SYSTEMS BIOLOGY OF THE PROTEASE WEB
Degradomics reveals new protein functions in vivo
Professor Overall has invented a suite of approaches and techniques, and developed software tools for the comprehensive analysis of proteases and their inhibitors on a system-wide scale. Only 340/565 human proteases (Nature Reviews Genetics) have known substrates and hence biological roles (Nature Reviews Molecular Cell Biology). Recognizing the importance of substrate-binding exosite domains on proteases, he was the first to use these as substrate ‘baits’ in a yeast two-hybrid screen (Science), at a time when protein disulphide cross-linkages were predicted to exclude the yeast two-hybrid approach for extracellular proteins. He showed that this was not a limitation in this yeast system, and that MMPs were ill-conceived drug targets with a paradigm shift that MMPs are unexpectedly beneficial in most diseases. He did so by demonstrating that virtually all human chemokines are specifically clipped by MMPs. Thus, MMPs are now regarded as key in orchestrating protective white blood cells in disease.
Defining protease substrate cleavage-site specificity is central to protease characterization and linkage to substrates. In this way he developed the only peptide library technique to simultaneously identify both the amino (P) and carboxyl (P’) amino acid residues flanking the cleavage site—Proteomic Identification of Cleavage Specificity (PICS) (Nature Protocols; Nature Biotechnology). With >14,000 cleavage sites identified by PICS in the protease-database MEROPS, its head curator described PICS as “the new gold-standard for profiling proteases”. In a recent application of PICS, he reported the unsuspected occurrence of an active metallopeptidase insertion in bacterial flagellin, the monomer of bacterial flagella that propels bacteria through biofilms and tissues. In identifying >1000 cleavage sites by PICS, he showed these metalloproteinase bearing flagellin assemble proteolytically active flagella and thereby nature’s largest (~20 µm) proteolytic polymer in >200 diverse bacterial species, e.g. the pathogen Clostridium haemolyticum (Nature Communications).
In conventional proteomics sample preparation for mass spectrometry, 100,000s of trypsin-generated peptides of a proteome dominated by abundant proteins dilutes the terminal peptides of protein N- and C-ends and protease-generated ‘neo’-termini, rendering cleaved peptides rarely detectable. This is especially problematic for low abundant signalling proteins like chemokines, cytokines and transcription factors, impairing insight into disease-relevant proteolytic events. Professor Overall’s technique, Terminal Amino Isotopic Labelling of Substrates (TAILS), circumvents these issues in a simple but powerful high-throughput method. TAILS purifies protein N-terminal peptides and cleaved neo-N-terminal peptides using innovative polymers-for-proteomics to simultaneously identify cleavage sites and hence substrates in native proteomes (Nature Biotechnology; Nature Protocols). Protein C-termini are difficult to label chemically en route to identification, which is further hampered by their absence of basic residues following trypsin digestion. Dr. Overall discovered a new protease in Archaea, LysargiNaseTM, to address this (Nature Methods). LysargiNase-digested proteins release C-terminal peptides that retain an N-terminal lysine or arginine enabling their ready detection in shotgun proteomics analyses and with higher coverage by C-terminal peptide enrichment by his C-TAILS technique (Nature Methods).
To analyse natural and neo-termini, he developed publicly-available software: WebPICS, CLIPPER, and the Termini-orientated protein Function INferred Database (TopFIND) (Nature Methods) with >290K termini/>33K cut-sites, receiving >4K hits p.a. PathFINDer and TopFINDer map substrates to protease pathways, which revealed a highly connected protease web in humans (PLoS Biology), so also highlighting the problems in interpreting knockout or over expression studies, as well as in drug targeting of proteases.
Dr. Overall has applied his degradomics platform to mechanistically dissect the roles of cathepsins in pancreatic cancer (Cell Reports), proteolytic pathways in skin inflammation (Science Signaling), anti-inflammatory activities of macrophage MMP12 in arthritis (Cell Reports) and in autoimmunity including lupus (Nature Communications), and MMP12’s unexpected potent antiviral roles (Nature Medicine). In this latter work he made a remarkable discovery that secreted MMP12 is a moonlighting protease (Nature Reviews Drug Discovery) that re-enters cells and traffics to the nucleus as a novel transcription factor regulating ~200 genes. By increasing transcription of IkBα—an inhibitor of the pro-inflammatory transcription factor NFkB—he found MMP12 was indispensable for IFNα secretion. He further identified multiple substrates of MMP12 whose gene transcription was repressed by nuclear MMP12—thereby demonstrating concerted dual-negative regulation of both protein substrates and their genes, highlighting a novel mechanism of a rapid cellular inhibition and removal of targeted cellular viral defence substrates.
In related work, he found that macrophage MMP12 first stimulates secretion, then over time inactivates, anti-viral interferon-α (Nature Medicine). MMP12 also inactivates interferon-γ by removing the receptor binding site (Nature Communications), providing feedback that drives transition from pro-inflammatory IFN-γ–activated (“M1”) macrophages to tissue-reparative immunosuppressant (“M2”) macrophages. In discovering new major roles of MMPs in regulation of signalling proteins, his studies reveal deeper complexity in the regulation of the extracellular matrix and cell signalling environment than just degradation.
Recently, Professor Overall explored the roles of the intracellular protease MALT1, an essential transducer in lymphocyte antigen receptor signalling and immune activation (Nature Communications). His team found that independent of proteolytic cleavage, non-proteolytic protein-protein interactions by MALT1 initiates NFkB activation, whereas at late stages of NFkB activation, MALT1 cleaves HOIL1 in the Linear Ubiquitin Assembly Complex (LUBAC) to downregulate essential linear ubiquitination of pathway mediators to halt NFkB signalling. Thereby, he upended the concept that MALT1 is solely an enhancer of antigen-driven signalling—with far-reaching consequences for our understanding of immunobiology that has major implications in MALT1 drug targeting for lymphoma. The multidisciplinary team he has assembled and leads have now developed a series of potent allosteric inhibitor drugs of MALT1, which bind at Trp580—the same site as the immunodeficient patient’s MALT1 Trp580Ser mutation. In an elegant use of chemical biology, Overall then found that inhibitor treatment rescued protein stability of the mutant MALT1, to restore MALT protein levels to normal in the patient’s cells. His inhibitor treatment also rescued NFkB and JNK signalling in the patient’s immune cells as well as substrate cleavage upon inhibitor washout (Nature Chemical Biology). Thus, his new molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring precision therapies to increase mutant enzyme activity and for treating similar molecularly-defined human disorders.
TAILS repeatedly demonstrate the unexpected in vivo abundance, from 45–60%, of stable cleaved protein ‘proteoforms’ co-existing with their full-length parent protein in cells and tissues. This reveals pervasive, functionally relevant protein processing in normal and diseased tissues. In view of the numerous examples of protease altered functions of bioactive proteins, the widespread occurrence of truncated proteins in proteomes has revealed a level of control of protein function that is often not considered in cell biology—yet one that is important with profound implications for molecular pathogenesis and patient diagnosis
Research Trainees and Presentations
Since 2007 we have disseminated our findings in 107 papers, including 10 in Nature Journals, Science Journals and 2 invited reviews in high impact Nature Review journals, and by 88 invited plenary talks at conferences and 59 invited seminars. Since 2007 20 post-doctoral scientists, 6 Ph.D. and M.Sc. students, 2 undergraduate thesis projects, and 8 Work-Study undergraduates have been trained in the Overall Lab. Since 2000, 8 post-doctoral trainees and 3 graduate students have held international or national fellowships and scholarships. Our trainees are encouraged to present at national and international meetings and since 2000 Overall Lab trainees have presented 219 talks and posters and of the 37 trainees in my lab at UBC, 31 of whom remain in science: 2 are Departmental Chairs, 4 are Full or Associate Professors, 4 are Assistant Professors or equivalent, 8 are scientists in industry, and 13 are at the next stage in training, e.g., Ph.D., post-doctoral fellows or research associates.
The Overall Laboratory has helped shape the current view of MMPs as key regulators of multiple signaling pathways that are integral to innate immunity rather than just dowdy degraders of the extracellular matrix (Butler & Overall 09 Nature Rev Drug Disc). We were at the forefront in this revision of in vivo roles for MMP with the first use of yeast 2-hybrid substrate screens for protease substrate discovery, that identified chemokines and CCN cytokines as novel MMP substrates (McQuibban et al 00 Science). Next we adapted proteomics for substrate discovery, but soon recognized inadequacies for the specialized tasks of substrate and cleavage site identification (López-Otín & Overall 02 Nature Rev Mol Cell Biol; Overall & Blobel 07 Nature Rev Mol Cell Biol). So, we initiated the new field of degradomics in 2000 to describe all genomic and proteomic investigations of proteases, their inhibitors and substrates. Following this paper in Science (McQuibban et al 00 Science) Dr. Carlos López-Otín (Chair of the 2007 MMP Gordon Research Conference) and Dr. Chris Overall wrote an invited review in Nature Reviews Molecular Cell Biology formally introducing the term degradomics and describing approaches to study proteolysis on a system-wide scale (López-Otín & Overall 02 Nature Rev Mol Cell Biol). This was updated by another paper in Nature Reviews Molecular Cell Biology, co-authored with Dr. Carl Blobel (Chair of the 2009 MMP Gordon Research Conference), describing proteomic and other innovative techniques to link proteases with substrates (Overall & Blobel 07 Nature Rev Mol Cell Biol). In Nature Reviews Genetics we annotated the complete human and mouse protease and inhibitor degradomes (Puente et al 03 Nature Rev Genetics).
We are leading the development of degradomics and its application to protease substrates. Initially, our quantitative proteomics experiments used isotope-coded affinity tags (ICAT) for substrate discovery (Tam et al 04 PNAS; Dean et al 07 Mol Cell Biol) and to analyze effects of MMP inhibitor drugs on cancer cells (Butler et al 07, 08 Mol Cell Biol). We then identified MMP substrates in cell culture (Dean & Overall 07 MCP; Prudova et al 10 MCP; Morrison et al 11 JBC; a.d.-Keller et al 12) using 4- or 8-plex iTRAQ labels. We developed PICS (proteomic identification of protease cleavage sites) (Schilling & Overall 08 Nature Biotech; Schilling et al 11a, b Nature Protocols) to profile the cleavage site specificity of 16 MMPs, most astacins (Becker-Pauly et al 11 MCP) and proteases of 4 classes using our new powerful web-based data processing site, WebPICS (Schilling et al 11), contributing >8000 cleavage sites to MEROPS, the protease database.
For identification of the substrate and the precise cleavage site, we developed the effective Terminal Amine Isotopic Labeling of Substrates (TAILS) for proteomics analysis of protease-generated (neo)-N-termini of natural substrates (Kleifeld et al 10 Nature Biotech; Doucet et al 10; Kleifeld et al 11 Nature Protocols). The tryptic peptides of proteomes are selectively removed leaving isotopic-labelled natural and neo-N-termini for MS/MS identification. To do so we patented a new class of polymer that we continue to enhance for proteomics (Beaudette et al 11). To complement N-TAILS we developed C-TAILS to identify the carboxy-termini of substrates and the C-terminome (Schilling et al 11, 10 Nature Methods). TAILS was improved using iTRAQ labels thereby enabling simultaneous analysis of up to 8 samples (Prudova et al 10 MCP). And now 10 samples by 10plex TMT Tags (Klein et al 14). We developed bioinformatics software (CLIPPER) for statistically valid identification of cleavage sites (a.d.-Keller et al 10, 12) and integrated our data into a KnowledgeBase of all protein termini and cleavage sites, TopFIND (Lange & Overall 12, 11 Nature Methods), and mathematically modeled the network of protease cascades and pathways (Fortelny et al 14, PLoS Biology) that interconnect to form the ‘protease web’ (Overall & Kleifeld 06a, Nature Rev Cancer). In validating the new protease substrates, limitations in Edman sequencing of cleavage products in gels triggered the refinement of MALDI-TOF methods for cleavage site identification (Starr & Overall 09) and a new sequencing method for proteins in solution: Amino Terminal Orientated Mass spectrometry of Substrates (ATOMS) (Doucet & Overall 11a,b).
Little is known of the key transcriptomic, proteomic, and proteolytic modification differences between the cells and extracellular signaling networks in human disease on a system-wide scale. We have now perfected these approaches for in vivo application and analyzed wild type vs. Mmp-/- mice in a variety of murine models of inflammation including skin (a.d.-Keller et al 13 Science Signaling) and arthritis in Mmp8-/- (Cox et al 10) and Mmp12-/- mice (Bellac et al 14). Thereby, we uncovered beneficial activities of MMPs that dampen inflammation, e.g., inactivation of most CCL monocyte chemokines by MMPs (Starr et al 12a, b JBC), including CCL7 in arthritis (McQuibban et al 00 Science), and of all CXCL PMN chemokines by MMP12 (Dean et al 08 Blood). This stirred our thinking to conceive drug anti-target (Overall & Kleifeld 06 Nature Rev Cancer; Dufour & Overall 13) vs. drug target concepts (Overall & López-Otín 02 Nature Rev Cancer). We also reported that the macrophage secreted MMP12 translocates to the nucleus of viral infected cells, where it binds the IκBα promoter, up-regulating its transcription. IκBα activation was essential for IFNα secretion and survival. MMP12 also clears systemic IFNα, forming a negative feedback loop. This is blocked by a drug we patented that does not cross the cell membrane and thus spares the anti-target activities of MMP12 and so is protective in vivo against viral infection (Marchant et al 14 Nature Medicine).
1. Quancard, J., Klein, T., Fung, S-Y., Renatus, M., Hughes, N., Israël, L., Priatel, J.J., Kang, S., Blank, M.A., Viner, R.I., Blank, J., Schlapbach, A., Erbel, P., Kizhakkedathu, J., Villard, F., Hersperger, R., Turvey, S.E., Eder, J., Bornancin, F., and Overall, C.M. 2019. An Allosteric MALT1 Inhibitor is a Molecular Corrector Rescuing Function in an Immunodeficient Patient. Nature Chemical Biology, doi 10.1038/s41589-018-0222-1.
2. Zeglinski, M.R., Butler, G.S., Overall C.M.^, and Granville, D.J.^ 2019. Therapeutic Modulation of Granzymes and Matrix Metalloproteases: Rerouting Dogma. Nature Reviews Drug Discovery, Invited Review, Submitted. ^Co-Senior Author.
3. Dufour, A., Bellac, C.L, Eckhard, U., Solis, N., Klein, T., Kappelhoff, R., Fortelny, N., Jobin, P., Rozmus, J., Mark, J., Pavlidis, P., Dive, V., Barbour, S.J., and Overall, C.M. 2018. C-Terminal Truncation of IFN-γ Inhibits Proinflammatory Macrophage Responses and is Deficient in Autoimmune Disease. Nature Communications 9: 2416, 1–18. doi: 10.1038/s41467-018-04717-4.
4. Klein, T., Eckhard, U., Dufour, A., Solis, N., and Overall, C.M. 2018. Proteolytic Cleavage—Mechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification. Chemical Reviews 118, 1137-1168. [IF47.93].
5. Fortelny, N., Overall, C.M., Pavlidis, P., and Cohen Freue, G.V. 2017. Can we Predict Protein from mRNA Levels? Nature 547, E19-E22. doi: 10:1038/nature23293
6. Eckhard, U., Bandukwala, H., Mansfield, M.J., Marino, G., Cheng, J., Wallace, I., Holyoak, T., Charles, T.C., Austin, J., Overall, C.M.*, and Doxey, A.C.* 2017. Discovery of a Proteolytic Flagellin Family in Diverse Bacterial Phyla that Assembles Enzymatically Active Flagella. Nature Communications 8:521, 1-9. DOI: 10.1038/s41467-017-00599-0. * Joint Shared Senior Authors.
7. Fortelny, N., Butler, G.S., Overall, C.M.* and Pavlidis, P.* 2017. Protease-inhibitor Interaction Predictions: Lessons on the Complexity of Protein-protein Interactions. Molecular Cellular Proteomics 16.6, 1038-1051, *Joint Shared Senior Authors. Featured Editors Pick.
8. Scott, N.E., Rogers, L.D., Prudova, A., Brown, N.F., Fortelny, N., Overall, C.M., and Foster, L.J. 2017 Interactome Disassembly During Apoptosis Occurs Independent of Caspase Cleavage. Molecular Systems Biology 13, 906, 1-22, doi: 10.15252/msb.20167067
9. Prudova, A., Gocheva, V., Keller, U. a-d, Eckhard, U., Olson, O., Akkari, L., Butler, G.S., Fortelny, N., Lange, P.F., Mark, J., Joyce, J., and Overall, C.M. 2016. TAILS N-terminomics and Proteomics Show Protein Degradation Dominates Over Proteolytic Processing by Cathepsins in Pancreatic Tumors. Cell Reports 16, 1762-1773, Featured cover. http://dx.doi.org/10.1016/j.celrep.2016.06.086
10. Klein, T., Fung, S.Y., Renner, F., Blank, M.A., Dufour, A., Kang, S., Bolger-Munro, M., Scurll, J.M., Priatel, J.J., Schweigler, P., Melkko, S., Gold, M.S., Viner, R.I., Régnier, S.H., Turvey, S.E., and Overall, C.M. 2015. The Paracaspase MALT1 Cleaves HOIL1 Reducing Linear Ubiquitination by LUBAC to Dampen Lymphocyte NF-κB Signalling Nature Communications 6, 8777. 1-17. (Featured Article and Featured in Nature Immunology)
11. 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.
12. 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 43 (Database issue), D290-297.
13. 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.
14. 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)
15. 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).
16. 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.
17.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 Photo).
18. Lange, P. and Overall, C.M. 2011. TopFIND, a Knowledgebase Linking Protein Termini with Function. Nature Methods 8, 703-704.
19. 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.
20. 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.
21. 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.
22. Kleifeld, O., Doucet, A., auf dem Keller, U., Prudova, A., Schilling, O., 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.
23. 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.
24. Schilling, O. and Overall, C.M. 2008. Proteome-derived Database Searchable Peptide Libraries for Identifying Protease Cleavage Sites. Nature Biotechnology 26, 685-694.
25. 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.
26. Wolf, K., Wu, Y.I., Liu, Y., Geiger, J., Tam, E., Overall, C.M., Stack, M.S., Friedl, P. 2007. Multi-step Pericellular Proteolysis Controls for the Transition from Individual to Collective Cancer Cell Invasion. Nature Cell Biology 9, 893-904.
27. Overall, C.M. and Kleifeld, O. 2006. Validating MMPs as Drug Targets and Anti-targets for Cancer Therapy. Nature Reviews Cancer 6, 227-239.
28. 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.
29. 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.
30. 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 SDF-1 Causes Neurodegeneration. (*Joint Senior Authors) Nature Neuroscience 6, 1064-71.
31. 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.
32. 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.
33. 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.
34. McQuibban, G.A., Gong, J.-H., Tam, E., McCulloch, C.A.G., Clark-Lewis, I., and Overall, C.M. 2000. Inflammation Dampened by Gelatinase A Cleavage of MCP-3. Science 289, 1202-1206.