Evaluation of metabolic labeling for comparative proteomics in breast cancer cells.

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.

Modular autonomy, ligand specificity, and functional cooperativity of the three in-tandem fibronectin type II repeats from human matrix metalloproteinase 2.

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-a-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-Pro-Pro)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.

Highly altered protein expression profile in the adriamycin resistant MCF-7 cell line.

The protein expression pattern in the cytosol fraction of the adriamycin resistant MCF-7 cell line (MCF-7/ADR) was compared to that of the parental MCF-7 cell line using two-dimensional gel electrophoresis and mass spectrometry. Twenty proteins with altered abundances were identified and studied in MCF-7/ADR. Both up regulation and down regulation are characterized. The most striking differences were found for proteins that were uniquely expressed in this cell line and not detectable in the parental MCF-7 cell line. These proteins include annexin I, the neuronal ubiquitin carboxyl hydrolase isoenzyme L-1 (also known as PGP9.5), glutathione-S-transferase pi class, nicotinamide N-methyltransferase, and interleukin-18 precursor. On the other hand, catechol-O-methyltransferase was expressed in the parental cell line, but was not detected in the adriamycin resistant cell line. This protein expression pattern was unique to MCF-7/ADR and not observed in MCF-7 cell lines selected for resistant to etoposide, mitoxantrone or melphalan.

Proteomic phenotyping: metastatic and invasive breast cancer.

The adriamycin resistant breast cancer cell line (MCF-7/ADR) is a subject of ongoing debate concerning its origin and or source. Previous studies in our laboratory showed that MCF-7/ADR has a unique cytosolic protein expression pattern when compared to that of the parental MCF-7 cell line and other drug resistant MCF-7 cell lines. Protein expression patterns obtained using two-dimensional gel electrophoresis and mass spectrometry indicated that this MCF-7/ADR cell line shares some similarities with the metastatic breast cancer cell lines MDA-MB. Further comparisons with available two-dimensional gel electrophoresis maps in the literature indicate that MCF-7/ADR has a protein expression signature even closer to of the ductal infiltrating breast carcinoma cell line 8701. These observations suggest that MCF-7/ADR cells might have originated in a selection of ductal infiltrating carcinoma cells, which were present among the original MCF-7 cell population. These ductal infiltrating carcinoma cells may possess an intrinsic adriamycin resistance phenotype.

Peptide ligands for the fibronectin type II modules of matrix metalloproteinase 2 (MMP-2).

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 THSHQWRHHQFPAPT, 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.

The col-1 module of human matrix metalloproteinase-2 (MMP-2): structural/functional relatedness between gelatin-binding fibronectin type II modules and lysine-binding kringle domains.

Human matrix metalloproteinase-2 (MMP-2) contains three in-tandem fibronectin type II (FII) repeats that bind gelatin. Here, we report the NMR solution structure of the first FII module of MMP-2 (col-1). The latter is described as a characteristic, globular FII fold containing two beta-sheets, a stretch of 3(1)-helix, a turn of alpha-helix, and an exposed hydrophobic surface lined with aromatic residues. We show that col-1 binds (Pro-Pro-Gly)6, a mimic of gelatin, with a Ka of approx. 0.42 mm(-1), and that its binding site involves a number of aromatic residues as well as Arg34, as previously found for the second and third homologous repeats. Moreover, the affinity of the in-tandem col-1+2 construct (col-12) toward the longer ligand (Pro-Pro-Gly)12 is twice that for (Pro-Pro-Gly)6, as expected from mass action. A detailed structural comparison between FII and kringle domains indicates that four main conformational features are shared: two antiparallel beta-sheets, a central 3(1)-helix, and the quasiperpendicular orientation of the two proximal Cys-Cys bonds. Structure superposition by optimizing overlap of cystine bridge areas results in close juxtaposition of their main beta-sheets and 31-helices, and reveals that the gelatin binding site of FII modules falls at similar locations and exhibits almost identical topological features to those of the lysine binding site of kringle domains. Thus, despite the minor (<15%) consensus sequence relating FII modules to kringles, there is a strong folding and binding site structural homology between the two domains, enforced by key common conformational determinants.

The two-domain NK1 fragment of plasminogen: folding, ligand binding, and thermal stability profile.

The two-domain fragment N+K1 (rNK1) [Glu(1)-Glu(163)] of human plasminogen was expressed in E. coli as a hexahistidine-tagged fusion protein and chromatographically purified. The (1)H NMR spectrum supports proper folding of the K1 component within the refolded rNK1 construct (rNK1/K1). The functional properties of rNK1/K1 were investigated via intrinsic fluorescence titration with kringle-specific omega-aminocarboxylic acid ligands. The affinities closely match those previously measured for the isolated K1, which indicates that the N-domain does not significantly affect the interaction of ligands with the lysine binding site of K1. Far-UV CD spectra recorded for the N-domain suggest conformational plasticity and flexibility for the module. Two classes of spectra, referred to as types A and B, were identified with the type A spectrum reflecting a higher secondary structure content than that estimated for the type B spectrum. Subtracting the CD spectrum of rK1 from that of rNK1 yields a spectrum (Delta) which reflects the conformation of the N-domain within the rNK1 construct (rNK1/N). Delta resembles the type A spectrum, suggesting that rNK1/N adopts a relatively more ordered conformation, stabilized by the adjacent rNK1/K1 domain. In contrast, thermal unfolding curves determined via CD indicate that the rNK1/N slightly lowers the melting temperature (T(m)) of rNK1/K1. Independence of the two domains within rNK1 was tested by monitoring the thermal unfolding of rNK1/K1 when in the presence of the kringle-specific ligand AMCHA, which left the rNK1/N T(m) essentially unaffected, while increasing that of the rNK1/K1 by approximately 10 degrees C.

Differential protein expression in the cytosol fraction of an MCF-7 breast cancer cell line selected for resistance toward melphalan.

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.