Biomedical subjects

B I Ratnikov

Publications and source records attributed to B I Ratnikov.

8 recordsLinked to original sources

PubMed2005

Integrin activation by talin.

The development and integrity of the cardiovascular system depends on integrins, a family of adhesion receptors, vitally important for homeostasis of animal species from fruit fly to man. Integrins are critical players in cell migration, cell adhesion, cell cycle progression, differentiation, and apoptosis. Consequently, integrins have a major impact on the patterning and functions of the blood and cardiovascular system. Integrins undergo conformational changes, which alter their affinity for ligands through a process operationally defined as integrin activation. Integrin activation is important for platelet aggregation, leukocyte extravasation, and cell adhesion and migration, thus influencing such processes as hemostasis, inflammation and angiogenesis. Recently, a series of studies have begun to define the mechanism of integrin activation by demonstrating that binding of a cytoskeletal protein, talin, to integrin beta subunit cytoplasmic tail is a last common step in integrin activation. These findings indicate that talin is likely to be at the center of converging signaling pathways regulating integrin activation.

Amino Acid Sequence↗

PubMed2001

Characterization of matrix metalloproteinase-26, a novel metalloproteinase widely expressed in cancer cells of epithelial origin.

Identification of expanding roles for matrix metalloproteinases (MMPs) in complex regulatory processes of tissue remodelling has stimulated the search for genes encoding proteinases with unique functions, regulation and expression patterns. By using a novel cloning strategy, we identified three previously unknown human MMPs, i.e. MMP-21, MMP-26 and MMP-28, in comprehensive gene libraries. The present study is focused on the gene and the protein of a novel MMP, MMP-26. Our findings show that MMP-26 is specifically expressed in cancer cells of epithelial origin, including carcinomas of lung, prostate and breast. Several unique structural and regulatory features, including an unusual 'cysteine-switch' motif, discriminate broad-spectrum MMP-26 from most other MMPs. MMP-26 efficiently cleaves fibrinogen and extracellular matrix proteins, including fibronectin, vitronectin and denatured collagen. Protein sequence, minimal modular domain structure, exon-intron mapping and computer modelling demonstrate similarity between MMP-26 and MMP-7 (matrilysin). However, substrate specificity and transcriptional regulation, as well as the functional role of MMP-26 and MMP-7 in cancer, are likely to be distinct. Despite these differences, matrilysin-2 may be a suitable trivial name for MMP-26. Our observations suggest an important specific function for MMP-26 in tumour progression and angiogenesis, and confirm and extend the recent findings of other authors [Park, Ni, Gerkema, Liu, Belozerov and Sang (2000) J. Biol. Chem. 275, 20540--20544; Uría and López-Otín (2000) Cancer Res. 60, 4745--4751; de Coignac, Elson, Delneste, Magistrelli, Jeannin, Aubry, Berthier, Schmitt, Bonnefoy and Gauchat (2000) Eur. J. Biochem. 267, 3323--3329].

Amino Acid Sequence↗

PubMed2001

Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP). The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2). Both activation and autocatalytic maturation of pro-MMP-2 in trans suggest that MT1-MMP should exist as oligomers on the cell surface. To better understand the functions of MT1-MMP, we designed mutants with substitutions in the active site (E240A), the cytoplasmic tail (C574A), and the RRXR furin cleavage motifs (R89A, ARAA, and R89A/ARAA) of the enzyme. The mutants were expressed in MCF7 breast carcinoma cells that are deficient in both MMP-2 and MT1-MMP. Our results supported the existence of MT1-MMP oligomers and demonstrated that a disulfide bridge involving the Cys(574) of the enzyme's cytoplasmic tail covalently links MT1-MMP monomers on the MCF7 cell surface. The presence of MT1-MMP oligomers also was shown for the enzyme naturally expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and double (R89A/ARAA) furin cleavage site mutants of MT1-MMP were processed in MCF7 cells into the mature proteinase capable of activating pro-MMP-2 and stimulating cell locomotion. This suggested that furin cleavage is not a prerequisite for the conversion of pro-MT1-MMP into the functionally active enzyme. A hydroxamate class inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MMP in MCF7 cells but not in HT1080 cells. This implied that a matrixin-like proteinase sensitive to hydroxamates could be involved in a furin-independent, alternative pathway of MT1-MMP activation in breast carcinoma cells. The expression of the wild type MT1-MMP enhanced cell invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In contrast, both the C574A and E240A mutations render MT1-MMP inefficient in stimulating cell migration and invasion. In addition, the C574A mutation negatively affected cell adhesion, thereby indicating critical interactions involving the cytosolic part of MT1-MMP and the intracellular milieu.

Breast Neoplasms↗

PubMed2001

Matrix-dependent proteolysis of surface transglutaminase by membrane-type metalloproteinase regulates cancer cell adhesion and locomotion.

Cell invasion requires cooperation between adhesion receptors and matrix metalloproteinases (MMPs). Membrane type (MT)-MMPs have been thought to be primarily involved in the breakdown of the extracellular matrix. Our report presents evidence that MT-MMPs in addition to the breakdown of the extracellular matrix may be engaged in proteolysis of adhesion receptors on tumor cell surfaces. Overexpression of MT1-MMP by glioma and fibrosarcoma cells led to proteolytic degradation of cell surface tissue transglutaminase (tTG) at the leading edge of motile cancer cells. In agreement, structurally related MT1-MMP, MT2-MMP, and MT3-MMP but not evolutionary distant MT4-MMP efficiently degraded purified tTG in vitro. Because cell surface tTG represents a ubiquitously expressed, potent integrin-binding adhesion coreceptor involved in the binding of cells to fibronectin (Fn), the proteolytic degradation of tTG by MT1-MMP specifically suppressed cell adhesion and migration on Fn. Reciprocally, Fn in vitro and in cultured cells protected its surface receptor, tTG, from proteolysis by MT1-MMP, thereby supporting cell adhesion and locomotion. In contrast, the proteolytic degradation of tTG stimulated migration of cells on collagen matrices. Together, our observations suggest both an important coreceptor role for cell surface tTG and a novel regulatory function of membrane-anchored MMPs in cancer cell adhesion and locomotion. Proteolysis of adhesion proteins colocalized with MT-MMPs at discrete regions on the surface of migrating tumor cells might be controlled by composition of the surrounding ECM.

Cell Adhesion↗

PubMed1997

Identification of a region in the integrin beta3 subunit that confers ligand binding specificity.

Many integrin adhesion receptors bind ligands containing the Arg-Gly-Asp (RGD) peptide motif. Most integrins exhibit considerable specificity for particular ligands and can distinguish among the many conformations of RGD. In this study we identify the domain of the integrin beta subunit involved in determining ligand binding specificity. Chimeras of beta3 and beta5, the most homologous integrin beta subunits, were expressed with alphav on the surface of human 293 cells. The ligand binding phenotype of each chimera was assessed using the ligands Fab-9 and fibrinogen, both of which have a binding preference for alphavbeta3. The results of the study show that when exons C and D of the beta3 subunit (residues 95-233) are substituted into beta5, the chimera gained the ability to bind Fab-9 with an affinity close to that of wild-type alphavbeta3. This chimera was able to mediate cell adhesion to fibrinogen. Furthermore, the swap of only a 39-residue segment of this larger domain, beta3 residues 164-202, into the backbone of beta5 enabled the chimeric integrin to bind soluble Fab-9. This small domain is highly divergent among the integrin beta subunits, suggesting that it may play a role in determining ligand selection by all integrins.

Amino Acid Sequence↗

PubMed1997

Evidence that the integrin beta3 and beta5 subunits contain a metal ion-dependent adhesion site-like motif but lack an I domain.

The amino-terminal domain of each integrin beta subunit is hypothesized to contain an ion binding site that is key to cell adhesion. A new hypothesis regarding the structure of this site is suggested by the crystallization of the I domains of the integrin alphaL and alphaM subunits (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638; Qu, A., and Leahy, D. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10277-10281). In those proteins, an essential metal ion is bound by a metal ion-dependent adhesion site (MIDAS). The MIDAS is presented at the apex of a larger protein module called an I domain. The metal ligands in the MIDAS can be separated into three distantly spaced clusters of oxygenated residues. These three coordination sites also appear to exist in the integrin beta3 and beta5 subunits. Here, we examined the putative metal binding site within beta3 and beta5 using site-directed mutagenesis and ligand binding studies. We also investigated the fold of the domain containing the putative metal binding site using the PHD structural algorithm. The results of the study point to the similarity between the integrin beta subunits and the MIDAS motif at two of three key coordination points. Importantly though, the study failed to identify a residue in either beta subunit that corresponds to the second metal coordination group in the MIDAS. Moreover, structural algorithms indicate that the fold of the beta subunits is considerably different than the I domains. Thus, the integrin beta subunits appear to present a MIDAS-like motif in the context of a protein module that is structurally distinct from known I domains.

Amino Acid Sequence↗

PubMed1995

Identification of a growth factor for primary murine stroma as macrophage colony-stimulating factor.

Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. We took advantage of an assay that permits analysis of primary stroma-initiating cells (SICs) on the clonal level, and further characterized SICs and the factors that regulate SICs. Stroma formation in this assay is dependent on a high-molecular-weight factor secreted by the stromal cell line AC3.U. Here we show that this factor is identical to macrophage colony-stimulating factor (M-CSF), and that purified M-CSF is sufficient for induction of stroma formation. M-CSF, isolated from the line AC3.U, as well as from L929 cells and COS cells transfected with an expression vector encoding M-CSF, migrated in two peaks as 160- and 650-kD species after gel filtration. These molecular-weight species encompassed all stroma-inducing activity, and both stimulated macrophage colony formation. Affinity chromatography and blocking studies with antibodies specific for M-CSF and c-fms confirmed M-CSF as the sole factor in the supernatant of the stromal cell line AC3.U that promotes stroma formation. Culture of marrow, for as little as 1 week, depleted M-CSF-dependent SIC while increasing the incidence of replatable, factor-independent SIC. This suggests that culture changes the properties of SICs, perhaps by inducing differentiation into mature stromal cells. Thus, our results show a novel function of M-CSF as an important modulator of stroma formation.

Animals↗

PubMed1994

Clonal analysis of primary marrow stroma: functional homogeneity in support of lymphoid and myeloid cell lines and identification of positive and negative regulators.

Stromal cells play an important role in regulating early hematopoiesis. How stromal cells exert their different functions and the factors that regulate stromal cells themselves remain to be elucidated definitively, however. We describe here a limiting dilution assay for primary stroma colonies from murine marrow. This system permits a critical analysis of stromal cell function and regulation on the clonal level. We report that stroma formation was dependent on an activity secreted by the long-term cultured stromal line AC-3.U. Differential ultrafiltration of AC-3.U supernatant (SN) suggests that this potentially novel activity is represented by molecules with apparent molecular weights (m.w.) of > 100 < 300 kD and > 300 kD. In contrast to the AC-3.U activity, hydrocortisone (HC) acts as a negative regulator of stroma colony formation. We used the stroma colony assay to analyze potential stromal cell heterogeneity. We found that most, if not all, primary stromal colonies supported expansion of both myeloid and lymphoid cell lines. In contrast, long-term cultured stromal cell lines differed not only among lines, but also on the level of sublines, in their ability to sustain myeloid and lymphoid cells. This intraclonal variation suggests that the heterogeneity of cell lines can be a reflection of ongoing culture adaptation. The functional homogeneity of primary stromal colonies, together with their susceptibility to regulators, indicates that the performance of primary stroma is subject to external control. The establishment of a clonal assay system has paved the way to analyze the molecules that regulate primary stroma and thereby hematopoietic cells.

Animals↗