Dr. Overall’s Research

Résumé of Dr. Christopher M. Overall, BDS, BSc (Hons), MDS, PhD, FCAHS, FRSC

Numbers refer to the asterisked 20 Significant publications from his curriculum vitae.

1. General achievements and prior recognition

Professor Overall’s investigations and concepts of proteolytic enzymes and the target substrate proteins they cleave have profoundly changed our understanding of the widespread roles of proteases in health and disease and how they mould proteomes in which they are active. He is best known for two related but distinct scientific achievements. The first is his development of innovative methods for the global discovery of protease substrates in vivo. The most notable are cutting-edge proteomic techniques^15-20, which enabled the unbiased identification of protease cleavage sites and protein substrates in vivo for the first time. Thereby, he established the field of degradomics (Nature Reviews Molecular Cell Biology). The second is leveraging his suite of techniques to reveal new and often unexpected biological roles for proteases and their cleaved substrates in normal tissues and their aberrations in disease. His influence has been profound — without exaggeration, his techniques and concepts in proteolysis and proteomics have shaped the field in which he led this new area of investigation. Multiple groups globally now employ his approaches to unveil new roles for proteases and their cleaved substrates in vivo.

As with much of Dr. Overall’s work, his findings have had broad implications for drug targeting and have resolved perplexing results on drug side effects (Nature Reviews Cancer). Thus, he is at the forefront of formulating innovative concepts on drug targeting and the roles of proteolysis in disease and their interplay in controlling cell signalling pathways. His research has resulted in 314 papers, including 31 Nature (2), Science (2), and daughter journal (27) papers, most as senior PI. His papers are highly cited (>41,350 citations), yielding an h-index of 108. He has mentored 40 post-doctoral scientists and graduated 14 Ph.D. and 7 M.Sc. students to achieve their best—9 Full Professors, 5 Associate Professors, and 6 Assistant Professors. Indeed, Chris was awarded the UBC John McNeill Excellence in Health Research Mentorship Award in 2023.

Professor Overall holds influential roles on the executive of >10 international committees, and his peers elected him to organize and chair the premier conferences of his fields: the 2003 MMP and 2010 Protease Gordon Research Conferences and in 2017 he Co-Chaired the International Proteolysis Society (IPS) Meeting. International recognition of Professor Overall’s advances in proteomics and systems biology is reflected by his election in 2015 as Co-Chair and then as Chair (2019 –) of the Human Proteome Organization (HUPO) Chromosome-Centric Human Proteome Project (HPP). The HPP published the high-stringency draft of the human proteome in Nature Communications in 2020: he was the only Canadian author. In 2022, he was invited to represent UBC at the G7 Research Summit on One Health. He is the recipient of numerous recognitions, e.g., election to the Royal Society of Canada; he is a Yonsei Distinguished Scholar of Yonsei University, Korea, an Honorary Professor, Albert-Ludwigs University, Freiburg, DE and at UBC, he is a Distinguished University Scholar and received the 2006 Killam Research Prize. Among his many awards, several stand out, including the Canadian Institutes of Health Research Researcher of the Year (2002), the Helmholtz (2008), IPS Lifetime Achievement (2011), Matrix Biology Society of Australia and New Zealand Barry Preston (2012), and the IADR Distinguished Scientist (2013) Awards. His advances in proteomics have been recognized by the Canadian National Proteomics Network Tony Pawson Award (2014), the Proteomass Scientific Society Award (2017), the 2018 HUPO Discovery Award in Proteomics Sciences, and the 2022 Helmut Holzer Award. He has presented >296 keynote, plenary, and invited talks at international conferences and >250 seminars at universities, institutes, and companies.

2. Degradomics reveals new protein functions in vivo

Only 340/565 human proteases (Nature Reviews Genetics) have known substrates, and from this incomplete knowledge, >200 proteases lack biological roles (Nature Reviews Molecular Cell Biology). This inspired Dr. Overall to invent a suite of approaches¹⁴, techniques^15-20, and software^4,16 to identify protease substrates on a system-wide scale. 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 screen¹⁴ (Science)—at a time when protein disulphide cross-linkages were predicted to exclude the yeast two-hybrid approach for extracellular proteins. Dr. Overall showed that this was not a limitation in this yeast system¹⁴ with a paradigm shift that MMPs are tissue-protective by orchestrating neutrophil and macrophage leukocyte responses through activation/inactivation of virtually all chemoattractant cytokines known as chemokines^10,13,14, and in subsequent research, by multiple cytokines^6,8, their binding proteins, and serpin inhibitors^11,12.

Defining protease substrate cleavage-site specificity is central to protease characterization, linkage to substrates and drug development. In this way, he pioneered the first 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)¹⁹ (Nature Biotechnology, Nature Protocols). 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 one application of PICS, he reported >4,300 cleavage sites in the MMP family, leading to structural insight into their active sites. Reported in Nature Communications⁷, he identified >1000 cleavage sites by an unsuspected metallopeptidase insertion in bacterial flagellin, the monomer of bacterial flagella that propels bacteria through biofilms and tissues. These metalloproteinase-bearing flagellin molecules assemble proteolytically active flagella (~20 µm) in >350 diverse bacterial species, e.g., the pathogen Clostridium haemolyticum.

In conventional proteomics sample preparation for mass spectrometry, 100,000s of trypsin-generated peptides of a proteome dominated by abundant proteins dilute the terminal amino (N) and carboxyl (C) terminal protein ends and protease-generated ‘neo’-termini, rendering these terminal peptides rarely detectable. This is especially problematic for low-abundant transcription factors and signalling proteins like cytokines and antiviral interferons, impairing insight into disease-relevant proteolytic events. Dr. Overall’s technique, Terminal Amino Isotopic Labelling of Substrates (TAILS)¹⁸, circumvents these issues in a simple but powerful high-throughput method. TAILS purifies protein N-terminal peptides and cleaved neo-N-terminal peptides using an innovative aldehyde-polymer to simultaneously identify cleavage sites and substrates in native proteomes (Nature Biotechnology; Nature Protocols). Monitoring the end fragments of the cut proteins, also known as terminomics, is a powerful new way to monitor disease activity.

Protein C-termini are difficult to label chemically en route to identification that is also hampered by their absence of C-terminal lysine or arginine residues following trypsin digestion. Dr. Overall discovered a new “trypsin mirror” protease in Archaea, LysargiNase^TM, to address this¹⁵ (Nature Methods). Cutting before lysine or arginine, LysargiNase-digested proteins release C-terminal peptides with an N-terminal lysine or arginine, facilitating detection by shotgun proteomics or by C-terminal peptide enrichment for broad coverage using C-TAILS technique¹⁷ (Nature Methods).

To specifically analyze natural and neo-termini, Dr. Overall developed publicly available software: WebPICS, CLIPPER, and the Termini-orientated protein Function INferred Database (TopFIND v4.1)¹⁶ (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 network in humans¹² (PLoS Biology)—so also highlighting the problems in interpreting knockout or overexpression studies, as well as in drug targeting of proteases.

Fundamental to understanding zoonotic virus pathobiology, by a One Health approach in the pandemic, Dr. Overall applied his techniques to discover >300 host cell substrates for the SARS-CoV-2 3CL^pro, main protease¹ (Cell Reports) and its cleavage site specificity (J Virology). Thereby, he discovered that the cytosolic lectin, galectin-8, is a novel intracellular sensor of SARS-CoV-2 spike protein, but galectin-8 cleavage by SARS-CoV-2 3CL^pro defeats autophagic destruction of galectin-8-tagged virus. In expanding the degradome and interactome of 3CL^{pro 20} (Nature Communications), he revealed mechanisms by which this viral protease bypasses cellular control of signal transduction by interacting with and disrupting protein assemblies in the adherens junction, cytoskeleton and centrosome of infected human lung epithelial cells. Notably, 3CL^pro drives the formation of intercellular immune-privileged conduits known as tunnel nanotubes (TNT). Immunolocalization of virions with 3CL^pro substrates within TNTs suggests that 3CL^pro activity accelerates TNT formation for “stealthy” viral transmission, which impedes the effectiveness of immune recognition and vaccines intended to reduce viral transmission.

Employing degradomics, Dr. Overall has mechanistically dissected the crosstalk between proteolytic pathways in multiple systems including complement in skin inflammation⁸ (Science Signaling), lysosomal proteases in pancreatic cancer⁹ (Cell Reports), MMPs in HIV-Associated Dementia¹⁰ (Nature Neuroscience), anti-inflammatory activities of macrophage MMP12 in arthritis¹¹ (Cell Reports) and autoimmunity, e.g., lupus⁶ (Nature Communications), and MMP12’s unexpected potent antiviral roles⁵ (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 transcription factor regulating ~200 genes. By increasing transcription of I_κB_α—an inhibitor of the pro-inflammatory NF_κB—he found MMP12 was indispensable for IFN_α 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 mechanism of rapid cellular inhibition and removal of key cellular proteins for antiviral defence.

In related work, Dr. Overall found that macrophage MMP12 first stimulates secretion and then, over time, inactivates anti-viral interferon-α⁵ (Nature Medicine). Similarly, MMP12 inactivates interferon-γ, also by removing the receptor binding site⁶ (Nature Communications), providing feedback that drives the transition from pro-inflammatory IFN-γ-activated macrophages (formerly termed “M1”) to tissue-reparative immunosuppressant (“M2”) macrophages. In discovering new and major roles of MMPs in regulating signalling proteins, his studies reveal deeper complexity in regulating the extracellular matrix and the cell signalling environment than mere degradation.

In a groundbreaking example of clinical application, Professor Overall explored the roles of the intracellular protease MALT1, an essential transducer in lymphocyte antigen receptor signalling and immune activation² (Nature Communications). He found that independent of proteolytic cleavage, non-proteolytic protein-protein interactions by MALT1 initiate NF_κB activation, whereas, at late stages of NF_κB signalling, MALT1 cleaves HOIL1 in the Linear Ubiquitin Assembly Complex (LUBAC) to downregulate essential linear ubiquitination of pathway mediators to halt NF_κB signalling². By developing the algorithm GO-2-Substrates, he recently predicted and validated new substrates to double the MALT1 substrate repertoire⁴; 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 lymphomas. The multidisciplinary team he assembled also reported in Nature Chemical Biology³ the development of a series of potent nanomolar allosteric inhibitors of MALT1 that bind at Trp580. Remarkably, this is the same site as an immunodeficient patient’s MALT1 Trp580Ser mutation, whose markedly diminished levels of the mutant MALT1 led to 50% reduced NF_κB signalling and immunodeficiency. In an elegant use of chemical biology, Dr. Overall demonstrated that treatment with this inhibitor stabilized the mutant MALT1, restoring MALT protein levels to normal in the patient’s lymphocytes. Moreover, inhibitor treatment also restored NF_κB and JNK signalling in the patient’s B and T lymphocytes and, during treatment pauses, rescued substrate cleavage. Thus, a new low molecular weight pharmacological molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring therapies to restore enzyme activity to treat similar molecularly defined disorders.

3. Scientific impact

Dr. Overall’s techniques and concepts in proteolysis and proteomics have shaped our understanding of the widespread roles of proteases and triggered a new area of investigation in terminomics/degradomics. In accelerating our appreciation of the unexpected prevalence of precise proteolytic processing in vivo and its effects on protein function, he has been instrumental in transforming our understanding of mechanisms of disease pathogenesis. TAILS has repeatedly demonstrated the unanticipated abundance, from 45–60%, of stable cleaved protein ‘proteoforms’ co-existing with their full-length parent protein in cells and tissues. Given the numerous examples of proteolytically-induced altered functions of bioactive proteins, the pervasive occurrence of truncated proteins in proteomes reveals new levels of control of protein function with profound implications for molecular pathogenesis and patient diagnosis. As presented in six Nature Reviews articles and other reviews and editorials. Dr. Overall’s research and concepts continue to shape the fields of proteomics, proteolytic enzymes, and antiviral and molecular immunology toward a deeper understanding of new drivers of inflammatory, immune, and infectious diseases that are seminal for developing precise diagnostics and new treatments.

Publications

314 Career total, with an h-index = 108 and >41,350 citations—including 70 >100 – 199, 29 >200 – 499, 13 >500 – 999, 3 >1,200 – 1,500, and 1>1,700, including 31 high-impact Nature (2), Science (2), Cell and daughter journal (27) papers, most as senior PI. Trainees are underlined, ^Co-Senior Author,
* Selected as one of the 20 most significant publications.

1. * Pablos, I., Machado, Y., de Jesus, H.C.R., Mohamud, Y., Kappelhoff, R., Lindskog, C., Vlok, M., Bell, P.A, Butler, G.S., Grin, P.M., Cao, Q.T., Nguyen, J.P., Solis, N., Abbina, S., Rut, W., Vederas, J.C., Szekely, L., Szakos, A., Drag, M., Kizhakkedathu, J., Mossman, K., Hirota, J., Jan, E., Lou, H., Banerjee, A., and Overall, C.M. 2021. Mechanistic Insights into COVID-19 by Global Analysis of the SARS-CoV-2 3CL^pro Substrate Degradome. Cell Reports 37, Oct 26;37(4):109892. doi: 10.1016/j.celrep.2021.109892. Citations as of Sep. 21, 2024: 81
2. * 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, C.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. doi:10.1038/ncomms9777. Featured Article and Featured in Nature Immunology; Highlighted by Faculty of 1000 as a high significance paper. Citations as of Sep. 21, 2024: 164
3. * 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 15, 304 – 313. Citations as of Sep. 21, 2024: 59
4. * Bell, P.A., Scheuermann, S., Renner, F., Pan, C.L., Lu, H.Y., Turvey, S.E., Bornancin, F., Régnier, C.H., and Overall, C.M. 2022. Integrating Knowledge of Protein Sequence with Protein Function for the Prediction and Validation of New MALT1 Substrates. Computational and Structural Biotechnology Journal 20, 4,717 – 4,732 Citations as of Sep. 1, 2023: 5
5. * 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., and 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. Citations as of Sep. 21, 2024: 264
6. * 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. Citations as of Sep. 21, 2024: 76
7. * 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.
   Citations as of Sep. 21, 2024: 42
8. * 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 Signalling 6: rs2, 1 – 15. doi: 10.1126/scisignal.2003512. Featured cover.
   Citations as of Sep. 21, 2024: 107
9. * Prudova, A., Gocheva, V., auf dem Keller, U., 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, 1,762 – 1,773. Featured cover. Citations as of Sep. 21, 2024: 79
10. * Zhang, K., McQuibban, G.A., 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 and Communicating Authors.
    Nature Neuroscience 6, 1064 – 1071. Citations as of Sep. 21, 2024: 378
11. * Bellac, C.L., Dufour, A., Krisinger, M.J., Loonchanta, A., Starr, A.E., auf dem Keller, U., Lange, P.F., Goebeler, V., Kappelhoff, R., Butler, G.S., Burtnick, L.D., Conway, E.M., Roberts, C.R., and Overall, C.M. 2014. Macrophage Matrix Metalloproteinase-12 Dampens Inflammation and Neutrophil Influx in Arthritis.
    Cell Reports 9, 618 – 632. Citations as of Sep. 21, 2024: 108
12. * 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.
    Citations as of Sep. 21, 2024: 178
13. * 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. Citations as of Sep. 21, 2024: 297
14. * 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 Monocyte Chemoattractant Protein-3.
    Science 289, 1202 – 1206. Selected by the Faculty of 1000 Biology for its significance.
    Citations as of Sep. 21, 2024: 978
15. * Huesgen, P.F., Lange, P.F., Rogers, L.D., Solis, N., Eckhard, U., Kleifeld, O., Goulas, T., Gomis-Rüth, F.X., and Overall, C.M. 2015. LysargiNase Mirrors Trypsin for Protein C-Terminal and Methylation-Site Identification. Nature Methods 12, 55 – 58. Citations as of Sep. 21, 2024: 156
16. * Lange, P. and Overall, C.M. 2011. TopFIND, a Knowledgebase Linking Protein Termini with Function. Nature Methods 8, 703 – 704. Citations as of Sep. 21, 2024: 102
17. * 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 (Chemical & Engineering News). Citations as of Sep. 21, 2024: 165
18. * 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 Labelling of Terminal Amines in Complex Samples Identifies Protein N-Termini and Protease Cleavage Products.
    Nature Biotechnology 28, 281 – 288. Citations as of Sep. 21, 2024: 567
19. * Schilling, O. and Overall, C.M. 2008. Proteome-Derived Database-Searchable Peptide Libraries for Identifying Protease Cleavage Sites. Nature Biotechnology 26, 685-694. Designated in the Exceptional Category by the Faculty of 1000 Biology for its significance. Citations as of Sep. 21, 2024: 445
20. * Butler, G.S., Vlok, M., Cesar Ramos de Jesus, H., Kaushal B., Machado, Y., Pablos, I.M., Solis, N., Kappelhoff, R., Bell, P.A., Nore, L., Grin, P., Nguyen, J.P., Cao, Q.T., Lamar, T., Vuong, W., Webster, S.J., Vederas, J.C., Hirota., J.A., Banerjee, A., Jan, E., and Overall, C.M. 2024. SARS-CoV-2 Main Protease, 3CLpro, Drives Cytoskeletal Reorganization and Tunnelling Nanotube Formation for Stealth Intercellular Infection. Nature Communications, conditional acceptance. https://doi.org/10.21203/rs.3.rs-3918469/v1.