Home About us Contact
|
Home About us Contact | ||
|
|
||
Metalloprotease
Kinds of Metalloprotease Terms modified by Metalloprotease Selected AbstractsNovel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruptionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2007Ben Davies Abstract Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the ,-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates. Mol. Reprod. Dev. 74: 539,553, 2007. © 2006 Wiley-Liss, Inc. [source] ADAM 12 as a first-trimester maternal serum marker in screening for Down syndromePRENATAL DIAGNOSIS, Issue 10 2006Jennie Laigaard Abstract Background A Disintegrin And Metalloprotease 12 (ADAM 12) is a glycoprotein synthesised by placenta and it has been shown to be a potential first-trimester maternal serum marker for Down syndrome (DS) in two small series. Here we analyse further, the potential of ADAM 12 as a marker for DS in a large collection of first-trimester serum samples. Materials and Methods The concentration of ADAM 12 was determined in 10,14-week pregnancy sera from 218 DS pregnancies and 389 gestational age-matched control pregnancies, which had been collected as part of routine prospective first-trimester screening programs (DS = 105) or as part of previous research studies (DS = 113). ADAM 12 was measured using a semi-automated time resolved immunofluorometric assay and median values for normal pregnancies were established by polynomial regression. These medians were then used to determine population distribution parameters for DS and normal pregnancy groups. Correlation with previously established PAPP-A and free ,-hCG multiple of the medians (MoMs) and delta nuchal translucency (NT) were determined and used to model the performance of first-trimester screening with ADAM 12 in combination with other first-trimester markers at various time periods across the first trimester. The benefits of a contingent testing model incorporating early measurement of PAPP-A and ADAM 12 were also explored. Results The maternal serum concentration of ADAM 12 was significantly reduced (p = 0.0049) with an overall median MoM of 0.79 in the DS cases and a log10 MoM SD of 0.3734 in the DS cases and 0.3353 in the controls. There was a significant correlation of ADAM 12 MoM in DS cases with gestational age (r = 0.375) and the median MoM increased from 0.50 at 10,11 weeks to 1.38 at 13 weeks. ADAM 12 was correlated with maternal weight (r(controls) = 0.283), PAPP-A (r(controls) = 0.324, r(DS) = 0.251) but less so with free ,-hCG (r(controls) = 0.062, r(DS) = 0.049) and delta NT (r(controls) = 0.110, r(DS) = 0.151). ADAM 12 was significantly (p = 0.026) lower in smokers (0.87 vs 1.00) and elevated in Afro-Caribbean women compared to Caucasian women (1.34 vs 1.00). Population modelling using parameters from this and an earlier study showed that a combination of ADAM 12 and PAPP-A measured at 8,9 weeks and combined with NT and free ,-hCG measured at 12 weeks could achieve a detection rate of 97% at a 5% false-positive rate or 89% at a 1% false-positive rate. PAPP-A and ADAM 12 alone at 8,9 weeks could identify 91% of cases at a 5% false-positive rate. Using this as part of a contingent-screening model to select an intermediate risk group of women for NT and free ,-hCG at 11,12 weeks would enable the detection of 92% of cases with a 1% false-positive rate at a cost of providing NT and free ,-hCG for 6% of women with 94% of women having completed screening by the 10th week of pregnancy. Conclusion ADAM 12 in early first trimester is a very efficient marker of DS. In combination with existing markers, it offers enhanced screening efficiency in a two-stage sequential first-trimester screening program or in a contingent-screening model, which may have benefits in health economies where universal access to high quality ultrasound is difficult. More data on early first-trimester cases with DS are required to establish more secure population parameters by which to assess further the validity of these models. Copyright © 2006 John Wiley & Sons, Ltd. [source] ChemInform Abstract: Synthesis and Biological Evaluation of Inhibitors of Botulinum Neurotoxin Metalloprotease.CHEMINFORM, Issue 41 2009Chenbo Wang Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Activation of Tolloid-like 1 gene expression by the cardiac specific homeobox gene Nkx2,5DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2009Inna Sabirzhanova Mammalian Tolloid-like 1 (Tll-1) is a pleiotropic metalloprotease that is expressed by a small subset of cells within the precardiac mesoderm and is necessary for proper heart development. Following heart tube formation Tll-1 is expressed by the endocardium and regions of myocardium overlying the region of the muscular interventricular septum. Mutations in Tll-1 lead to embryonic lethality due to cardiac defects. We demonstrate that the Tll-1 promoter contains Nkx2,5 binding sites and that the Tll-1 promoter is activated by and directly binds Nkx2,5. Tll-1 expression is ablated by a dominant negative Nkx2,5 or by mutation of the Nkx2,5 binding sites within the Tll-1 promoter. In vivo, Tll-1 expression is decreased in the hearts of Nkx2,5 knockout embryos when compared with hemizygous and wild-type embryos. These results show that Nkx2,5 is a direct activator of Tll-1 expression and provide insight into the mechanism of the defects found in both the Tll-1 and Nkx2,5 knockout mice. [source] Novel metalloprotease,disintegrin, meltrin , (ADAM35), expressed in epithelial tissues during chick embryogenesisDEVELOPMENTAL DYNAMICS, Issue 3 2004Mitsuko Watabe-Uchida Abstract Members of the ADAM (adisintegrin and metalloprotease) family are involved in fertilization, morphogenesis, and pathogenesis. Their metalloprotease domains mediate limited proteolysis, including ectodomain shedding of membrane-anchored growth factors and intercellular-signaling proteins, and their disintegrin domains play regulatory roles in cell adhesion and migration. In screening for cDNAs encoding chicken ADAM proteins expressed during muscle development, we identified Meltrin , as a novel member of this family. To elucidate its functions, we investigated its expression during development by using antibodies raised against its protease domain. In the somites, Meltrin , protein was specifically expressed in the myotomal cells, which delaminate from the dermomyotome to form epithelial sheets. It was also found in the surface ectoderm, lens placodes, otic vesicles, and the gut epithelia. Basolateral localization of Meltrin , in these epithelial cells suggests its unique roles in the organization of the epithelial tissues and development of the sensory organs and the gut. Developmental Dynamics 230:557,568, 2004. © 2004 Wiley-Liss, Inc. [source] GDNF hyperalgesia is mediated by PLC,, MAPK/ERK, PI3K, CDK5 and Src family kinase signaling and dependent on the IB4-binding protein versicanEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Oliver Bogen Abstract The function of the isolectin B4 (IB4+)-binding and GDNF-dependent Ret (Ret+)-expressing non-peptidergic subpopulation of nociceptors remain poorly understood. We demonstrate that acute administration of GDNF sensitizes nociceptors and produces mechanical hyperalgesia in the rat. Intrathecal IB4,saporin, a selective toxin for IB4+/Ret+ -nociceptors, attenuates GDNF but not NGF hyperalgesia. Conversely, intrathecal antisense to Trk A attenuated NGF but not GDNF hyperalgesia. Intrathecal administration of antisense oligodeoxynucleotides targeting mRNA for versican, the molecule that renders the Ret-expressing nociceptors IB4-positive (+), also attenuated GDNF but not NGF hyperalgesia, as did ADAMTS-4, a matrix metalloprotease known to degrade versican. Finally, inhibitors for all five signaling pathways known to be activated by GDNF at GFR,1/Ret: PLC,, CDK5, PI3K, MAPK/ERK and Src family kinases, attenuated GDNF hyperalgesia. Our results demonstrate a role of the non-peptidergic nociceptors in pain produced by the neurotrophin GDNF and suggest that the IB4-binding protein versican functions in the expression of this phenotype. [source] Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activitiesEXPERIMENTAL DERMATOLOGY, Issue 8 2010Annica Hedberg Please cite this paper as: Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activities. Experimental Dermatology 2010; 19: e265,e274. Abstract:, Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB × NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice. [source] Golgi reassembly stacking protein 55 interacts with membrane-type (MT) 1-matrix metalloprotease (MMP) and furin and plays a role in the activation of the MT1-MMP zymogenFEBS JOURNAL, Issue 15 2010Christian Roghi Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a proteinase involved in the remodelling of extracellular matrix and the cleavage of a number of substrates. MT1-MMP is synthesized as a zymogen that requires intracellular post-translational cleavage to gain biological activity. Furin, a member of the pro-protein convertase family, has been implicated in the proteolytic removal of the MT1-MMP prodomain sequence. In the present study, we demonstrate a role for the peripheral Golgi matrix protein GRASP55 in the furin-dependent activation of MT1-MMP. MT1-MMP and furin were found to co-localize with Golgi reassembly stacking protein 55 (GRASP55). Further analysis revealed that GRASP55 associated with the cytoplasmic domain of both proteases and that the LLY573 motif in the MT1-MMP intracellular domain was crucial for the interaction with GRASP55. Overexpression of GRASP55 was found to enhance the formation of a complex between MT1-MMP and furin. Finally, we report that disruption of the interaction between GRASP55 and furin led to a reduction in pro-MT1-MMP activation. Taken together, these data suggest that GRASP55 may function as an adaptor protein coupling MT1-MMP with furin, thus leading to the activation of the zymogen. Structured digital abstract ,,MINT-7897990: Furin (uniprotkb:P09958) and GRASP55 (uniprotkb:Q9H8Y8) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897801: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT2-MMP (uniprotkb:P51511) by two hybrid (MI:0018) ,,MINT-7897821: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT3-MMP (uniprotkb:P51512) by two hybrid (MI:0018) ,,MINT-7897577: GRASP55 (uniprotkb:Q9R064) and MT1-MMP (uniprotkb:P50281) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897366: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0915) with GRASP55 (uniprotkb:Q9H8Y8) by anti bait coimmunoprecipitation (MI:0006) ,,MINT-7897617, MINT-7897659, MINT-7897681, MINT-7897702, MINT-7897725, MINT-7898032, MINT-7898011, MINT-7897907, MINT-7897884: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT1-MMP (uniprotkb:P50281) by two hybrid (MI:0018) ,,MINT-7898002: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0914) with Furin (uniprotkb:P09958) by anti bait coimmunoprecipitation (MI:0006) ,,MINT-7897500: MT1-MMP (uniprotkb:P50281) and Giantin (uniprotkb:Q14789) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897750, MINT-7897394: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT1-MMP (uniprotkb:P50281) by anti tag coimmunoprecipitation (MI:0007) ,,MINT-7897562: MT1-MMP (uniprotkb:P50281) and GRASP55 (uniprotkb:Q9H8Y8) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897512: TGN46 (uniprotkb:O43493) and MT1-MMP (uniprotkb:P50281) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897921, MINT-7897975: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with Furin (uniprotkb:P09958) by two hybrid (MI:0018) ,,MINT-7898052, MINT-7897410: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0915) with GRASP55 (uniprotkb:Q9R064) by anti bait coimmunoprecipitation (MI:0006) ,,MINT-7897951: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with PC7 (uniprotkb:Q16549) by two hybrid (MI:0018) ,,MINT-7897866: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT5-MMP (uniprotkb:Q9Y5R2) by two hybrid (MI:0018) ,,MINT-7897633: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with TGFA (uniprotkb:P01135) by two hybrid (MI:0018) ,,MINT-7897551: GRASP55 (uniprotkb:Q9H8Y8) and Giantin (uniprotkb:Q14789) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897938: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with PC5/6B (uniprotkb:Q04592) by two hybrid (MI:0018) [source] Properties of the hatching enzyme from Xenopus laevisFEBS JOURNAL, Issue 18 2001Ting-Jun Fan Using an anti-(glutathione S -transferase,UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti-(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40-kDa form. The 60-kDa HE had a high hydrolytic and UEVE-solubilizing activity, and its activities against Boc-Leu-Gly-Arg-7-amino-4-methylcoumarin (-NH-Mec) and UEVE were inhibited by anti-(GST UVS.2) Ig in a dose-dependent manner. The 60-kDa form was easily autodigested into a 40-kDa form. The 40-kDa molecule alone had no detectable UEVE-solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60-kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60-kDa molecule into 40-kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co-ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc-Leu-Gly-Arg-NH-Mec was optimal at pH of 7.4, and with an apparent Km value of 200 µmol·L,1 at 30 °C. The HE is very sensitive to trypsin-specific inhibitors such as leupeptin, (4-amidino-phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N -,-tosyl- l -lysylchloromethane (Tos-Lys-CH2Cl), indicates that it is a trypsin-type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease-specific ,HExHxxGFxHE' sequence in the deduced HE amino-acid sequence, indicates that this HE is a Zn2+ metalloprotease. [source] Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomalleiFEMS MICROBIOLOGY LETTERS, Issue 1 2000May-Ann Lee Abstract Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia. It is responsible for melioidosis in human and animals. The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase. The isolation of virulence genes and the study of their functions will contribute to our understanding of bacterial pathogenesis. Previous studies have implicated protease as a contributing virulence factor in the pathogenesis of some bacteria. Three out of 5000 clones screened from a genomic DNA library of B. pseudomallei were found to express protease activity. The clones were found to have the same sequence. The nucleotide sequence revealed an open reading frame (designated as metalloprotease A, mprA) encoding a 500-amino acid protein, MprA, with an estimated molecular mass of 50,241 Da. The predicted amino acid sequence shares homology with the subtilisin family of serine proteases. [source] Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformansFEMS YEAST RESEARCH, Issue 4 2007Richard A. Eigenheer Abstract Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or ,-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography,mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans. [source] Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4IMMUNOLOGY, Issue 3 2001Tsutomu Yoshikawa Summary Mast cell chymase plays important roles in inflammation and tissue remodeling. Here we show that mast cell chymase also functions as an enhancer of immunoglobulin production. In the culture of murine spleen cells stimulated with lipopolysaccharide and interleukin-4, purified rat chymase (rat mast cell protease-I; RMCP-I), at physiological concentrations, enhanced immunoglobulin E (IgE) and IgG1 syntheses but not IgG3 synthesis. The enhancement was also evident when spleen cells depleted of T cells and macrophages were employed as responding cells. Enzymatic activity of RMCP-I was required to enhance IgE and IgG1, because two inhibitors for chymotryptic enzymes, chymostatin and Y-40613, a novel chymase inhibitor, suppressed the enhanced immunoglobulin production, and phenylmethylsulphonyl fluoride, an irreversible inhibitor for serine proteases, totally abolished the enhancing effect. Furthermore, a specific inhibitor for Zn2+ -dependent metalloproteases, GI 129471, could also completely inhibit the production of IgE and IgG1 that was enhanced by RMCP-I, suggesting that a metalloprotease also played an essential role in the immunoglobulin production. Our results together with others show that proteases from mast cell granules have important function not only in the efferent phase but also in the afferent phase of immune responses. [source] CX3CL1/fractalkine shedding by human hepatic stellate cells: contribution to chronic inflammation in the liverJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8a 2009Katia Bourd-Boittin Abstract Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-, induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro. We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases. [source] Purification and characterization of solvent-tolerant, thermostable, alkaline metalloprotease from alkalophilic Pseudomonas aeruginosa MTCC 7926JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 9 2009Ulhas Patil Abstract BACKGROUND: Microbial proteases are becoming imperative for commercial applications. The protease secreted by Pseudomonas aeruginosa MTCC 7926, isolated from solvent-contaminated habitat was purified and characterized for activity at various edaphic conditions. The purified alkaline protease was investigated for dehairing of animal skin, anti-staphylococcal activity and processing of X-ray film. RESULTS: The protease was 24-fold purified by ammonium sulfate fractionation, sephadex G-100 gel filtration and DEAE-cellulose, with 36% recovery. KM and Vmax, using casein were 2.94 mg mL,1 and 1.27 µmole min,1, respectively. The apparent molecular mass by SDS-PAGE was 35 kDa. Alkaline protease was active at pH 6,11 and temperature 25,65 °C. Its activity was (a) 86.8% in 100 mmol L,1 NaCl, (b) >95% in metal ions (Mn2+, Ca2+, Mg2+, Fe2+) for 1 h, (c) >90% in bleaching agents and chemical surfactants, (d) 135.4 ± 2.0% and 119.9 ± 6.2% with rhamnolipid and cyclodextrin, respectively, (e) stable in solvents for 5,30 days at 27 °C, and (f) inhibited by EDTA, indicating metalloprotein. CONCLUSION: This work showed that purified protease retained its activity in surfactants, solvents, metals, and bleaching agents. The enzyme is an alternative for detergent formulations, dehairing of animal skin, X-ray film processing, treatment of staphylococcal infections and possibly non-aqueous enzymatic peptide synthesis. Copyright © 2009 Society of Chemical Industry [source] Purification and characterization of a 630 kDa bacterial killing metalloprotease (KilC) isolated from plaice Pleuronectes platessa (L.), epidermal mucusJOURNAL OF FISH DISEASES, Issue 5 2008T Tvete Abstract Antibacterial chemicals in the mucus of fish such as lysozyme, lectins, peptides and proteases provide an efficient first line of defence against pathogens. This study shows that there are at least three antibacterial proteins in plaice skin mucus in addition to lysozyme. One of these proteins is responsible for approximately 74% of the antibacterial activity and is a 630 kDa protease complex designated KilC (bacterial killing metalloprotease C). Purified KilC kills the bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa efficiently. The protease activity of KilC is dependent upon the divalent cation Mg2+ and shows pH dual optima of 5.0 and 8.0. The enzyme has a temperature optimum of 25 °C and is made up of at least five different sized peptides. Studies with protease inhibitors show that the catalytic site of KilC may be cysteine- or serine protease-like. KilC may kill bacterial cells by acting directly upon the bacteria or by producing low molecular weight bioactive compounds such as peptides. [source] Areca nut extract-treated gingival fibroblasts modulate the invasiveness of polymorphonuclear leukocytes via the production of MMP-2JOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 1 2009Hsuan-Hsuan Lu Background:, Areca nut chewing is associated with an increase in the incidence of oral neoplastic or inflammatory diseases. Aberrations in matrix metalloprotease (MMP) expression are associated with the pathogenesis of oral diseases. This study investigated the potential effects of areca nut extract (ANE) on human gingival fibroblasts and the consequential impacts on inflammatory pathogenesis. Methods:, Analyses of senescence marker, cell viability, changes of the cell cycle, and cell granularity in gingival fibroblasts together with an assessment of the invasiveness of polymorphonuclear (PMN) leukocytes after treatment with the supernatant of ANE-treated gingival fibroblasts were performed to characterize the phenotypic impacts. Western blotting and gelatin zymography were used to assay the expression and activity of MMP-2. Results:, Chronic subtoxic (<10 ,g/ml) ANE treatment resulted in premature growth arrest, appearance of senescence-associated ,-galactosidase activity and various other senescence-associated phenotypes in gingival fibroblasts. Gingival fibroblasts established from older individuals had a higher propensity to become ANE-induced senescent gingival fibroblasts. An activation of MMP-2 was identified in senescent cells. PMN leukocytes treated with the supernatant of ANE-induced senescent cells exhibited a significant increase in invasiveness, which was abrogated by both a MMP-2 blocker and a MMP-2 nullifying antibody. Conclusions:, This study provides evidence whereby MMP-2 secreted from ANE-induced senescent gingival fibroblasts would facilitate the invasiveness of PMN leukocytes, which could be associated with the oral inflammatory process in areca chewers. [source] Alcohol Stimulates Activation of Snail, Epidermal Growth Factor Receptor Signaling, and Biomarkers of Epithelial,Mesenchymal Transition in Colon and Breast Cancer CellsALCOHOLISM, Issue 1 2010Christopher B. Forsyth Background:, Alcohol consumption is associated with the risk of progressive cancers including colon and breast cancer. The mechanisms for the alcohol-induced aggressive behavior of these epithelial cancer cells have not been fully identified. Epithelial,mesenchymal transition (EMT) is a developmental program recently shown to play a role in cancer progression and metastases. We hypothesized that alcohol might promote cancer progression by inducing EMT in cancer cells and tested this hypothesis by assessing alcohol-stimulated changes in phenotypic markers of EMT as well as the EMT transcription factor Snail and its related cell signaling. Methods:, Colon and breast cancer cell lines and a normal intestinal epithelial cell line were tested as well as colonic mucosal biopsy samples from alcoholic subjects. Cells were treated with alcohol and assessed for EMT-related changes using immunofluorescent microscopy, western blotting, reporter assays, RT-PCR, and knockdown of Snail with siRNA. Results:, We show alcohol upregulated the signature EMT phenotypic marker vimentin as well as matrix metalloprotease (MMP)-2, MMP-7, and MMP-9 and cell migration in colon and breast cancer cells,all characteristics of EMT. Alcohol also stimulated nuclear localization of Snail phosphorylated at Ser246, transcription from a Snail reporter plasmid, and Snail mRNA expression by RT-PCR. Snail siRNA knockdown prevented alcohol-stimulated vimentin expression. In vivo, Snail expression was significantly elevated in colonic mucosal biopsies from alcoholics. Also, we found alcohol stimulated activation of epidermal growth factor receptor (EGFR) signaling and an EGFR inhibitor blocked alcohol-induced cell migration and Snail mRNA expression. Conclusions:, Collectively, our data support a novel mechanism for alcohol promoting cancer progression through stimulating the EMT program in cancer cells via an EGFR-Snail mediated pathway. This study reveals new pathways for alcohol-mediated promotion of cancer that could be targeted for therapy or prevention of alcohol-related cancers. [source] Novel ADAMTS-13 mutations in an adult with delayed onset thrombotic thrombocytopenic purpuraJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2006Z. TAO Summary.,Background:,Thrombotic thrombocytopenic purpura (TTP) is associated with congenital and acquired deficiency of ADAMTS-13, a metalloprotease that cleaves von Willebrand factor (VWF) and reduces its adhesive activity. Mutations throughout the ADAMTS13 gene have been identified in congenital TTP patients, most of whom have initial episodes during infancy or in early childhood. Patients and methods:,We report the case of an adult male who was diagnosed with idiopathic thrombocytopenic purpura at age 34, and with TTP 14 years later. The patient was compound heterozygous for an 18 bp in-frame deletion (C365del) in the disintegrin domain and a point mutation of R1060W in the seventh thrombospondin domain of the ADAMTS-13 gene. Conclusions:In vitro studies found that C365del and R1060W severely impair ADAMTS-13 synthesis in transfected Hela cells, whereas the deletion mutant also failed to cleave VWF under static and flow conditions. [source] Therapeutic potential of sulfamides as enzyme inhibitorsMEDICINAL RESEARCH REVIEWS, Issue 6 2006Jean-Yves Winum Abstract Sulfamide, a quite simple molecule incorporating the sulfonamide functionality, widely used by medicinal chemists for the design of a host of biologically active derivatives with pharmacological applications, may give rise to at least five types of derivatives, by substituting one to four hydrogen atoms present in it, which show specific biological activities. Recently, some of these compounds started to be exploited for the design of many types of therapeutic agents. Among the enzymes for which sulfamide-based inhibitors were designed, are the carbonic anhydrases (CAs), a large number of proteases belonging to the aspartic protease (HIV-1 protease, ,-secretase), serine protease (elastase, chymase, tryptase, and thrombin among others), and metalloprotease (carboxypeptidase A (CPA) and matrix metalloproteinases (MMP)) families. Some steroid sulfatase (STS) and protein tyrosine phosphatase inhibitors belonging to the sulfamide class of derivatives have also been reported. In all these compounds, many of which show low nanomolar affinity for the target enzymes for which they have been designed, the free or substituted sulfamide moiety plays important roles for the binding of the inhibitor to the active site cavity, either by directly coordinating to a metal ion found in some metalloenzymes (CAs, CPA, STS), usually by means of one of the nitrogen atoms present in the sulfamide motif, or as in the case of the cyclic sulfamides acting as HIV protease inhibitors, interacting with the catalytically critical aspartic acid residues of the active site by means of an oxygen atom belonging to the HNSO2NH motif, which substitutes a catalytically essential water molecule. In other cases, the sulfamide moiety is important for inducing desired physico-chemical properties to the drug-like compounds incorporating it, such as enhanced water solubility, better bioavailability, etc., because of the intrinsic properties of this highly polarized moiety when attached to an organic scaffold. This interesting motif is thus of great value for the design of pharmacological agents with a lot of applications. © 2006 Wiley Periodicals, Inc. Med Res Rev [source] Cytokines, matrix metalloproteases, angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patientsALLERGY, Issue 5 2009A. Leonardi Background:, To detect the presence of multiple mediators and growth factors in tears of vernal keratoconjunctivitis (VKC) patients with active disease using stationary phase antibody arrays. Methods:, Tears were collected from 12 normal subjects (CT) and 24 active VKC patients. Tears were centrifuged and successively probed using three microwell plate arrays specific for: (i) cytokines: interleukin (IL)-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-13, interferon-, and tumour necrosis factor-,; (ii) growth factors: basic fibroblast growth factor (bFGF), platelet-derived growth factor, thrombopoietin, angiopoietin-2, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), keratocyte growth factor, tissue inhibitor of metalloprotease (TIMP)-1 and heparin-binding epithelial growth factor (HB-EGF) and (iii) matrix metalloprotease (MMP)-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1 and TIMP-2. Results:, Interleukin-8 signals were detected in all CT and highly detected in all VKC samples. The Th2-type cytokines, IL-4, IL-5 and IL-10 were detected only in tears of VKC patients. Signals for bFGF, HB-EGF, VEGF and HGF were detected in 41,87% of VKC samples and in few CT samples. Only TIMP-1 and TIMP-2 were found in all normal and patient tear samples, whereas MMP-1, MMP-2, MMP-3, MMP-9 and MMP-10 were highly present in all VKC samples. Conclusions:, Stationary phase antibody array methodology was useful for the screening of various cytokines, growth factors and MMPs in tears. These analyses identified in tears of VKC patients previously unreported factors including MMP-3 and MMP-10 and multiple proteases, growth factors and cytokines, which may all play an important role in the pathogenesis of conjunctival inflammation. [source] Serine proteases from two cell types target different components of a complex that governs regulated intramembrane proteolysis of pro-,K during Bacillus subtilis developmentMOLECULAR MICROBIOLOGY, Issue 3 2005Ruanbao Zhou Summary Upon starvation Bacillus subtilis undergoes a developmental process involving creation of two cell types, the mother cell and forespore. A signal in the form of a serine protease, SpoIVB, is secreted from the forespore and leads to regulated intramembrane proteolysis (RIP) of pro-,K, releasing active ,K into the mother cell. RIP of pro-,K is carried out by a membrane-embedded metalloprotease, SpoIVFB, which is inactive when bound by BofA and SpoIVFA. We have investigated the mechanism by which this complex is activated. By expressing components of the signalling pathway in Escherichia coli, we reconstructed complete inhibition of pro-,K RIP by BofA and SpoIVFA, and found that SpoIVB serine protease activity could partially restore RIP, apparently by targeting SpoIVFA. Pulse-chase experiments demonstrated that SpoIVFA synthesized early during B. subtilis sporulation is lost in a SpoIVB-dependent fashion, coincident with the onset of pro-,K RIP, supporting the idea that SpoIVB targets SpoIVFA to trigger RIP of pro-,K. Loss of BofA depended not only on SpoIVB, but also on CtpB, a serine protease secreted from the mother cell. CtpB appeared to cleave BofA near its C-terminus upon coexpression in E. coli, and purified CtpB degraded BofA. We propose that RIP of pro-,K involves a three-step proteolytic cascade in which SpoIVB first cleaves SpoIVFA, CtpB then cleaves BofA and finally SpoIVFB cleaves pro-,K. [source] cDNA-AFLP reveals genes differentially expressed during the hypersensitive response of cassavaMOLECULAR PLANT PATHOLOGY, Issue 2 2005BENJAMIN P. KEMP SUMMARY The tropical staple cassava is subject to several major diseases, such as cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis. Disease-resistant genotypes afford the only practical solution, yet despite the global importance of this crop, little is known about its defence mechanisms. cDNA-AFLP was used to isolate cassava genes differentially expressed during the hypersensitive reaction (HR) of leaves in response to an incompatible Pseudomonas syringae pathovar. Seventy-eight transcript-derived fragments (TDFs) showing differential expression (c. 75% up-regulated, 25% down-regulated) were identified. Many encoded putative homologues of known defence-related genes involved in signalling (e.g. calcium transport and binding, ACC oxidases and a WRKY transcription factor), cell wall strengthening (e.g. cinnamoyl coenzyme A reductase and peroxidase), programmed cell death (e.g. proteases, 26S proteosome), antimicrobial activity (e.g. proteases and ,-1,3-glucanases) and the production of antimicrobial compounds (e.g. DAHP synthase and cytochrome P450s). Full-length cDNAs including a probable matrix metalloprotease and a WRKY transcription factor were isolated from six TDFs. RT-PCR or Northern blot analysis showed HR-induced TDFs were maximally expressed at 24 h, although some were produced by 6 h; some were induced, albeit more slowly, in response to wounding. This work begins to reveal potential defence-related genes of this understudied, major crop. [source] PECAM-1 and gelatinase B coexist in vascular cuffs of multiple sclerosis lesionsNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 1 2006I. Nelissen In multiple sclerosis (MS), the matrix metalloprotease (MMP) gelatinase B/MMP-9 and platelet endothelial cell adhesion molecule (PECAM)-1 have both been implicated in trans-endothelial infiltration of leucocytes into the brain, but their functional connection has not yet been investigated. We investigated the expression of gelatinase B and PECAM-1 in ,post mortem brains of MS patients by immunohistochemistry. Because increased soluble PECAM-1 serum levels have been observed in MS patients, we also tested in vitro whether this could be due to cleavage of PECAM-1 by gelatinase B or matrilysin-1/MMP-7. Constitutive expression of PECAM-1 was found on brain endothelial cells, whilst in active MS lesions cell-bound PECAM-1 was highly up-regulated on foamy macrophages in perivascular infiltrates and co-localized with gelatinase B. However, human THP-1 monocyte-bound or soluble recombinant PECAM-1 were both resistant to proteolytic cleavage by gelatinase B or matrilysin-1 in vitro, as demonstrated by Western blot analysis and flow cytometry. These results suggest that PECAM-1 and gelatinase B may complement each other during the transmigration of the blood,brain barrier by mononuclear cells. [source] ADAM12: a novel first-trimester maternal serum marker for Down syndromePRENATAL DIAGNOSIS, Issue 13 2003Jennie Laigaard Abstract Objectives The concentration of bioavailable insulin-like growth factor (IGF) I and II is important to foetal growth. It is regulated by insulin-like growth factor binding proteins (IGFBP) 1 through 6. Proteolytic cleavage of IGFBP-3 takes place in human pregnancy serum; accordingly, IGFBP-3 serum levels decrease markedly during pregnancy. ADAM12 (A disintegrin and metalloprotease) is an IGFBP-3 and IGFBP-5 protease and is present in human pregnancy serum. The goal of this study was to determine whether ADAM12 concentration in maternal serum is a useful indicator of foetal health. Methods We developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of ADAM12 in serum. The assay range was 42 to 667 µg/L. Recombinant ADAM12 was used as the standard for calibration. Results We found that ADAM12 was highly stable in serum. Serum concentration increased from 180 µg/L at week 8 of pregnancy to 670 µg/L at 16 weeks, and reached 12 000 µg/L at term. In 18 first-trimester Down syndrome pregnancies, the concentration of ADAM12 was decreased, thus the median multiple of mean (MoM) value was 0.14 (0.01,0.76). A detection rate for foetal Down syndrome of 82% for a screen-positive rate of 3.2% and a 1:400 risk cut-off was found by Monte Carlo estimation using ADAM12 and maternal age as screening markers. Conclusion ADAM12 is a promising marker for Down syndrome. Copyright © 2003 John Wiley & Sons, Ltd. [source] Insights into the anthrax lethal factor,substrate interaction and selectivity using docking and molecular dynamics simulationsPROTEIN SCIENCE, Issue 8 2009Georgios A. Dalkas Abstract The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn-dependent, highly specific metalloprotease with a molecular mass of ,90 kDa that cleaves most isoforms of the family of mitogen-activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK-based synthetic peptide provided structure-activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF-MEK/MKK interaction along the catalytic channel up to a distance of 20 Å from the zinc atom. This residue-specific view of the enzyme-substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot-spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction. [source] Curcumin Suppresses the Paclitaxel-Induced Nuclear Factor-,B in Breast Cancer Cells and Potentiates the Growth Inhibitory Effect of Paclitaxel in a Breast Cancer Nude Mice ModelTHE BREAST JOURNAL, Issue 3 2009Hee Joon Kang MD Abstract:, Most anticancer agents activate nuclear factor kappa B (NF-,B), which can mediate cell survival, proliferation, and metastasis. Curcumin has been shown to inhibit the growth of various cancer cells, without toxicity to normal cells. The antitumor effects of curcumin could be due in part to the inactivation of NF-,B. We hypothesize that blocking NF-,B activity may augment paclitaxel cancer chemotherapy. In this study, we investigated whether the inactivation of NF-,B by curcumin would enhance the efficacy of paclitaxel for inhibiting breast cancer growth in vitro and in vivo. We confirmed that curcumin inhibited paclitaxel-induced activation of NF-,B and potentiated the growth inhibitory effect of paclitaxel in MDA-MB-231 breast cancer cells. The combination of curcumin with paclitaxel elicited significantly greater inhibition of cell growth and more apoptosis, compared with either agent alone. In an experimental breast cancer murine model using MDA-MB-231 cells, combination therapy with paclitaxel and curcumin significantly reduced tumor size and decreased tumor cell proliferation, increased apoptosis, and decreased the expression of matrix metalloprotease 9 compared with either agent alone. These results clearly suggest that a curcumin,paclitaxel combination could be a novel strategy for the treatment of breast cancer. [source] Vibrio vulnificus infection and metalloproteaseTHE JOURNAL OF DERMATOLOGY, Issue 9 2006Shin-ichi MIYOSHI ABSTRACT Vibrio vulnificus,is ubiquitous in aquatic environments; however, it occasionally causes serious and often fatal infections in humans. These include invasive septicemia contracted through consumption of raw seafood, as well as wound infections acquired through contact with brackish or marine waters. In most cases of septicemia, the patients have underlying disease(s), such as liver dysfunction or alcoholic cirrhosis, and the secondary skin lesions including cellulitis, edema and hemorrhagic bulla appear on the limbs. Although V. Vul,produces various virulent factors including polysaccharide capsule, type IV pili, hemolysin and proteolytic enzymes, the 45-kDa metalloprotease may be a causative factor of the skin lesions, because the purified protease enhances vascular permeability through generation of chemical mediators and also induces serious hemorrhagic damage through digestion of the vascular basement membrane. As well as other bacteria, V. Vul,can regulate the protease production through the quorum-sensing system depending on bacterial cell density. However, this system operates efficiently at 25°C, but not at 37°C. Therefore, V. vulnificus may produce sufficient amounts of the protease only in the interstitial tissue of the limbs, in which temperature is lower than the internal temperature of the human body. [source] Serum Matrix Metalloprotease-1 and Vascular Endothelial Growth Factor,A Predict Cardiac Allograft RejectionAMERICAN JOURNAL OF TRANSPLANTATION, Issue 1 2009S. Aharinejad Cardiac allograft rejection is currently diagnosed from endomyocardial biopsies (EMB) that are invasive and impractical to repeat. A serological marker could facilitate rejection monitoring and minimize EMB-associated risks. We investigated the relation of serum matrix metalloprotease (MMP)-1 and vascular endothelial growth factor (VEGF)-A concentrations to cardiac allograft rejection, using 1176 EMBs and serum samples obtained from 208 recipients. Acute cellular rejection was diagnosed in 186 EMBs. Mean week 1 and week 2 serum MMP-1 concentrations predicted rejection (p = 0.001, AUC = 0.80). At the optimal cut-off level of ,7.5 ng/mL, MMP-1 predicted rejection with 82% sensitivity and 72% specificity. Initial serum MMP-1 <5.3 ng/mL (lowest quartile) was associated with rejection-free outcome in 80% of patients. Both MMP-1 (p < 0.001, AUC = 0.67,0.75) and VEGF-A (p < 0.01, AUC = 0.62,0.67) predicted rejection on the next EMB, while rejection at EMB was identified only by VEGF-A (p < 0.02, AUC = 0.70,0.77). Patients receiving combined cyclosporine-A and everolimus had the lowest serum MMP-1 concentrations. While serum MMP-1 predicts rejection-free outcome and VEGF-A identifies rejection on EMB, both markers predict rejection in follow-up of cardiac transplant recipients. Combination of serum MMP-1 and VEGF-A concentration may be a noninvasive prognostic marker of cardiac allograft rejection, and could have important implications for choice of surveillance and immunosuppression protocols. [source] Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater Prawn Macrobrachium rosenbergii (De Man) PostlarvaeAQUACULTURE RESEARCH, Issue 5 2009Mohamed Ayaz Hasan Chisty Abstract Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL15 giant freshwater prawn (Macrobrachium rosenbergii). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0,73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases. [source] Nuclear autoantigen CENP-B transactivation of the epidermal growth factor receptor via chemokine receptor 3 in vascular smooth muscle cellsARTHRITIS & RHEUMATISM, Issue 9 2009Geneviève Robitaille Objective We have previously found that the CENP-B nuclear autoantigen, which is specifically targeted by autoantibodies in the limited cutaneous form of systemic sclerosis, behaved as a potent migratory factor for human pulmonary artery smooth muscle cells (PASMCs). Other recent studies have shown that several disease-associated autoantigens induced cell migration by interacting with various chemokine receptors. Prompted by this hypothesis, we undertook this study to determine whether CENP-B interacts with chemokine receptors on the surface of human PASMCs, to explore the relevant signaling pathways, and to characterize the effects of anti,CENP-B binding on SMC stimulation. Methods To demonstrate the expression of specific chemokine receptors by human PASMCs at both the messenger RNA and protein levels, reverse transcription,polymerase chain reaction, immunoblotting, and flow cytometry analyses were performed. Desensitization studies and specific inhibitors were used to further identify the CENP-B target on the surface of human PASMCs. Results Our data strongly suggested that CENP-B used chemokine receptor 3 (CCR3) to mediate human PASMCs signaling. Moreover, several lines of evidence indicated that CENP-B binding subsequently stimulated the cross-talk between CCR3 and epidermal growth factor receptor (EGFR) via a matrix metalloprotease,dependent mechanism that involved the processing of heparin-binding EGF-like growth factor. Transactivation of the EGFR through CCR3 was found to be a critical pathway that elicits MAP kinase activation and secretion of cytokines such as interleukin-8. Finally, anti,CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular smooth muscle cells. Conclusion The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti,CENP-B autoantibodies. [source] | |||||||||||||||||||||||||||||||