012 Barry Preston Award (Lifetime Achievement Award in Matrix Biology) Matrix Biology Society Australia & NZ; 2013 Distinguished Scientist Award, IADR; 2014 the Tony Pawson Award for Outstanding Contribution and Leadership to the Canadian Proteomics Community; elected member of HUPO Council; elected Executive Committee Member of the HUPO Chromosome-Centric Human Proteome Project (C-HPP).


Selected peer-reviewed publications
40 papers in past three years; 71 since 2010, career total 210.

1. Huesgen, P.F., Lange, P.F., Rogers, L.D., Solis, N., Eckhardt, U., Kleifeld, O., Goulas, T., Gomis-Rüth, F.X., and Overall, C.M. 2015. LysargiNase Mirrors Trypsin for Protein C-Termini and Methylation Sites Identification. Nature Methods 12, 55-58.
2. Fortelny, N., Yang, Sh., Pavlidis, P., Lange, P.F., and Overall, C.M. 2015. Proteome TopFIND 3.0 and TopFINDer: Database and Analysis Tools for the Association of Protein Termini to Pre- and Post-translational Events. Nucleic Acids Research (Database Issue), in press.
3. Marchant, D.J., Bellac, C., Moraes, T.J., Wadsworth, S.J., Dufour, A., Butler, G.S., Bilawchuk, L.M., Hendry, R.G., Robertson, A.G., Cheung, C.T., Ng, J., Ang, L., Luo, Z., Heilbron, K., Norris, M.J., Duan, W., Bucyk, T., Karpov, A., Devel, L., Georgiadis, D., Hegele, R.G., Luo, H., Granville, D.J., Dive, V., McManus, B.M., Overall, C.M. 2014. A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity. Nature Medicine 20, 493-502. doi 10.1038/nm.3508. (Featured Article in News and Views).
4. Fortelny, N., Cox, J.H., Kappelhoff, R., Starr, A.E., Lange, P.F., Pavlidis, P., and Overall, C.M. 2014. Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web.
PLoS Biology 12, e1001869. doi:10.1371/journal.pbio.1001869. (Featured Weekly Editors Pick).
5. Cruz, R., Huesgen, P., Riley, S.P., Wlodawer, A., Faro, C., Overall, C.M., Martinez, J.J., and Simoes, I. (2014) RC1339/APRc from Rickettsia conorii Is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes. PLoS Pathogens 10, e1004324. doi: 10.1371/journal.ppat.1004324.
6. Bellac, C.L., Dufour, A., Krisinger, M.J., Roberts, C.R., Loonchanta, A., Butler, G.S., Starr, A.E., Lange, P.F., auf dem Keller, U., Goebeler, V., Kappelhoff, R., Burtnick, L.D., Conway, E.M., and Overall, C.M. 2014. Macrophage Matrix Metalloproteinase-12 Dampens Inflammation and Neutrophil Influx in Arthritis.
Cell Reports 9, 618-632.
7. Prudova, A., Serrano, K., Eckhard, U., Fortelny, N., Devine, D., and Overall, C.M. 2014. TAILS N-Terminomics of Human Platelets Reveals Pervasive Metalloproteinase-Dependent Proteolytic Processing in Storage.
e-Blood, 124 (26), e49-e60.
8. Lange, P., Huesgen, P.F., Nguyen, K., and Overall, C.M. 2014. Annotating N termini for the Human Proteome Project: N termini and N-acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome. Journal of Proteomics Research 13, 2028–44
9. auf dem Keller, U., Prudova, A., Eckhard, U., Fingleton, B., and Overall, C.M. 2013. Systems-Level Analysis of Proteolytic Events in Increased Vascular Permeability and Complement Activation in Skin Inflammation. Science Signaling 6, rs2, 1-15: DOI: 10.1126/scisignal.2003512.
10. Dufour, A. and Overall, C.M. 2013. Missing the Target: Matrix Metalloproteinase Anti-Targets in Inflammation and Cancer. Trends in Pharmacological Sciences 34, 233-242. Invited Review (Cover).
11. Lange, P. and Overall, C.M. 2013. The TAILS of Proteins: Protein Termini Tell Tales of Proteolysis and Protein Function. Current Opinion in Chemical Biology 17, 73-82. Edited by Bogyo, M. and Rudd, P. Published by Elsevier (Amsterdam, Netherlands) j.cbpa.2012.11.025.
12. Wilson, C.H., Indarto, D., Doucet, A., Pogson, L.D., Pitman, M.R., McNicholas, K., Menz, R.I., Overall, C.M.*, and Abbott, C.A.* 2013. Identifying Natural Substrates for Dipeptidyl Peptidases 8 and 9 using Terminal Amine Isotopic Labeling of Substrates (TAILS) Reveals in Vivo Roles in Cellular Homeostasis and Energy Metabolism. * Joint Senior Authors. Journal of Biological Chemistry 288, 13,936-13,949. Paper of the Week.
Selected by the Faculty of 1000 Biology for its significance.
13. Broder, C., Arnold, P., Vadon-Le Goff, S., Konerding, M., Bahr, K., Müller, S., Overall, C.M., Bond, J.S., Koudelka, T., Tholey, A., Hulmes, D.J., Moali, C., Becker-Pauly, C. 2013. Metalloproteases Meprin α and Meprin β are C- and N-Procollagen Proteinases Important for Collagen Assembly and Tensile Strength. Proceedings National Academy of Sciences (U.S.A.) 110,14219-14224.
14. Lange, P., Huesgen, P. and Overall, C.M. 2012. TopFIND 2.0—Linking Protein Termini with Proteolytic Processing and Modifications Altering Protein Function. Nucleic Acids Research, 40, D351-61.
15. Starr, A.E., Dufour, A., Maier, J., and Overall, C.M. 2012. Biochemical Analysis of Matrix Metalloproteinase Activation of Chemokines CCL15 and CCL23 and Increased Glycosaminoglycan Binding of CCL16. Journal of Biological Chemistry 287, 5,848-5,860.
16. Lange, P. and Overall, C.M. 2011. TopFIND, a Knowledgebase Linking Protein Termini with Function.
Nature Methods 8, 703-704.
17. Kleifeld, O., Doucet, A., Prudova, A., auf dem Keller, U., Gioia, M., Kizhakkedathu, J., and Overall, C.M. 2011. System-Wide Proteomic Identification of Protease Cleavage Products by Terminal Amine Isotopic Labeling of Substrates. Nature Protocols 6, 1578-1611.
18. Schilling, O., Huesgen, P.F., Barré, O., auf dem Keller, U., and OveralI, C.M. 2011. Characterization of the Prime and Non-Prime Active Site Specificities of Proteases by Proteome-derived Peptide Libraries and Tandem Mass Spectrometry. Nature Protocols 6, 111-120.
19. Morrison, C.J., Mancini, S., Kappelhoff, R., Cipollone, J., Roskelley C., and Overall, C.M. 2011. Microarray and Proteomic Analysis of Breast Cancer Cell and Osteoblast Co-cultures: The Role of Osteoblast Matrix Metalloproteinase (MMP)-13 in Bone Metastasis. Journal of Biological Chemistry 286, 34,271-34,285.
20. Doucet, A., and Overall, C.M. 2011. Broad Coverage Identification of Multiple Proteolytic Cleavage Sites Using Proteomics as a Complement to Edman Sequencing. Mol Cell Proteomics 10, M110.003533 1-12.
21. Becker-Pauly, C., Barré, O., Schilling, O., auf dem Keller, U., Broder, C., Schütte, A., Kappelhoff, R., Stöcker, W. Overall, C.M. 2011. Proteomic Analyses Reveal an Acidic Prime Side Specificity for the Astacin Metalloprotease Family Reflected by Physiological Substrates. Mol Cell Proteomics 10, M111.009233 1-19.
22. Schilling, O., Barré, O., Huesgen, P.F., and Overall, C.M. 2010. Proteome-wide Analysis of Protein Carboxy Termini: C Terminomics. Nature Methods 7, 508-511. Featured in C&EN.
23. Kleifeld, O., Doucet, A., auf dem Keller, U., Prudova, A., Schilling, O., Kainthan, R.K., Starr, A., Foster, L.J., Kizhakkedathu, J.N., and Overall, C.M. 2010. Isotopic labeling of Terminal Amines in Complex Samples Identifies Protein N-termini and Protease Cleavage Products. Nature Biotechnology 28, 281-288.
24. auf dem Keller, U., Bellac, C., Li, Y., Lou, Y., Lange, P., Ting, R., Harwig, C., Kappelhoff, R., Dedhar, S., Adam, M., Ruth, T.J., Bernard, F., Perrin, D., Overall, C.M. 2010. Novel MMP Inhibitors: [18F]-Marimastat-Aryltrifluoroborate as a Probe for in vivo PET Imaging in Cancer. Cancer Research 70, 7,562-7,569.
25. Prudova, A., auf dem Keller, U., Butler, G.A., and Overall, C.M. 2010. Multiplex N-Terminome Analysis of MMP-2 and MMP-9 Degradomes by iTRAQ-TAILS Quantitative Proteomics. Mol Cell Proteomics 9, 894-911. Cox, J.H., Starr, A.E., Kappelhoff, R., Yan, R., Roberts, C.R., Overall, C.M. 2010. Matrix Metalloproteinase-8 Deficiency Exacerbates Inflammatory Arthritis Through Delayed Neutrophil Apoptosis and Reduced Caspase-11 Expression. Arthritis and Rheumatism 62, 3,645-3,655. Editorial Highlighted.
26. auf dem Keller, U., Prudova, A., Gioia, M., Butler, G.S., and Overall, C.M. 2010. Quantitative N-Terminome Analysis and Identification of Protease Cleavage Products. Mol Cell Proteomics 9, 912-27.
27. Butler, G.S. and Overall, C.M. 2009. Proteomic Identification of Multitasking Proteins in Unexpected Locations Complicates Drug Targeting. Nature Reviews Drug Discovery 8, 935-948. Featured Cover.
28. Schilling, O. and Overall, C.M. 2008. Proteome-derived Database Searchable Peptide Libraries for Identifying Protease Cleavage Sites. Nature Biotechnology 26, 685-694.
29. Doucet, A., Butler, G.S., Rodríguez, D., Prudova, A., and Overall, C.M. 2008. Metadegradomics: Towards Quantitative Degradomics of Proteolytic Post-Translational Modifications of the Cancer Secretome. Molecular Cellular Proteomics 7, 1925-1951.
30. Dean, R.A., Cox, J.H., Bellac, C.L., Doucet, A., Starr, A.E., and Overall, C.M. (2008). Macrophage-specific Metalloelastase (MMP-12) Truncates and Inactivates ELR+ CXC Chemokines and Generates CCL2, 7, 8, and 13 Antagonists: Potential Role of the Macrophage in Terminating PMN Influx. Blood 112, 3444-3453.
31. Overall, C.M. and Blobel, C.P. 2007. In Search of Partners: Linking Extracellular Proteases to Substrates. Nature Reviews Molecular Cell Biology 8, 245-257.
32. Wolf, K., Wu, Y.I., Liu, Y., Geiger, J., Tam, E., Overall, C.M., Stack, M.S., Friedl, P. 2007. Pericellular Proteolysis Controls for the Transition from Individual to Cancer Cell Invasion. Nature Cell Biology 9, 893-04.
33. Dean, R.A. and Overall, C.M. 2007. Proteomic Discovery of Metalloprotease Substrates in the Cellular Context by iTRAQ Labeling Reveals A Diverse MMP-2 Substrate Degradome. Mol Cell Proteomics 6, 611-623.
34. Dean, R.A., Butler, G.S., Hamma-Kourbali, Y., Delbé, J., Brigstock, D.R., Courty, J., and Overall, C.M. 2007. Identification of Candidate Angiogenic Factors Processed by Matrix Metalloproteinase-2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/ Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis. Molecular Cellular Biology 27, 8454-8465.
35. Overall, C.M. and Kleifeld, O. 2006. Validating MMPs as Drug Targets and Anti-targets for Cancer Therapy. Nature Reviews Cancer 6, 227-239.
36. Tam, E.M., Morrison, C.M., Wu, Y., Stack, S., and Overall, C.M. 2004. Membrane Protease Proteomics: Isotope Coded Affinity Tag/Tandem Mass Spectrometry Identification of Undescribed MT1-MMP Substrates, Proceedings National Academy of Sciences U.S.A. 101, 6917-6922.
37. Puente, X.S., Sanchez, L.M., Overall, C.M., and López-Otín, C. 2003. Human and Mouse Proteases: A Comparative Genomic Approach. Nature Reviews Genetics 4, 544-558.
38. Zhang, K., McQuibban, G.S., Silva, C., Butler, G.S., Johnston, J.B., Holden, J., Clark-Lewis, I., Overall, C.M.*, and Power, C.* 2003. HIV-Induced Metalloproteinase Processing of the Chemokine Stromal Cell Derived Factor-1 Causes Neurodegeneration. (*Joint Senior Authors) Nature Neuroscience 6, 1064-71.
39. Balbín, M., Fueyo, A., Tester, A.M., Pendás, A. M., Pitiot, A.S., Astudillo, A., Overall, C.M., Shapiro, S. and López-Otín, C. 2003. Loss of Collagenase-2 Confers Increased Skin Tumor Susceptibility to Male Mice. Nature Genetics 35, 252-257.
40. López-Otín, C. and Overall, C.M. 2002. Protease Degradomics: A New Challenge for Proteomics. Invited Review. Nature Reviews Molecular Cell Biology 3, 509-519.
41. Overall, C.M. and López-Otín, C. 2002. Strategies for MMP Inhibition in Cancer: Innovations for the Post-Trial Era. Invited Review. Nature Reviews Cancer 2, 657-672.
42. McQuibban, G.A., Gong, J.-H., Tam, E., McCulloch, C.A.G., Clark-Lewis, I., and Overall, C.M. 2000. Inflammation Dampened by Cleavage of Monocyte Chemoattractant Protein-3. Science 289, 1202-06.

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