Analysis of differential protein expression in the cytosol of melphalan-resistant and -susceptible MCF-7 cell lines has been carried out using a combination of two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics. Comparison of multiple digitized gel arrays detected several spots as candidates for differentially expressed proteins in melphalan-resistant MCF-7 cells. The up-regulated proteins included retinoic acid binding protein II, an isoform of the macrophage migration inhibition factor, and other unidentified proteins. The down-regulated proteins included calreticulin, cyclophin A, and an isoform of the 27 kD heat shock protein. Correlation of the differential expression of some of the proteins with acquired resistance of MCF7 cells to melphalan is discussed.

Protein expression patterns in the cytosol of MCF-7 cells resistant to adriamycin and to adriamycin/verapamil were compared to that of the parental MCF-7 cell line and to each other using metabolic labeling and two-dimensional gel electrophoresis. Growing the parental MCF-7 cell line in 13C6-arginine- and 13C6-lysine-enriched medium resulted in C-terminal labeling of all tryptic peptides. The culture media was optimized for the incorporation of these labeled amino acids under conditions that also supported cell growth. Protein abundances were found to be distinctive in MCF-7 cells resistant to adriamycin and those selected for resistance to both adriamycin and verapamil.

Matrix metalloproteinase 2 (MMP-2) contains three fibronectin type II (col) modules that contribute to its collagen specificity. We observed that the CD spectra of the separate col modules account for the CD and temperature profiles of the in-tandem col-123 construct. Thus, to the extent of not significantly perturbing the secondary structure and thermal stability characteristics of the neighboring units, the domains within col-123 do not interact. Via NMR, we investigated ligand binding properties of the three repeats within col-123: col-123/1 (the col-1 domain within col-123), col-123/2, and col-123/3. Interactions of col-123 with the collagen mimic peptide (Pro-Pro-Gly) 6 (PPG6) and propeptide segment PIIKFPGDVA (p33-42) were studied. While col-123/1 and col-123/2 bound PPG6, they interacted more weakly with p33-42. In contrast, col-123/3 exhibited a higher affinity for p33-42 than for PPG6. Thus, despite their structural homology, the col repeats of MMP-2 differ in substrate specificity. Furthermore the binding affinities toward the three in-tandem col repeats were close to those determined for the individual isolated domains or for col-12/1, indicating that vis-à -vis these ligands each module interacts essentially as an autonomous unit. Interestingly the domains within col-123 exhibited enhanced affinities for Hel3, a construct that contains ((Gly-ProPro) 12 ) 3 in triple helical configuration. Nevertheless the affinities were significantly higher for col-123/1 and col-123/2 relative to col-123/3 in line with their behaviors toward PPG6. This hints at a cooperative participation toward Hel3, which is a closer mimic of collagen, a hypothesis that is supported by the detected lower affinities of col-12/1, col-12/2, col-2, col-23/2, col-3, and col-23/3 for Hel3. Elevated expression of matrix metalloproteinase (MMP)1 in malignant tumors has been attributed to their ability to metastasize (1). In particular MMP-2 and MMP-9 are considered critical in that they both degrade collagen IV, thus facilitating penetration of the basement membranes by tumor cells (2, 3). Moreover MMP-2 and MMP-9 play important roles in development, tissue remodeling, and inflammation (for a review, see Ref. 4).Besides the catalytic and hemopexin domains common to most MMPs, MMP-2 and MMP-9 also contain three in-tandem fibronectin type II (FN2) modules that are positioned within the protease in the vicinity of the active site. In MMP-2, the FN2 modules are ascribed affinities to gelatin, type I and IV collagens, elastin, and laminin (5-10). NMR solution structures of the second FN2 domain of the bovine seminal fluid protein PDC-109 (PDC109/b) (11), the two FN2 repeats from human fibronectin (12-15), and the three FN2 modules from human MMP-2 (col-1, col-2, and col-3, respectively) (16 -18) have been reported. In addition, the x-ray crystallographic structure of the intact, ligand-free pro-MMP-2 is also known (19).It has been shown that fragments containing two or three consecutive FN2 modules from MMP-2 bind immobilized gelatin stronger ...

The interaction of matrix metalloproteinase 2 (MMP-2) with gelatin is mediated by three repeats homologous to fibronectin type II (FN2) modules, which are inserted in the catalytic domain in proximity of the active site. We screened a random 15-mer phage display library to identify peptides that interact with the FN2 modules of MMP-2. Interestingly, the selected peptides are not gelatin-like and do not share a common, obvious sequence motif. However, they contain a high proportion of aromatic residues. The interactions of two peptides, WHWRH0RIPLQLAAGR and THSHQWRHHQF-PAPT, with constructs comprising the in-tandem first and second and second and third FN2 modules of MMP-2 (Col-12 and Col-23, respectively) were characterized by NMR. Both peptides interact with Col-12 and Col-23 with apparent association constants in the mM ؊1 range. Peptide binding results in perturbation of signals from residues located in the gelatin-binding pocket and flexible parts of the molecule. Although the former finding suggests that the gelatin-binding site is involved in the contact, the interpretation of the latter is less straightforward and may well reflect both the direct and indirect effects of the interaction.Matrix metalloproteinase 2 (MMP-2, 1 gelatinase A), and the closely related MMP-9 (gelatinase B) are unique among the metalloproteinases in that three gelatin-binding fibronectin type II (FN2) modules (Col-1, Col-2, and Col-3) are inserted in their catalytic domain in the vicinity of the active site (1). The solution conformation of each FN2 repeat from human MMP-2 has been characterized via NMR spectroscopy (2-4). Moreover, the x-ray crystallographic structure of the intact human pro-MMP-2 has been reported (5).In the second FN2 module from each MMP-2 and MMP-9, residues that are important for the interaction with gelatin have been identified via site-directed mutagenesis (6, 7). Additionally, the ligand binding surfaces of all three modules of MMP-2 have been mapped from 1 H and 15 N NMR perturbations induced by (PPG) 6 and the longer chain analog, (PPG) 12 , synthetic peptide mimics of gelatin (2-4). In line with the crystallographic evidence, which shows that the FN2 modules in MMP-2 point away from each other (5), our NMR studies of the interaction between Col domains and (PPG) 6 and (PPG) 12 have shown that consecutive Col modules contain distinct ligand-binding sites in which affinities for these ligands are virtually identical to those of the individual domains (3,4,8).Although the affinity of the MMP-2 Col domains for collagenous ligands appears by now to be well established, less is known regarding the specificity of the interaction. In our previous studies we found that the peptide PIIKFPGDVA, which corresponds to segment 33-42 of the pro-MMP-2, interacts with the three Col domains of MMP-2 in a manner that mimics the interaction with the collagen-like (PPG) 6 and (PPG) 12 peptides (3, 4). Preference for binding to Col-3 was indicated, consistent with the x-ray crystallographic structure of the pro-MMP-2 (5). In ...

Human matrix metalloproteinase-2 (MMP-2) contains an array of three fibronectin type II (FII) modules postulated to interact with gelatin (denatured collagen).Here, we verify that the NMR solution structure of the third FII repeat (COL-3) is similar to that of the second FII repeat (COL-2); characterize its ligand-binding properties; and derive dynamics properties and relative orientation in solution for the two domains of the COL-23 fragment, a construct comprising COL-2 and COL-3 in tandem, with each domain possessing a putative collagen-binding site. Interaction of the synthetic gelatinlike octadecapeptide (Pro-Pro-Gly) 6 (PPG6) with COL-3 is weaker than with COL-2. We found that a synthetic peptide comprising segment 33-42 (peptide 33-42) from the MMP-2 prodomain interacts with COL-3 and, albeit with lower affinity, with COL-2 in a way that mimics PPG6 binding. COL-3 strongly prefers peptide 33-42 over PPG6, which suggests that intramolecular interactions with the prodomain could modulate binding of pro-MMP-2 to its gelatin substrate. In COL-23, the two modules retain their structural individuality and tumble independently. Overall, the NMR data indicate that the relative orientation of the modules in COL-23 is not fixed in solution, that the modules do not interact with one another, and that COL-23 is rather flexible. The binding sites face opposite each other, and their responses to, and normalized affinities for, the longer ligand PPG12 are virtually identical to those of the individual domains for PPG6, thus precluding cooperativity, although they may interact simultaneously with multiple sites of the extracellular matrix. (2) and, unique among the metalloproteinases, three in-tandem fibronectin type II (FII) modules, which are inserted in the catalytic domain in the vicinity of the active site. In its latent form, the prodomain folds over the active-site cleft and contributes a cysteine thiol group, which coordinates the catalytic zinc ion and, as indicated by the recent x-ray crystallographic model (3), inserts the side chain of Phe 37 into the hydrophobic pocket of the third FII domain. This interaction can be disrupted by proteolysis. Once the active site is free, MMP-2 undergoes autolytic cleavage, resulting in loss of the prodomain (1).The FII modules account for the affinity of MMP-2 for gelatin, type I and IV collagens, elastin, and laminin (4 -9). A number of residues involved in binding of small hydrophobic ligands to the related second FII module of the bovine seminal fluid protein PDC-109 (PDC-109/b) were inferred from 1 H NMR studies (10). In addition, several residues that are important for interaction with gelatin have been identified, via site-directed mutagenesis, in the second FII modules from MMP-2 (11) and MMP-9 (12). However, little is known as to how tandem arrays of FII domains interact with other molecules. Fragments containing two or three consecutive FII modules from MMP-2 exhibit significantly higher apparent affinities for immobilized gelatin than any of the single modules (5...