Molecular Demolition

The ability of cells to degrade and rearrange extracellular matrix proteins is crucial for an organism's growth and development. Nearly 40 years ago, Jerome Gross and Charles Lapiere discovered that when tadpole tail fins resorb, their skin releases an enzyme that degrades native collagen triple helixes in the underlying support matrix.1 This enzyme, called collagenase, is present in a wide variety of vertebrates, invertebrates, and plants. Critical as it is, collagenase is just one member of a large class of enzymes called metalloproteinases—protein-digesting enzymes whose catalytic activity depends on the presence of metal ions. Metalloproteinases can be inhibited by chelators, such as EDTA, which coordinate metal ions.2 Several zinc (Zn2+)-dependent endopeptidases exist, and are known collectively as matrix metalloproteinases (MMPs), or matricins.2 Both secreted and membrane-bound MMPs catalyze the breakdown of proteins located either on the cell's plasma membrane, or within the extracellular matrix (ECM).2 Researchers have come to recognize the critical role these enzymes play in regulating processes that range from development and morphogenesis to angiogenesis and metastasis. Biotech companies have stepped in to offer a wide range of reagents to facilitate the study of these diverse proteins. MMP Function The ECM,3 composed of proteoglycans, fibrous proteins such as collagen and elastin, and other secreted proteins, helps to define a tissue's physical properties. It also influences development, morphogenesis, and remodeling of neighboring tissue. A major task of MMPs in vivo is thought to be disruption of the ECM's structural organization during development, tissue resorption, and disease progression.2,4 This is accomplished by altering or degrading several ECM components, including collagens, laminins, fibronectins, elastins, and the proteoglycan protein core.5 MMPs also play a major role in wound healing and tissue repair. Several MMPs also possess a "sheddase" function, which converts membrane-bound cytokines, cytokine receptors, and adhesion molecules to their soluble (inactive) forms.6 Because they can degrade the ECM, MMPs influence cell migration, remodeling, and inflammatory responses. However, these enzymes may also be involved in the underlying etiology of invasive and inflammatory diseases, including cancer, rheumatoid arthritis, cirrhosis of the liver, fibrotic lung disease, multiple sclerosis, bacterial meningitis, and aortic aneurisms.6 MMPs in Cancer and Angiogenesis One characteristic of invasive processes like metastasis and angiogenesis is the degradation of the ECM and basal membranes, which are normally physical barriers to cell migration. MMPs may also modulate cell adhesion, which can allow cells to move through the ECM.7 The abnormal regulation of MMP synthesis and/or activation in tissue could stimulate ECM and cellular basement membrane degradation, clearing the way for metastasis. Published reports support this relationship. MMPs have been linked to the invasive and metastatic behavior of a wide variety of malignancies, and these enzymes are generally overexpressed in a variety of tumors.2,8 Also, the expression of several different MMPs has been correlated with the invasive capacities of certain tumors.9 Finally, the number of different MMPs detected in a tumor tends to increase with the tumor's progression.5 Membrane-type MMPs (MT-MMPs) such as MT1-MMP have been strongly implicated in oncogenesis. These enzymes localize to invasive fronts and specialized membrane extensions known as invadopodia, where ECM degradation and cellular invasion can occur.10 The soluble MMPs 1, 2, 3, 9, and 14 have also been implicated as agonists in tumorigenesis.8,10 Transgenic mice that overexpress certain MMPs generally develop more cancers than do control mice.2 In contrast, genetically engineered mice that lack a specific MMP generally produce fewer metastatic growths than do controls. Angiogenesis is a necessary component of tumor progression, as rapidly dividing tumor cells require a ready supply of oxygen and nutrients. During angiogenesis, blood vessel endothelial cells undergo a loosening of the ECM so that new blood vessels can form. Like cancer progression, angiogenesis may in part rely on the controlled activity of MMPs and their endogenous inhibitors. MMP enzymatic activity may stimulate the migration of endothelial cells during angiogenesis.11 For example, MT1-MMP is upregulated during endothelial cell induction and migration,11 is released from plasma membranes during angiogenesis, and is required for both the spontaneous formation of capillaries from endothelial cells and for collagen matrix remodeling by endothelial cells. Thus, certain MMPs may play a critical role in both tumor progression and angiogenesis. Structural Considerations Based on substrate specificities and structural considerations, MMPs are divided into six arbitrary groups: collagenases, gelatinases, stromelysins, MT-MMPs, non-mammalian MMPs, and other MMPs.10 Currently, this gene family consists of 25 secreted and cell-surface endoproteases in vertebrates, and includes 22 human homologs.2,10 All known MMPs share significant sequence homology and a common multidomain structure.2,12 Each family member contains a catalytic domain and a variable number of additional domains; at present, scientists have identified eight different modular domains. Matrilysin, composed of a signal peptide, propeptide, and catalytic domain, represents the minimal MMP enzyme. The MMP N-terminal domain usually contains a 20-amino-acid-long signal peptide that directs MMPs into the endoplasmic reticulum (ER) lumen for secretion. The adjoining domain is the roughly 80-amino-acid-long propeptide region, which contains a highly conserved motif—ProArgCysGly[Val/Asn]CysAsp—near the C-terminus in the inactive precursor protein. This propeptide sequence must be proteolytically removed to activate the metalloenzyme.13 The motif's first cysteine residue is invariant, and its sulfur atom stabilizes the inactive prometalloenzyme by occupying one of the catalytic Zn2+ ion's four coordination sites. MMP catalytic domains are approximately 170 amino acids long and include the Zn2+-binding motif, HisGluXBXHisXBGlyBXHisSer (where B represents a bulky hydrophobic residue and X represents any amino acid).2 This motif's three histidine side chains chelate the zinc ion. A conserved methionine turn (AlaMetTyrLeuPro) lies beneath the active site Zn2+, forming a hydrophobic pocket that the catalytic Zn2+ and three coordinating histidine residues occupy.9 X-ray crystallography studies show that MMP catalytic domains contain a five-stranded b-sheet, three a-helices, and bridging loops.14 This domain also contains a second, structural Zn2+, and two or three calcium ions that stabilize enzyme structure. Some MMPs (MMP-2 and MMP-9) also contain three fibronectin type II repeats within the catalytic domain, which may facilitate adherence to gelatin and collagen substrates.9 A proline-rich hinge region joins the catalytic domain to the C-terminal hemopexin domain. This domain is approximately 200 residues long and contains four repeats that share structural similarity with hemopexin, a heme-binding plasma glycoprotein that protects against oxygen damage. The hemopexin-like domain folds into a four-bladed b-propeller structure,12 and appears to functionally bind substrate. This domain is required for collagenases to cleave interstitial collagens, and may modulate substrate specificity. This domain may also influence the binding of endogenous inhibitory molecules known as "tissue inhibitors of matrix metalloproteinases" (TIMPs). Six MMPs contain an 80-to-90-amino-acid-long C-terminal transmembrane domain.2 This single-pass transmembrane domain anchors the enzymes to the plasma membrane, allowing these MMPs to proteolytically remove molecules, such as L-selectin, from plasma membranes, and to activate soluble MMP proenzymes during their secretion. Activation and Catalytic Activity MMPs are expressed primarily in connective tissue and bone marrow-derived cells.14 All MMPs are synthesized as pre-proenzymes, and are processed to inactive proenzymes known as zymogens.13 A "cysteine switch" regulates the onset of MMP enzymatic activity.9 A highly conserved cysteine residue in the MMP's propeptide domain acts as the catalytic zinc ion's fourth ligand, and functions to stabilize the catalytically inactive proenzyme. When this Cys-Zn2+ bond is intact, the switch is closed, and when the bond is disrupted, the switch is open. Both proteolysis and chemical perturbation of the propeptide can trigger a conformational change that disrupts (opens) the Cys-Zn2+ interaction, allowing the subsequent cleavage of the propeptide to generate mature metalloenzyme. In cells, proteolytic activation of MMP proenzymes occurs in two steps. First, an activator proteinase attacks an exposed loop in the MMP's propeptide domain. This cleavage destabilizes the Cys-Zn2+ interaction in the proenzyme. The resulting conformational change allows a second proteolytic cleavage, which removes the remainder of the N-terminal propeptide fragment to generate the mature, active enzyme. Removal of the entire MMP propeptide allows the catalytic Zn2+ to coordinate with water, rather than cysteine. This water is now available for substrate peptide bond hydrolysis;12 enzymatic activity is thought to proceed by general acid-base catalysis. The MMP's substrates include inactive MMP proenzymes. For example, membrane-bound MT1-MMP can activate MMP-2 proenzyme on the cell surface.2 MMP Regulation MMPs exhibit overlapping expression patterns and substrate specificities. However, the enzyme's activity is tightly regulated, and unwanted proteolysis and tissue destruction do not normally occur. For example, MMPs are involved in a self-regulating loop with ECM proteins; increased expression of ECM proteins leads to increased MMP catalytic activity, which maintains ECM homeostasis. In vivo, MMP enzymatic activities can be controlled by regulation of gene expression, cellular protein levels, secretory activity, proenzyme activation, and by endogenous inhibitors, which include the plasma protein a-macroglobulins and TIMPs.2 Except for MMP-2 (gelatinase A), which is constitutively expressed in cells, most matrix metalloproteinases are generally expressed at low levels in adult organisms. Various agents including growth factors, hormones, eicosanoids, phorbol esters, and pro-inflammatory cytokines can transcriptionally regulate these inducible endopeptidases during cell remodeling. Pro-inflammatory cytokines can induce MMP production via proto-oncogenes, such as members of the c-jun and c-fos families.6 On the other hand, glucocorticoids, retinoic acid, and interleukin-10 can negatively regulate expression of certain MMPs. A second control point for MMP activity is regulation of enzyme activation, which can occur either within or outside of the cell. MMP-11 (stromelysin-3), MMP-27 (epilysin), and several MT-MMPs contain an 11-amino-acid furin-processing motif.2 This binding site allows intracellular MMP proenzyme activation by the serine protease furin, which localizes to the trans-Golgi network, or by proteases such as plasmin at the cell surface.2 Thus, metalloproteinase catalytic activity can undergo activation before these proteins exit the cell. Intracellular MMP activity may allow these enzymes to proteolytically cleave and activate other MMPs (in the absence of endogenous inhibitors) either inside the cell, or at the cell surface. In contrast, soluble MMPs are generally synthesized and secreted as inactive proenzymes, which are then activated outside the cell, and may catalyze the breakdown of extracellular or cell-surface proteins.2 Negative Regulation MMP catalytic activity may also be regulated by specific endogenous inhibitors known as TIMPs.2,12 When bound to MMPs, these regulatory molecules inhibit the metalloenzyme's proteolytic activity.15 Currently, four classes of TIMPs have been identified in vertebrates, which vary in their abilities to inhibit different MMPs.2 TIMPs are soluble proteins whose molecular weights vary from approximately 21-30 kDa. These multifunctional enzymes form strong, reversible, noncovalent complexes with both proenzymes and enzymatically activated MMPs.2 To inhibit active MMPs, the TIMP's terminal amino group is thought to fill the fourth coordination site of the catalytic Zn2+ that was previously occupied by cysteine in the proenzyme. Structural studies have further shown that TIMPs affect the MMP catalytic domain, which results in inhibition of the enzyme's hydrolytic activity. TIMPs negatively regulate MMP activity during ECM turnover. In both endothelial cells and tumor cells, a reduction in cellular TIMP expression generally increases the capability of invading cells to dissolve the neighboring tissue's ECM. Under in vitro conditions, TIMPs can inhibit cell invasion, tumorigenesis, metastasis, and angiogenesis.16 TIMPS may also possess multiple cellular functions that are independent of their ability to inhibit MMPs. Free TIMPs—those that are not bound to MMPs—can exhibit growth-promoting (mitogenic) functions in several cell types.2 However, TIMP-2 can also suppress receptor tyrosine kinase activation of growth factors in the absence of MMPs.16 Because of the roles MMPs play in metastasis, MMP inhibition by TIMPs could prove to be a promising strategy for the inhibition of tumor growth. Recently, synthetic low molecular weight hydroxyamic acid-based MMP inhibitors have been developed. These inhibitors function as Zn2+ chelators, and do not show specificity for individual MMPs.5 Synthetic matrix metalloproteinase inhibitors include the broad-spectrum inhibitors Batimastat and Marimastat. These inhibitors have been extensively used in clinical trials for the treatment of various cancers.5 MMP research is currently a hot topic, owing to the central role these enzymes play in various developmental and disease processes. Consequently, the MMP literature is growing rapidly: PubMed recorded 1,000-1,300 new MMP-related references in each of the past three years. As a result, a variety of tools and reagents are available to assist scientists who study MMP function. These tools include purified and recombinant MMPs and TIMPs, antibodies, expression detection kits, enzyme assay kits, synthetic inhibitors, and MMP substrates. These reagents should ease the burdens of researchers and drug developers alike, as the former work to understand the tasks these enzymes play in vivo, and the latter strive to spin that knowledge into pharmaceutical gold. Gregory Smutzer (smutzer@hotmail.com) is a freelance writer in Lansdowne, Pa. Paul Billings, Department of Orthopedic Surgery, University of Pennsylvania School of Medicine, provided technical assistance. References 1. J. Gross, C.M. Lapiere, "Collagenolytic activity in amphibian tissues: A tissue culture assay," Proceedings of the National Academy of Sciences, 48:1014-22, 1962. 2. M.D. Sternlicht, Z. Werb, "How matrix metalloproteinases regulate cell behavior," Annual Review of Cell and Developmental Biology, 17:463-516, 2001. 3. D.A. Fitzgerald, "Integral connections," The Scientist, 15[16]:29, Aug. 20, 2001. 4. M. Aumailley, B. Gayraud, "Structure and biological activity of the extracellular matrix," Journal of Molecular Medicine, 76:253-265, 1998. 5. R. Hoekstra et al., "Matrix metalloproteinase inhibitors: Current developments and future perspectives," The Oncologist, 6:415-27, October 2001. 6. D. Leppert et al., "Matrix metalloproteinases: Multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis," Brain Research Reviews, 36:249-57, October 2001. 7. D.E. Kleiner, W.G. Stetler-Stevenson, "Matrix metalloproteinases and metastasis," Cancer Chemotheraphy and Pharmacology, 43(Suppl.):S42-S51, 1999. 8. D.V. Rozanov et al., "Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP)," Journal of Biological Chemistry (JBC), 276:25705-14, July 13, 2001. 9. H. Nagase, J.F. Woessner, Jr., "Matrix metalloproteinases," JBC, 274:21491-4, 1999. 10. W.C. Parks, R.P Mecham, eds. Matrix Metalloproteinases, San Diego: Academic Press, 1998, pp. 1-14. 11. B.G. Galvez et al., "Membrane type I-matrix metalloproteinase is activated during migration of human endothelial cells and modulates endothelial motility and matrix remodeling," JBC, 276:37491-500, Oct. 5, 2001. 12. J. F. Woessner, H. Nagase, Matrix Metalloproteinases and TIMPs, Oxford, UK: Oxford University Press, 2000, pp. 50-71. 13. H. Nagase, "Activation mechanisms of matrix metalloproteinases," Biological Chemistry, 378:151-160, 1997. 14. E. Morgunova et al., "Structure of human pro-matrix metalloproteinase-2: Activation mechanism revealed," Science, 284:1667-70, 1999. 15. E. Russo, "MMPS may provide clues to multiple ailments," The Scientist, 13[14]:6, July 5, 1999. 16. S.E. Hoegy et al., "Tissue inhibitor of metalloproteinases-2 (TIMP-2) suppresses TKR-growth factor signaling independent of metalloproteinase inhibition," JBC, 276:3203-14, Feb. 2, 2001. Supplemental Materials Matrix Metalloproteinase Research Tools

Molecular Demolition

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