Marta Toth

Assessment of gelatinases (MMP-2 and MMP-9) by gelatin zymography.

Gelatin zymography is a simple yet powerful method to detect proteolytic enzymes capable of degrading gelatin from various biological sources. It is particularly useful for the assessment of two key members of the matrix metalloproteinase family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), due to their potent gelatin-degrading activity. This polyacrylamide gel electrophoresis-based method can provide a reliable assessment of the type of gelatinase, relative amount, and activation status (latent, compared with active enzyme forms) in cultured cells, tissues, and biological fluids. The method can be used to investigate factors that regulate gelatinase expression and modulate zymogen activation in experimental systems. The system provides information on the pattern of gelatinase expression and activation in human cancer tissues and how this relates to cancer progression. Interpretation of the data obtained in gelatin zymography requires a thorough understanding of the principles and pitfalls of the technique; this is particularly important when evaluating enzyme levels and the presence of active gelatinase species. If properly used, gelatin zymography is an excellent tool for the study of gelatinases in biological systems.

Processing, shedding, and endocytosis of membrane type 1-matrix metalloproteinase (MT1-MMP).

Matrix metalloproteinases (MMPs) are multidomain zinc‐dependent proteolytic enzymes that play pivotal roles in many normal and pathological processes. Some members of the MMP family are anchored to the plasma membrane via specialized domains and thus are perfectly suited for pericellular proteolysis. Membrane‐anchoring also confers the membrane type‐MMPs (MT‐MMPs) a unique and complex array of regulatory processes that endow cells with the ability to control MT‐MMP‐dependent proteolytic activity independently of the levels of endogenous protease inhibitors. Emerging evidence indicates that mechanisms as diverse as autocatalytic processing, ectodomain shedding, homodimerization and internalization can all contribute to the modulation of MT‐MMP activity on the cell surface. How these distinct processes interact to attain the optimal level of enzyme activity in a particular setting and the molecular signals that trigger them constitute a new paradigm in MMP regulation. This review will discuss the recent findings concerning these diverse regulatory mechanisms in the context of MT1‐MMP (MMP‐14).

Cell surface association of matrix metalloproteinase-9 (gelatinase B).

Matrix metalloproteinase (MMP)-9 (gelatinase B) belongs to the MMP family of zinc-dependent endopeptidases that has been associated with tumor cell invasion and metastasis and tumor-induced angiogenesis. As a secreted MMP, pro-MMP-9 is released into the extracellular environment by both tumor and stroma cells, where it fulfills its proteolytic functions degrading both extracellular matrix (ECM) and non-ECM proteins. A major dilemma in our understanding of MMP-9 function is how the released protease is targeted to the right location and how its activity is controlled at the pericellular space. It has been proposed that MMP-9 interact with cell surface components and that this type of interaction positively regulates enzymatic activation and activity. However, recent evidence shows that association of MMP-9 with the cell surface is mediated by a distinct array of surface proteins that serve to regulate multiple aspects of the enzyme function including localization, inhibition and internalization. How these distinct mechanisms regulate the overall MMP-9 activity at the pericellular space remains an important goal in our understanding of MMP-9 function at the cell surface. Furthermore, the study of surface-associated MMP-9 imposes new conceptual and methodological challenges with particular consideration to the unique structural and functional characteristics of this key enzyme.

Characterization of the Monomeric and Dimeric Forms of Latent and Active Matrix Metalloproteinase-9 DIFFERENTIAL RATES FOR ACTIVATION BY STROMELYSIN 1

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K m values in the nanomolar range and relative low k catvalues (1.9 × 10−3 and 4.1 × 10−4s−1, for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1.

High Affinity Binding of Latent Matrix Metalloproteinase-9 to the α2(IV) Chain of Collagen IV

Association of matrix metalloproteinases (MMPs) with the cell surface and with areas of cell-matrix contacts is critical for extracellular matrix degradation. Previously, we showed the surface association of pro-MMP-9 in human breast epithelial MCF10A cells. Here, we have characterized the binding parameters of pro-MMP-9 and show that the enzyme binds with high affinity (K d ∼22 nm) to MCF10A cells and other cell lines. Binding of pro-MMP-9 to MCF10A cells does not result in zymogen activation and is not followed by ligand internalization, even after complex formation with tissue inhibitor of metalloproteinase-1 (TIMP-1). A 190-kDa cell surface protein was identified by ligand blot analysis and affinity purification with immobilized pro-MMP-9. Microsequencing and immunoblot analysis revealed that the 190-kDa protein is the α2(IV) chain of collagen IV. Specific pro-MMP-9 surface binding was competed with purified α2(IV) and was significantly reduced after treatment of the cells with active MMP-9 before the binding assay since α2(IV) is hydrolyzed by MMP-9. A pro-MMP-9·TIMP-1 complex and MMP-9 bind to α2(IV), suggesting that neither the C-terminal nor the N-terminal domain of the enzyme is directly involved in α2(IV) binding. The closely related pro-MMP-2 exhibits a weaker affinity for α2(IV) compared with that of pro-MMP-9, suggesting that sites other than the gelatin-binding domain may be involved in the binding of α2(IV) to pro-MMP-9. Although pro-MMP-9 forms a complex with α2(IV), the proenzyme does not bind to triple-helical collagen IV. These studies suggest a unique interaction between pro-MMP-9 and α2(IV) that may play a role in targeting the zymogen to cell-matrix contacts and in the degradation of the collagen IV network.

Pro-MMP-9 activation by the MT1-MMP/MMP-2 axis and MMP-3: role of TIMP-2 and plasma membranes

MMP-9 (gelatinase B) is produced in a latent form (pro-MMP-9) that requires activation to achieve catalytic activity. Previously, we showed that MMP-2 (gelatinase A) is an activator of pro-MMP-9 in solution. However, in cultured cells pro-MMP-9 remains in a latent form even in the presence of MMP-2. Since pro-MMP-2 is activated on the cell surface by MT1-MMP in a process that requires TIMP-2, we investigated the role of the MT1-MMP/MMP-2 axis and TIMPs in mediating pro-MMP-9 activation. Full pro-MMP-9 activation was accomplished via a cascade of zymogen activation initiated by MT1-MMP and mediated by MMP-2 in a process that is tightly regulated by TIMPs. We show that TIMP-2 by regulating pro-MMP-2 activation can also act as a positive regulator of pro-MMP-9 activation. Also, activation of pro-MMP-9 by MMP-2 or MMP-3 was more efficient in the presence of purified plasma membrane fractions than activation in a soluble phase or in live cells, suggesting that concentration of pro-MMP-9 in the pericellular space may favor activation and catalytic competence.

Potent Mechanism-based Inhibitors for Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (kon of 102 to 104 M-1 s-1 and the reversal of the process is slow (koff of 10-3 to 10-4 s-1). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.

Effects on Substrate Profile by Mutational Substitutions at Positions 164 and 179 of the Class A TEMpUC19 β-Lactamase from Escherichia coli

We investigated the effects of mutations at positions 164 and 179 of the TEMpUC19 β-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum β-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A β-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum β-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg164 → Asn, Arg164 → Ser, Asp179 → Asn, and Asp179 → Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum β-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K s ) invariably increased. Computation of the microscopic rate constants (k 2 andk 3) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k 3) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k 2) and deacylation (k 3) of the extended-spectrum β-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEMpUC19 β-lactamase. Our results indicated that severe steric interactions between the large 7β functionalities of the expanded-spectrum β-lactams and the Ω-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.

MMP25 (MT6-MMP) Is Highly Expressed in Human Colon Cancer, Promotes Tumor Growth, and Exhibits Unique Biochemical Properties

MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (Ki = 0.2 nm) over TIMP-2 (Ki = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.

Class D β-Lactamases: Are They All Carbapenemases?

ABSTRACT Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are enzymes of the utmost clinical importance due to their ability to produce resistance to carbapenems, the antibiotics of last resort for the treatment of various life-threatening infections. The vast majority of these enzymes have been identified in Acinetobacter spp., notably in Acinetobacter baumannii. The OXA-2 and OXA-10 enzymes predominantly occur in Pseudomonas aeruginosa and are currently classified as narrow-spectrum class D β-lactamases. Here we demonstrate that when OXA-2 and OXA-10 are expressed in Escherichia coli strain JM83, they produce a narrow-spectrum antibiotic resistance pattern. When the enzymes are expressed in A. baumannii ATCC 17978, however, they behave as extended-spectrum β-lactamases and confer resistance to carbapenem antibiotics. Kinetic studies of OXA-2 and OXA-10 with four carbapenems have demonstrated that their catalytic efficiencies with these antibiotics are in the same range as those of some recognized class D carbapenemases. These results are in disagreement with the classification of the OXA-2 and OXA-10 enzymes as narrow-spectrum β-lactamases, and they suggest that other class D enzymes that are currently regarded as noncarbapenemases may in fact be CHDLs.