Proteases in invasion: matrix metalloproteinases.

The role of proteases in general, and the matrix metalloproteinases in particular, in tumor invasion and metastasis is well established. However, the classic view that these enzymes simply provide a mechanism for the breakdown of connective tissue barriers has been challenged. This overview summarizes recent evidence to support the changing view of the role of matrix metalloproteinases in cancer progression. First we briefly review the central role of cell invasion in cancer progression and also the matrix metalloproteinase family members. We then focus on the emerging roles for these enzymes in cancer progression, including the role of matrix metalloproteinases in cell proliferation and release of growth factors, cell migration and in modification of the extracellular matrix to reveal cryptic sites that alter cell behaviour.

Variable forms of soluble guanylyl cyclase: protein-ligand interactions and the issue of activation by carbon monoxide.

Soluble guanylyl cyclase (sGC) is known to be activated by NO binding to the heme moiety; previous studies have shown that CO does not activate sGC to the same extent as NO. Resonance Raman spectroscopy reveals different heme pocket structures for soluble guanylyl cyclase prepared by alternate methods, all of which display activation by NO. In our preparation, and in the expressed protein sGC1, the resting Fe(II) state is mainly 6-coordinate and low-spin, and the CO adduct has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment. In contrast, the protein sGC2 is 5-coordinate, high-spin in the resting state, and the CO adduct has perturbed vibrational frequencies indicative of a negatively polarizing residue in the binding pocket. The differences may result from the need to reconstitute sGC1 or different isolation procedures for sGC1 versus sGC2. However, both sGC1 and sGC2 are activated by the same mechanism, namely displacement of the proximal histidine ligand upon NO binding, and neither one is activated by CO. If CO is an activator in vivo, some additional molecular component is required.

Matrix metalloproteinases. Novel targets for directed cancer therapy.

Matrix metalloproteinases (MMPs), or matrixins, are a family of zinc endopeptidases that play a key role in both physiological and pathological tissue degradation. Normally, there is a careful balance between cell division, matrix synthesis and matrix degradation, which is under the control of cytokines, growth factors and cell matrix interactions. The MMPs are involved in remodelling during tissue morphogenesis and wound healing. Under pathological conditions, this balance is altered: in arthritis, there is uncontrolled destruction of cartilage; in cancer, increased matrix turnover is thought to promote tumour cell invasion. The demonstration of a functional role of MMPs in arthritis and tumour metastasis raises the possibility of therapeutic intervention using synthetic MMP inhibitors with appropriate selectivity and pharmacokinetics. As the process of drug discovery focuses on structure-based design, efforts to resolve the 3-dimensional structures of the MMP family have intensified. Several novel MMP inhibitors have been identified and are currently being investigated in clinical trials. The structural information that is rapidly accumulating will be useful in refining the available inhibitors to selectively target specific MMP family members. In this review, we focus on the role of MMPs and their inhibitors in tumour invasion, metastasis and angiogenesis, and examine how MMPs may be targeted to prevent cancer progression.

Molecular regulation of cellular invasion--role of gelatinase A and TIMP-2.

Extracellular matrix (ECM) turnover is an event that is tightly regulated. Much of the coordinate (physiological) or discoordinate (pathological) degradation of the ECM is catalyzed by a class of proteases known as the matrix metalloproteinases (MMPs) or matrixins. Matrixins are a family of homologous Zn atom dependent endopeptidases that are usually secreted from cells as inactive zymogens. Net degradative activity in the extracellular environment is regulated by specific activators and inhibitors. One member of the matrixin family, gelatinase A, is regulated differently from other MMPs, suggesting that it may play a unique role in cell-matrix interactions, including cell invasion. The conversion from the 72 kDa progelatinase A to the active 62 kDa species may be a key event in the acquisition of invasive potential. This discussion reviews some recent findings on the cellular mechanisms involved in progelatinase A activation and, in particular, the role of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) and transmembrane containing metalloproteinases (MT-MMP) in this process.

Studies of the heme coordination and ligand binding properties of soluble guanylyl cyclase (sGC): characterization of Fe(II)sGC and Fe(II)sGC(CO) by electronic absorption and magnetic circular dichroism spectroscopies and failure of CO to activate the enzyme.

The mechanism of activation of soluble guanylyl cyclase by NO is poorly understood although it is clear that NO interacts with a heme group in the protein via formation of a heme-nitrosyl adduct. The objective of this study is to investigate the coordination environment of the heme in the enzyme spectroscopically in the presence of known heme ligands and to correlate the spectral characteristics with other heme proteins of known structure. Comparison of the electronic and magnetic circular dichroism (MCD) spectra for ferrous bovine soluble guanylyl cyclase (Fe(II)sGC) in the absence and presence of the common heme ligand CO with those of other hemoproteins suggests that histidine is an axial ligand to the heme iron in Fe(II)sGC. Further analysis indicates that Fe(II)sGC is predominantly bis-histidine ligated; the ratio of MCD signal intensity in the visible region to that in the Soret region is most consistent with an admixture of pentacoordinate and hexacoordinate ferrous heme in Fe(II)sGC at pH 7.8. Spectral changes upon CO binding have been correlated with the activity of the enzyme to determine the relationship between coordination structure and activity. Although CO clearly binds to Fe(II)sGC to form a six-coordinate adduct, it fails to significantly activate the enzyme regardless of heme content or CO concentration. In contrast, the extent of activation of sGC by NO is dependent on the heme content in the enzyme and on the concentration of NO. These observations are consistent with a mechanism for activation of soluble guanylyl cyclase in which the bond between the heme iron and the proximal histidine must be broken for activation to take place.

Reconstructed 19 kDa catalytic domain of gelatinase A is an active proteinase.

Matrix metalloproteinases share high protein sequence homology and have defined domain structures. Gelatinases have a unique 19 kDa fibronectin-like insert in the catalytic domain. A synthetic gene was made to express the catalytic domain of human gelatinase A (GCD), in which two polypeptide fragments of the catalytic domain were joined with deletion of the insert. The synthetic gene was highly expressed in Escherichia coli, and the 19 kDa GCD was purified to homogeneity after in vitro refolding. The GCD showed activity at a pH range of 5.5-9 in cleavage of the thiopeptolide Ac-Pro-Leu-Gly-thioester-Leu-Leu-Gly-OEt with optimal activity at neutral pH (Km = 134 microM and kcat = 16 s-1 at pH 7.0). The activity required both zinc and calcium ions, but high concentration of zinc ion showed inhibition. Several stromelysin catalytic domain inhibitors inhibited the GCD with similar specificity. The GCD cleaved the fluorogenic peptides Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 and Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 with catalytic efficiency close to full length human gelatinase A. The reconstructed GCD cleaves not only thiopeptolide and peptide substrates but also protein substrates such as gelatin. These results are consistent with the notion that gelatinases have the same structure for the catalytic domain as other matrix metalloproteinases like stromelysins and collagenases.