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1.
Prolidase (EC 3.4.13.9) is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. An increase in enzyme activity is correlated with increased rates of collagen turnover indicative of extracellular matrix (ECM) remodeling, but the mechanism linking prolidase activity and ECM is poorly understood. Thus, the effect of ECM-cell interaction on intracellular prolidase activity is of special interest. In cultured human skin fibroblasts, the interaction with ECM and, more specifically, type I collagen mediated by the β1 integrin receptor regulates cellular prolidase activity. Supporting evidence comes from the following observations: 1) in sparse cells with a low amount of ECM collagen or in confluent cells in which ECM collagen was removed by collagenase (but not by trypsin or elastase) treatment, prolidase activity was decreased; 2) this effect was reversed by the addition of type I collagen or β1 integrin antibody (agonist for β1 integrin receptor); 3) sparse cells (with typically low prolidase activity) showed increased prolidase activity when grown on plates coated with type I collagen or on type IV collagen and laminin, constituents of basement membrane; 4) the relative differences in prolidase activity due to collagenase treatment and subsequent recovery of the activity by β1 integrin antibody or type I collagen treatment were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells, as shown by Western immunoblot analysis. Thus, we conclude that prolidase activity responded to ECM metabolism (tissue remodeling) through signals mediated by the integrin receptor. J. Cell. Biochem. 67:166–175, 1997. Published 1997 Wiley-Liss, Inc. 相似文献
2.
Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis 总被引:6,自引:0,他引:6
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Parsons M Keppler MD Kline A Messent A Humphries MJ Gilchrist R Hart IR Quittau-Prevostel C Hughes WE Parker PJ Ng T 《Molecular and cellular biology》2002,22(16):5897-5911
Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination. 相似文献
3.
Matthew J. Rock Paul Holden William A. Horton Daniel H. Cohn 《Molecular and cellular biochemistry》2010,338(1-2):215-224
Cartilage oligomeric matrix protein (COMP) is a pentameric ~524 kDa multidomain extracellular matrix protein and is the fifth member of the thrombospondin family. COMP is abundantly expressed in proliferating and hypertrophic chondrocytes of the growth plate, articular cartilage, synovium, tendon, and ligament. The spatial localization of COMP highlights its importance in the phenotypes of pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), COMP disorders that are characterized by disproportionate short stature, brachydactyly, scoliosis, early-onset osteoarthritis, and joint hypermobility. In this study, the role of COMP in ligament was investigated with a series of cell attachment assays using ligament cells binding to COMP. A dose-dependent cell attachment activity was found, which was inhibited by a peptide containing the SFYVVMWK amino acid sequence derived from the globular C-terminal domain of COMP. This activity was independent of the recently described RGD-dependent attachment activity. Function-blocking antibodies to CD47 and αVβ3 integrin reduced cell attachment to COMP, implicating the participation of these cell surface molecules in COMP cell binding. Immunofluorescence studies showed that cell attachment to COMP induced the formation of lamellae containing F-actin microspikes associated with fascin. We propose that COMP promotes cell attachment via two independent mechanisms involving cell surface CD47 and αVβ3 integrin and that a consequence of cell attachment to COMP is the specific induction of fascin-stabilized actin microspikes. 相似文献
4.
Restrictin: a chick neural extracellular matrix protein involved in cell attachment co-purifies with the cell recognition molecule F11. 总被引:13,自引:0,他引:13
We report here the characterization of restrictin, a novel chick neural extracellular matrix glycoprotein associated with the cell recognition molecule F11. Immunoaffinity chromatography using monoclonal antibody 23-13 directed to restrictin yield a major relative molecular mass band at 170 x 10(3) and minor bands at 160, 180, 250 and 320 x 10(3) which are immunologically related to each other. Neural cells attach on immobilized restrictin in a short-term adhesion assay. This adhesion can be blocked specifically by monoclonal or polyclonal antibodies to restrictin but not by antibodies to F11 or by the peptide GRGDSP. Antibodies to restrictin do not interfere with the fasciculation of retinal axons and the isolated restrictin does not stimulate the outgrowth of axons. In the developing nervous system, restrictin is localized in very restricted regions and is found within areas of F11 expression. The timing and pattern of expression of restrictin and its cell attachment activity suggest that it participates in developmental events of the nervous system. 相似文献
5.
Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin. 相似文献
6.
D G Woodside A Obergfell L Leng J L Wilsbacher C K Miranti J S Brugge S J Shattil M H Ginsberg 《Current biology : CB》2001,11(22):1799-1804
Syk protein tyrosine kinase is essential for immune system development and function [1]and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs)[1]. Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase. 相似文献
7.
Ranajay Saha Surajit Rakshit Samir Kumar Pal 《Journal of molecular recognition : JMR》2013,26(11):568-577
Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo‐Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady‐state and picosecond‐resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N‐dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo‐Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β‐CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature‐dependent circular dichroism studies explore the change in the secondary structure of Apo‐Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
8.
《Seminars in cell biology》1995,6(6):367-376
Neutrophil Proteinases have the capacity to degrade almostevery component of the extracellular matrix. In marked contrast to the wealth of available data about the structure and activity of these proteinases when they are free in solution, there has been relatively little information about the mechanisms by which neutrophils use and control their proteolytic enzymes in an extracellular milieu that is replete with proteinase inhibitors. However, recent data have provided insights into several mechanisms that permit these enzymes to evade inhibition: (1) compartmentalization; (2) localized inactivation of proteinase inhibitors; (3) tight binding of enzymes to substrates; and (4) binding of proteinases to the neutrophil's cell surface. 相似文献
9.
10.
The interaction of retinol-binding protein with its plasma-membrane receptor. 总被引:4,自引:1,他引:3
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125I-labelled retinol-binding protein (RBP) bound to specific receptors in human placental brush-border membranes. Binding at 22 degrees C reached equilibrium within 15 min, but prolonged incubation caused a subsequent decline. Scatchard analysis of the equilibrium binding data at 22 degrees C and 15 min showed high-(3.0 +/- 2.7 x 10(-9) M) and low-(9.5 +/- 3.5 x 10(-8) M) affinity binding components. 125I-RBP, bound to membranes at 22 degrees C for 15 min and subsequently dissociated with excess unlabelled RBP, exhibited biphasic dissociation kinetics consisting of fast and slow components of release. In contrast, Scatchard analysis and dissociation kinetics of the binding that had taken place at 37 degrees C for 1 h showed the fast-dissociating/low-affinity binding component, but little of the slow-dissociating/higher-affinity binding component. When 125I-RBP, after incubation with membranes at 37 degrees C for 1 h, was re-isolated and subjected to dissociation kinetic analysis using a fresh batch of membranes, the fast-dissociating phase was unchanged, but the slow phase was almost absent. The complex kinetics were interpreted in terms of a heterogeneity in RBP consisting of high- and low-affinity binding forms. The higher-affinity-binding form is thought to be converted into the lower-affinity state on binding to the receptor. Transthyretin inhibited 125I-RBP binding to the membrane, suggesting that free, rather than transthyretin-associated, RBP bound to the receptor. The RBP receptor was trypsin-, heat- and thiol-group-specific-reagent sensitive and was highly specific for RBP. 相似文献
11.
Paul E Di Cesare Frank S Chen Matthias Moergelin Cathy S Carlson Michael P Leslie Roberto Perris Carrie Fang 《Matrix biology》2002,21(5):461-470
Recent work indicates that cartilage oligomeric matrix protein (COMP) plays an important role in extracellular matrix assembly and matrix-matrix protein interactions. In order to identify the proteins in extracellular matrix that interact with COMP, we used an ELISA-based solid-phase binding assay, which revealed a specific, high-affinity interaction between COMP and fibronectin. This interaction is concentration-dependent and saturable, and appears to occur under physiologically relevant conditions. Electron microscopy after negative staining and fragment binding analysis using the solid-phase assay revealed a predominant binding site for the COMP C-terminal globular domain to a molecular domain approximately 14 nm from the N-terminal domain of fibronectin, which can be inhibited by the presence of a polyclonal antibody specific for the C-terminal heptadecapeptide of COMP. This interaction is further demonstrated in vivo by colocalization of both COMP and fibronectin in the chondrocyte pericellular matrix by laser confocal microscopy of chondrocytes grown in agarose culture, and by appositional and colocalization of these proteins in the growth plate of primates by immunohistochemistry. 相似文献
12.
Ricciardelli C Russell DL Ween MP Mayne K Suwiwat S Byers S Marshall VR Tilley WD Horsfall DJ 《The Journal of biological chemistry》2007,282(14):10814-10825
Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility. 相似文献
13.
G Y Ishioka A G Lamont D Thomson N Bulbow F C Gaeta A Sette H M Grey 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(8):2446-2451
The T cell independence of complex polysaccharide Ag has suggested the possibility that carbohydrates may be incapable of T cell recognition because of a failure to interact with MHC restriction elements and/or a failure of MHC/carbohydrate complexes to interact with and be recognized by Ag-specific TCR. We have used two approaches to obtain information about T cell recognition of carbohydrate. First, we have determined the capacity of a series of oligosaccharides and glycolipids to bind a murine class II MHC molecule, IAd. No significant binding was observed with the 26 compounds tested, but the limitation to these studies was that there was a relatively limited collection of synthetic carbohydrate and glycolipid structures of limited complexity available for analysis. The second approach involved the study of the effect of glycosylation of a known peptide T cell epitope (OVA 323-339) on MHC binding of the peptide and on T cell recognition. Three patterns of effects were observed: 1) no effect on either binding or T cell recognition. This pattern was observed when the carbohydrate was located at residues removed from the core MHC-binding region. When the carbohydrate was located within the core MHC-binding regions, either 2) glycosylation destroyed both MHC binding and T cell recognition; or 3) glycosylation did not ablate MHC binding or T cell recognition. In this latter instance, there was evidence to indicate that the carbohydrate moiety was an important part of the antigenic determinant recognized by T cells. 相似文献
14.
Structure of the pericellular matrix: Association of heparan and chondroitin sulfates with fibronectin-procollagen fibers 总被引:2,自引:0,他引:2
Klaus Hedman Staffan Johansson Tapio Vartio Lena Kjellén Anttl Vaheri Magnus Höök 《Cell》1982,28(3):663-671
Immunofluorescent staining of a pericellular matrix produced by cultured human embryonic skin fibroblasts showed a codistribution among fibronectin, heparan sulfate proteoglycans and part of the chondroitin sulfate in a fibrillar network. Isolated matrix in an “intact” form could be scraped off the dish after detergent solubilization of the cells. On centrifugation In cesium chloride density gradients, most sulfated glycosaminoglycans and matrix proteins remained associated and were recovered at a density of 1.34 g/cm3 (≥2 M CsCI). However, when 4 M guanidine hydrochloride was included in the gradient medium, the components dissociated, suggesting that the sulfated glycosaminoglycans are bound to matrix proteins by strong noncovalent linkages. Interactions between sulfated glycosaminoglycans produced by the fibroblasts and fibronectin could also be demonstrated by affinity chromatography on immobilized plasma fibronectin and by immunoprecipitation of fibronectin in conditioned culture medium, which resulted in a coprecipitation of the sulfated glycosaminoglycans. In these two systems, the fibronectin glycosaminoglycan bonds were broken at 0.2 M salt and were apparently weaker than the bonds responsible for the structural integrity of the matrix. These findings Implicate heparan and chondroitin sulfate proteoglycans as Integral components of the pericellular matrix fibers and suggest that the association of the proteoglycans with the fibronectin-procollagen matrix is stabilized by multiple molecular Interactions. 相似文献
15.
The role of recombinant stem cell factor in early B cell development. Synergistic interaction with IL-7. 总被引:13,自引:0,他引:13
I K McNiece K E Langley K M Zsebo 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(11):3785-3790
The cDNA for stem cell factor was recently isolated from Buffalo rat liver cells (BRL-3A) and recombinant rat stem cell factor produced from Escherichia coli (rrSCF164). rrSCF164 synergizes with rhIL-7 to stimulate pre-B clonal growth in agar culture of mouse bone marrow cells, and in this study we have characterized the role of rrSCF164 in B cell development. The combination of rrSCF164 plus rhIL-7 stimulated increased colony numbers compared with the sum of colonies stimulated by rrSCF164 and rhIL-7 alone. Also, increased cell proliferation per colony was stimulated by the combination of rrSCF164 plus rhIL-7 compared with rhIL-7 or rrSCF164 alone. The colonies formed with rrSCF164 plus rhIL-7 and rhIL-7 alone contained exclusively pre-B cells, which expressed B220 Ag and cytoplasmic mu-chain, but were negative for surface Ig expression. Morphological examination of the cells in the colonies showed blast-like characteristics. rrSCF164 alone and in combination with rhIL-7 stimulated generation of B220+ cells in liquid culture of B220- cells, whereas rhIL-7 alone had no stimulatory effect on B220- cells. Both stem cell factor mRNA and bioactivity were detected in a mouse bone marrow-derived stromal cell line, termed OZ-11. We propose that stem cell factor is a stromal-derived factor that synergizes with IL-7 to stimulate the proliferation and differentiation of pro-B cells to pre-B cells, which become responsive to IL-7 alone. 相似文献
16.
Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. 总被引:20,自引:4,他引:16
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We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment. 相似文献
17.
Quantitative binding studies of [G-3H]streptogramin A and [G-3H]streptogramin B with high-salt-washed ribosomes were carried out in the presence of a minimum of 10% (v/v) ethanol because of the antibiotic insolubility in water. It was observed that the presence of streptogramin A increases the affinity of [G-3H]streptogramin B for the ribosome. Thus the dissociation constant for [G-3H]-streptogramin B interaction with the ribosome is Kd=13.3 nM in the presence of streptogramin A and Kd=59 nM in its absence. Furthermore the values for the dissociation constants for [G-3H]-streptogramin B interaction in the presence of 50% (v/v) ethanol, were Kd=0.13 micronM in the presence of streptogramin A and Kd=0.70 micronM in its absence. This increased affinity of [G-3H]streptogramin B in the presence of streptogramin A can explain the synergistic effects of mixtures of streptogramins A and B at the ribosome level. 相似文献
18.
Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae. 总被引:14,自引:4,他引:10
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In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent. 相似文献
19.
Steroid hormone receptor localization in the nuclear matrix: interaction with acceptor sites 总被引:4,自引:0,他引:4
E R Barrack 《Journal of steroid biochemistry》1987,27(1-3):115-121
20.
DnaA protein/DNA interaction. Modulation of the recognition sequence 总被引:13,自引:0,他引:13