overall lab research interests

research themes

  • Matrix metalloproteinase (MMP) substrate discovery using proteomic and yeast two-hybrid approaches.
  • Protease degradomics and associated mass spectrometric and protease chip technology development.
  • Structural and functional analysis of MMPs and tissue inhibitor of metalloproteases (TIMPs).
  • De novo protease construction: Engineering novel catalytic activity onto inert scaffolds.
  • Chemokine processing by MMPs and its role in cancer, HIV, and inflammation.
  • Basic research in these themes is applied to understanding the molecular diagnosis and pathogenesis of cancer, chronic inflammatory diseases including arthritis and periodontitis, and HIV.

main approaches

Degradomics

  • Degradomics is the application of genomic and proteomic techniques to elucidate protease and protease-substrate repertoires-or 'degradomes'-on an organism-wide scale. This system-wide approach promises to reveal new roles for proteases in vivo, new diagnostic indicators of disease, and new drug targets to treat disease. We are developing novel mass spectrometric approaches to identify cleaved substrates in tissues and cells, and targeted isotope coded affinity tags (TICAT) and protease chips to identify active proteases in tissues and cells and in disease such as the cancer degradome and arthritis degradome.

    Recent advances include:
  • ICAT analysis of protease transfected cells (Tam et al 2004)(Overall et al 2004)
  • CLIP-CHIP: A dedicated complete human protease and inhibitor 70-mer oligonucleotide microarray chip. [poster](Overall et al 2004)
  • CLIP-TAILS: A new proteomic approach of substrate discovery - Terminal Affinity Isotope Labelling of Substrates.
  • Anotation of the human and mouse DEGRADOMES [Degradome Page](Puente et al 2003)
Protein engineering
  • The MMP system in man is comprised of 23 proteases and 4 TIMPs.
    We focus on four important MMPs and two TIMPs:
    • MMP-2 (gelatinase A)
    • MMP-8 (neutrophil collagenase)
    • MT1-MMP (a cell membrane collagenase and MMP-2 activator)
    • MT2-MMP (a cell surface protease and cell membrane activator of MMP-2)
    • TIMP-2 and TIMP-4
  • To perform mechanistic studies of MMP function, activation and inhibition we use engineered and wild-type recombinant domains and proteases that are expressed in a variety of heterologous hosts such as E. coli, P. pastoris, and mammalian cells utilizing fermenters at a Pilot Plant scale.
  • Site directed mutations are designed by 3-D modelling and the mutant proteins characterized to map binding sites and activity determinants. Protease and protease domain interactions are characterized using a wide variety of biochemical and cell culture techniques including enzyme kinetic, structural, and mass spectrometric analyses. Other techniques utilize membrane preparations, chemical cross-linking, fluorescence anisotropy, Kd determination, affinity chromatography, and ELISA.
  • To understand the mechanistic aspeccts of protease activity we are building new proteases by engineering active sites onto inert protein scaffolds.

100 L fermenter used for E. coli expression of recombinant MMP domains and mutants, Overall Lab, UBC

100 L fermenter used for expression
of MMPs in E. coli or Pichia pastoris
Other fermenters used include
35 and 10 L bioreactors [pic].

New perspectives on the in vivo role of MMPs

  • With 187 enzymes in man, the metalloproteases are the largest of the five protease classes.
  • MMPs are traditionally thought to degrade the extracellular matrix in normal turnover and disease. Although MMP-2 is pivotal for penetration of basement membrane type IV collagen to facilitate cancer metastasis, new evidence implicates MMP-2 and other MMPs in the control of many cellular processes and immune cell functions by proteolytic processing of bioactive molecules, such as cell surface receptors and adhesion molecules, cytokines, growth factors, Fas ligand and, as we have recently shown, chemokines.
  • We believe these new functions of MMPs will prove to be as important in vivo as the proposed roles of MMPs in matrix remodeling. Discovering new MMP substrates and mechanistically dissecting their function and regulation by proteolysis in vivo is a major focus of THE OVERALL LAB. We are developing a number of new proteomic techniques (degradomics) to discover new MMP protease substrates in normal tissues and disease, such as the cancer degradome and the arthritis degradome.
Proteolytic processing: Proteases were initially characterized as nonspecific degradative enzymes associated with protein catabolism. However, proteases also achieve precise cellular control of multiple biological processes through limited proteolysis of protein targets, termed proteolytic processing, by the highly specific catalysis of peptide bonds, that regulates the fate and activity of a wide range of critical bioactive proteins.

MMP degradomics and degradomes

  • Only by considering individual proteases as a part of a system and proteolytic systems as a whole can the impact of the protease degradome on the substrate degradome in vivo be understood and its perturbations recognized in disease. The hierarchical importance of proteases within a system is affected by specific activity and redundancy, expression levels, temporal/spatial distribution, activation, turnover, and inhibition properties that influence proteolytic potential in vivo. Understanding this is a critical issue for drug development.
  • Innovative strategies are needed to identify new protease substrates. We have termed this field of study degradomics (López-Otín & Overall 2002) and a major focus of THE OVERALL LAB is the development of new proteomic and yeast two-hybrid genetic techniques to identify the protease and protease substrate repertoires, or degradomes, in man.
  • 'Exosite scanning' is a substrate-screening method we developed based on the hypothesis that proteins that bind exosites might be protease substrates. Exosite scanning with disulphide-bond-containing domains of extracellular proteases as bait for extracellular substrates was first demonstrated in my laboratory with the identification of unexpected cytokine substrates for MMP-2, and the discovery of new roles for MMPs in the regulation of inflammation and HIV infection (McQuibban et al 2000, McQuibban et al 2002, Zhang et al 2002).
  • 'Inactive catalytic domain capture' (ICDC) is a technique that we have also developed using inactive catalytic domain mutants as baits in yeast two-hybrid screens and as immobilized native protein baits for genetic and proteomic profiling, respectively, to identify new MMP substrates (López-Otín & Overall 2002, Overall et al 2004).
  • "ICAT (Isotope Coded Affinity Tag) Labelling of protease-transfected cells followed by multi-dimensional liquid chromatography tandem mass spectrometry (MD/LC MS/MS) has led to the rapid identification of new protease substrates (Tam et al 2004, Overall et al 2004).
  • To elucidate the MMP protease degradomes we are developing functional degradomic approaches including the use of TICAT (targeted isotope coded affinity tags), mass spectrometry, and affinity probes for protease activity profiling to distinguish active enzymes from their inactive-precursor or inhibitor-bound forms. In addition, we are developing substrate chips and protease-specific chips to capture and identify specific proteases from complex biological samples.

Degradomics: All genomic and proteomic investigations and techniques regarding the genetic and functional identification and characterization of proteases, their substrates and inhibitors in an organism.
Degradomes: The degradome is the complete set of proteases expressed at specific time by a cell, tissue or organism. The degradome of a protease is its substrate repertoire. (López-Otín & Overall, 2002)

MMPs in Cancer

  • The levels of the secreted MMPs in the peri-tumour stroma and membrane type-MMPs (MT-MMPs) on the tumor cell surface correlate with tumor grade. MMP substrates in cancer include bioactive mediators and matrix proteins.
  • The in vivo mechanisms of MMP activation and the protein interactions involved in the assembly of MMP activation complexes and cell surface-MMP rafts critical for focal proteolysis and tumor cell invasion are largely unknown. Structure/function and mechanistic investigations in THE OVERALL LAB focus on MT1-MMP, MT2-MMP, TIMP-2 and TIMP-4 and their roles in MMP activation.
  • MT-MMPs are implicated in focal proteolysis and, in an amplification cascade, activate MMP-2. We were the first to report the cellular activation of MMP-2 (Overall & Sodek 1990). This entails the docking of the MMP-2 zymogen to MT1-MMP via binding of the TIMP-2 C-terminal domain and tail to the MMP-2 hemopexin carboxyl (C) domain. We mapped the TIMP-2 binding site by mutagenesis (Overall et al 1999), defined the role of the TIMP-2 and TIMP-4 C-terminal tails in binding (Overall et al 2000, Kai et al 2002), identified the role of TIMP-4 in regulating these processes (Bigg et al 2001), and discovered a non-TIMP-2 pathway of MMP-2 activation by MT2-MMP (Morrison et al 2001).
3D model of human MMP-2 showing the binding sites of TIMP-2 mapped by mutagenesis in the Overall Lab, UBC
Sites mutated in the MMP-2 hemopexin C domain to define the TIMP-2 docking site
  • Yeast two-hybrid hits have also led us to investigate the role of MT-MMP cytoplasmic tail-binding proteins in gene transcription and how these targets pertain to cancer progression.
  • Cancer cells utilize chemokine receptors for targeting metastatic cells to different organs. The effect of MMP cleavage on chemokine gradients in this process is a new area of investigation in my laboratory.

MMP inhibitor drugs for cancer: A guiding paradigm of the past 20 years has been that general proteolytic catabolism of tissue provides tumor cells with pivotal access to the vascular and lymphatic systems, thereby facilitating cancer dissemination. Over the past two decades targeting proteases to mitigate these processes has been the prevailing rationale for developing anti-proteolytic agents to treat cancer. However, it is now apparent that this simplistic view is too narrow and that targeting proteases has to be done with extreme precision.

Proteolytic processing by MMPs influences many basic processes essential in cancer including cell proliferation, adhesion and dispersion, migration, differentiation, angiogenesis, apoptosis, and host defense cell evasion. While this presents a broad range of targets for MMP inhibitor drugs to control cancer, inadvertent growth-promoting effects on cancer cells, suppression of host defense processes and clinical side effects are challenges. Protease-specific and substrate-specific anti-exosite inhibitors are urgently needed. (Overall & López-Otín 2002)

MMP substrate interactions

  • Understanding the structure and function of MMPs and the interactions with their substrates is essential for understanding the mechanics of MMP proteolysis, inhibition, and the influence of MMPs on cell behaviour.
  • Peptide bond cleavage specificity is determined by the nature of the MMP active site and by substrate-binding exosites. Exosites on the hemopexin C domain of the collagenases and MT1-MMP perform collagen unwinding or "triple helicase" activity that is an absolute requirement for native collagen cleavage (Tam et al 2002). In searching for novel substrates of MMP-2 we reported in Science (McQuibban et al 2000) that the hemopexin C domain of MMP-2 also binds chemokines, leading to their efficient cleavage and inactivation. We are mapping the chemokine exosite by mutagenesis and defining the role of exosites in MMP function using engineered substrates.
  • We have also found that collagen and elastin substrates bind to the gelatinase-specific fibronectin type II modules, which form the collagen binding domain (CBD) (Steffensen et al 1995), and proposed that the CBD drives a unique mechanism of collagen triple helicase activity. Our mutagenesis studies have mapped the collagen binding site on the CBD and identified single residues that, when mutated, knockout collagen binding.

Collagen binding site on the third fibronectin type II module of human matrix metalloproteinase 2 (MMP-2). The pit on this surface is remarkably stable and mutagenesis studies conducted in the Overall Lab show this is critical for binding collagen alpha-chains.

Collagen binding site in CBD3 of MMP-2
Click here to view alternative NMR structures of the third fibronectin type II module of human matrix metalloproteinase 2 (MMP-2). The pit on this surface is remarkably stable and mutagenesis studies conducted in the Overall Lab show this is critical for binding collagen alpha-chains.

Molecules in Motion
 Click here to view CBD movie

Alternative NMR structures of the third fibronectin type II repeat of human MMP-2 showing the stability of the hydrophobic pit identified by our mutagenesis studies to be pivotally important in binding collagen.
  • In other projects, we have generated over 40 mutations of the active sites of several MMPs to study the structure function relationships of the active site subsites and the catalytic zinc ion environment.
  • New information on the active site and in substrate recognition is essential for the design of new, highly selective inhibitors that may specifically block single MMPs. We plan to develop exosite inhibitors that do not bind the active site, but may block binding and cleavage of selected substrates by MMPs, without affecting cleavage of other targets.

Chemokine processing by MMPs

  • Chemokine substrate discovery: Using mass spectrometry chemokines are screened for cleavage by recombinant MMPs expressed in THE OVERALL LAB (MMPs 1, 2, 3, 7, 8, 9, 13, 14, 15 and 18). Cleaved chemokine products are characterized by receptor binding, Scatchard analysis, calcium mobilization, transwell migration assays, and in vivo models of inflammation (McQuibban et al 2000, 2002).
  • Using engineered chemokine substrates we are mapping the location of binding site on the hemopexin C domain of MMPs.
  • These studies have revealed exciting new roles for MMPs in regulating chemokine activity and thereby inflammatory responses in vivo (McQuibban et al 2002), with other connections found to CD34+ stem cell mobilization (McQuibban et al 2001), HIV infection and HIV associated dementia (Zhang et al 2002), and we are now persuing PMN chemokines.
  • Most recently we have established MMP-2 and MMP-8 knock-out mouse colonies to study the role of these MMPs in processing chemokines in various animal models including cancer, dementia and other neurodegenerative diseases, inflammation, arthritis, lung fibrosis and wound healing.
 Graph showing MMP cleavage of SDF-1 increases HIV-1 infection of CD4+ cells

Inflammation Dampened by Gelatinase A Cleavage of Monocyte Chemoattractant Protein-3:
McQuibban et al, Science 289, 1202-1206
 Chemokines are a ~54 member super-family of chemoattractant cytokines responsible for directing and maintaining leukocyte traffic throughout the body. Monocyte chemoattractant proteins (MCPs) are pivotal stimulators of monocyte and lymphocyte chemotaxis and function. By exosite scanning we discovered that MCP-3 represents an entirely novel substrate class not previously described for MMPs. MCP-3 binds the hemopexin C domain of MMP-2 with a dissociation constant (Kd) of 0.4 x 10-6 M. Mass spectrometry and N-terminal sequencing of the MCP-3 degradation fragments revealed that the scissile bond was Gly4-Ile5, the preferred scissile bond in gelatin for this enzyme. We designated this cleavage product MCP-3 (5-76). Enzyme kinetic analyses revealed that MMP-2 cleavage of MCP-3 was extremely efficient and with a kcat/Km of ~8,000 M-1sec-1, MCP-3 is a considerably better substrate than gelatin. In calcium flux and trans-well migration assays, MMP-2-mediated cleavage of MCP-3 not only resulted in loss of bioactivity as evident from the abrogation of CC receptor (CCR)-induced intracellular calcium mobilization and loss of cell chemotaxis, but also generated a potent receptor antagonist for native MCPs that bind CCR-1, -2 and -3. Moreover, this effect is amplified since cleaved MCP-3 antagonizes other chemokines that bind these receptors such as macrophage inflammatory protein-1alpha. Hence, MCP-3 (5-76) was shown to be a broad spectrum CCR antagonist in vitro. In mouse models of inflammation, the MMP-cleaved MCP-3 almost totally eliminated monocyte infiltration in subcutaneous blisters in a concentration-dependent manner and, by fluorescent activated cell sorting analysis, reduced mononuclear infiltration in zymosan-induced peritonitis by 40% up to 4 h after administration of the antagonist. Importantly, the pathophysiological relevance of this was also demonstrable in man. We isolated MMP-2/MCP-3 complexes from synovial fluid of arthritis patients and conclusively identified the cleaved MCP-3 fragment in rheumatoid synovial fluids using a neoepitope antibody strategy, providing direct evidence for the involvement of MMPs in inactivating MCP-3 in human disease and in modulating inflammation.

What benefits will be gained from these studies?

  • Understanding the structure function relationships of the MMP active site and exosite domains by protein engineering will provide fundamental information on the mechanistic aspects of MMP activation, activity, and inhibition in the extracellular compartment and on the cell surface.
  • The growing awareness that MMPs cleave a wide variety of bioactive substrates in vivo, the relatively minor phenotypic alterations in normal matrix turnover in most MMP-knockout mice, the general absence of effects on normal connective-tissue remodeling during long-term exposure to synthetic MMP inhibitors in animal models and patients in MMP-inhibitor drug trials, and the existence of efficient intracellular pathways of extracellular matrix turnover indicate that MMPs are not important for normal matrix remodeling as generally assumed. Indeed the general lack of in vivo evidence for extracellular matrix-protein cleavage by MMPs indicates other important roles for these proteinases. Hence, our focus on bioactive substrate discovery and processing by MMPs with resulting regulatory effects on cellular responses and host defense systems in physiological and pathological processes is directed to discovering novel in vivo roles for MMPs and new drug targets and lead compounds.
  • Degradomic analysis of the protease systems active in biological samples at a system-wide scale using activity profiling and substrate chips will provide a new level of information not available today. System-wide analysis of the MMP family will provide data both on the synergy and functional redundancy of different proteases, and on their relative roles in different tissues or diseases. This should provide insights into how a cell or tissue responds in terms of initiating protease action in different physiological and pathological processes. Hence, MMP degradomic studies will identify new protease targets to control disease and MMP-processed bioactive mediators, such as cleaved chemokines, that may be useful to treat disease.

Selected Publications from THE OVERALL LAB

Bigg, H.F., Morrison, C.J., Butler, G.S., Bogoyevitch, M.A., Wang, Z., Soloway, P.D., and Overall, C.M. 2001. Tissue Inhibitor of Metalloproteinases-4 (TIMP-4) Inhibits, but does not Support, the Activation of Gelatinase A via Efficient Inhibition of Membrane Type 1-Matrix Metalloproteinase. Cancer Res. 61, 3610-3618. [Reprint (PDF)]

Kai, H.S.-T., Butler, G.S., Morrison, C.J., King, A.E., Pelman, G.R., and Overall, C.M. 2002.
Utilization of a Novel Recombinant Myoglobin Fusion Protein Expression System to Characterize the Tissue Inhibitor of Metalloproteinase (TIMP)-4 and TIMP-2 C-terminal Domain and Tails by Mutagenesis. THE IMPORTANCE OF ACIDIC RESIDUES IN BINDING THE MMP-2 HEMOPEXIN C DOMAIN. J. Biol. Chem. 277, 48696-48707. [Reprint (PDF)]

López-Otín, C. and Overall, C.M. 2002. Protease Degradomics: A New Challenge for Proteomics. Nature Reviews Molecular Cell Biol. 3, 509-519. [Online (HTML)] [Reprint (PDF)] [Nature Reviews Molecular Cell Biology Homepage]

McQuibban, G.A., Butler, G.S., Gong, J.-H., Bendall, L., Power, C., Clark-Lewis, I., and Overall, C.M. 2001. Matrix Metalloproteinase Activity Inactivates the Chemokine Stromal-Cell Derived Factor-1. J. Biol. Chem. 276, 43503-43508. [Reprint (PDF)]

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. [Reprint (PDF)]

McQuibban, G.A., Wong, J.P., Gong, J.-H., Wallace, J.L., Clark-Lewis, I., and Overall, C.M. 2002. Matrix Metalloproteinase Processing of Monocyte Chemoattractant Proteins Generates CC Chemokine Receptor Antagonists With Anti-inflammatory Properties In vivo. Blood 100, 1160-1167.[Reprint (PDF)]

Morrison, C.J., Butler, G.S., Bigg, H.F., Roberts, C.R., Soloway, P.D., and Overall, C.M. 2001. Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway. J. Biol. Chem. 276, 47402-47410. [Reprint (PDF)]

Overall, C.M. and Sodek, J. 1990. Concanavalin A Produces a Matrix Degradative Phenotype in Human Fibroblasts: Induction and Endogenous Activation of Collagenase, 72-kDa Gelatinase, and Pump-1 is Accompanied by the Suppression of TIMP. J. Biol. Chem. 265, 21141-21151. [Abstract (PDF)]

Overall, C.M., King, A.E., Sam, D.K., Ong, A.D., Lau, T.T.Y., Wallon, U.M., DeClerck, Y.A., and Atherstone, J. 1999. Identification of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Binding Site on the Hemopexin Carboxyl Domain of Human Gelatinase A by Site-Directed Mutagenesis: The Hierarchical Role in Binding TIMP-2 of the Unique Cationic Clusters of Hemopexin Modules III and IV. J. Biol. Chem. 274, 4421-4429. [Reprint (PDF)]

Overall, C.M., Tam, E.M., Kappelhoff, R., Connor, A., Ewart, T., Morrison, C.J., Puente, X., López-Otín,C., and Seth, A. 2004. Protease Degradomics: Mass Spectrometry Discovery of Protease Substrates and the CLIP-CHIP, a Dedicated DNA Microarray of all Human Proteases and Inhibitors. Biol. Chem. 385, 493-504. [Reprint (PDF)]

Overall, C.M., Tam, E., McQuibban, G.A., Morrison, C.J., Wallon, U.M., Bigg, H.F., King, A. E., and Roberts, C.R. 2000. Domain Interactions in the Gelatinase A:TIMP-2:MT1-MMP Activation Complex: The Ectodomain of the 44-kDa Form of Membrane Type-1 Matrix Metalloproteinase Does Not Modulate Gelatinase A Activation. J. Biol. Chem. 275, 39497-39505. [Reprint (PDF)]

Overall, C.M., and López-Otín, C. 2002. Strategies for MMP Inhibition in Cancer: Innovations for the Post-Trial Era. Nature Reviews Cancer 2, 657-672. [Reprint (PDF)]

Puente, X.S., Sanchez, L.M., Overall C.M., and López-Otín, C. 2003. Human and Mouse Proteases: A Comparative Genomic Approach. Nature Rev. Genetics 4, 544-558. [Reprint (PDF)]

Steffensen, B., Wallon, U.M., and Overall, C.M. 1995. Extracellular Matrix Binding Properties of Recombinant Fibronectin Type II-like Modules of 72-kDa Gelatinase/Type IV Collagenase: High Affinity Binding to Native Type I Collagen but not Native Type IV Collagen. J. Biol. Chem. 270, 11555-11566. [Reprint (PDF)]

Tam, E.M., Wu, Y.I., Butler, G.S., Stack, M.S., and Overall, C.M. 2002. Collagen binding properties of the MT1-MMP hemopexin C domain: The ectodomain of the 44-kDa autocatalytic fragment of MT1-MMP inhibits cell invasion by disrupting native type I collagen cleavage. J. Biol. Chem. 277, 39005-39014. [Reprint (PDF)]

Tam, E.M., Morrison, C.J., Wu, Y.I., Stack, M.S., and Overall, C.M. 2004. Membrane Protease Proteomics: Isotope-coded Affinity Tag MS Identification of Undescribed MT1-Matrix Metalloproteinase Substrates. Proc. Natl. Acad. Sci. USA 101, 6917-6922. [Reprint (PDF)]

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 Cleavage of the Chemokine SDF-1a Causes Neurodegeneration Nature Neurosci. 6, 1064-1071. [Reprint (PDF)]