overall lab research interests
substrate discovery using proteomic and yeast two-hybrid approaches.
degradomics and associated mass spectrometric and protease chip technology
and functional analysis of MMPs and tissue inhibitor of metalloproteases
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.
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
et al 2004)
- CLIP-CHIP: A
dedicated complete human protease and inhibitor 70-mer
oligonucleotide microarray chip. [poster](Overall et
- CLIP-TAILS: A
new proteomic approach of substrate discovery - Terminal
Affinity Isotope Labelling of Substrates.
of the human
and mouse DEGRADOMES [Degradome
Page](Puente et al 2003)
MMP system in man is comprised of 23 proteases and 4 TIMPs.
We focus on four important MMPs and two TIMPs:
(a cell membrane collagenase and MMP-2 activator)
(a cell surface protease and cell membrane activator
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
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.
understand the mechanistic aspeccts of protease activity
we are building new proteases by engineering active
sites onto inert protein scaffolds.
L fermenter used for expression
of MMPs in E. coli or Pichia pastoris
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
- 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.
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.
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,
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).
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).
elucidate the MMP protease degradomes we are developing
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
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.
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.
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
MMP substrate interactions
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.
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.
binding site in CBD3 of MMP-2
here to view CBD
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
Chemokine processing by MMPs
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
- Most recently
we have established MMP-2 and
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
Dampened by Gelatinase A Cleavage of Monocyte Chemoattractant
McQuibban et al, Science
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?
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
- 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
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
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
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
C. and Overall, C.M. 2002. Protease Degradomics: A New Challenge
for Proteomics. Nature Reviews Molecular
Cell Biol. 3, 509-519. [Online
Reviews Molecular Cell Biology Homepage]
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.
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
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
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
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
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
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
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
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.
X.S., Sanchez, L.M.,
Overall C.M., and López-Otín,
Human and Mouse Proteases: A Comparative Genomic Approach. Nature
Rev. Genetics 4, 544-558. [Reprint (PDF)]
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.
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
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
Natl. Acad. Sci. USA 101, 6917-6922. [Reprint
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.