Tissue patterns of gene expression were analyzed by measuring mRNA levels and incorporation of radioactive amino acids for cystatin C and P,-microglobulin, the two extracellular proteins in the brain with the highest ratio of concentration in cerebrospinal fluid over that in blood plasma.The primary structure of rat cystatin C mRNA from choroid plexus was determined by nucleotide sequencing of cloned cDNA and the tissue patterns of gene expression were analysed by RNA blot analysis and in situ hybridization. Cystatin C was found to be composed of 120 amino acids and to contain a potential site for Nlinked glycosylation. The tissue with the highest cystatin C mRNA level was the choroid plexus of the brain. Cystatin C mRNA was also detected in lower levels in other areas of the brain, testis, epididymis, seminal vesicles, prostate, ovary, submandibular gland, and, in trace amounts, in liver. Choroid plexus pieces in culture secreted radioactive cystatin C when incubated with radioactive leucine.Rat Pz-microglobulin cDNA was cloned and identified by nucleotide sequencing and comparison of the obtained sequence with that of mouse and human fl,-microglobulin cDNA. Tissue levels of P,-microglobulin mRNA in the rat were measured by hybridization to rat fi,-microglobulin cDNA. The highest levels of f12-microglobulin mRNA were observed in liver and choroid plexus. Other parts of the brain and testis contained lower levels of P,-rnicroglobulin mRNA. Note. The novel nucleotide sequence data published here have been deposited with thc EMBL sequence data bank and are available under the accession numbers XI6956 and X16957. barrier [7] or a selective breakdown of plasma proteins [8] during transcellular transport from the bloodstream to the brain have also been proposed to explain the different patterns of protein concentrations in cerebrospinal fluid and plasma. A high intracellular concentration of transthyretin in the choroid plexus [9] is compatible with the assumption that this plasma protein is synthesized within the brain. Using hybridisation to specific cDNA probes, very strong expression of the genes for transthyretin [lo-171, transferrin [lo, 18, 191 and ceruloplasmin [20] was recently observed in choroid plexus. Polyadenylated RNA extracted from brain was shown to direct the incorporation of radioactive amino acids into several plasma proteins immunoprecipitated from cell-free protein synthesizing systems [21].The two extracellular brain proteins with the highest ratios of the concentration in cerebrospinal fluid over that in blood plasma are cystatin C and p,-microglobulin. Recently, the primary structures of the cDNAs for human cystatin C [22] and mouse [23] and human [24] microglo globulin were elucidated. For the experiments reported in this paper this information was used for the synthesis of specific oligonucleotide probes, which were found to cross-hybridize with the corresponding mRNAs in the rat. cDNA clones for both rat p2-microglobulin and rat cystatin C were isolated and their structure characte...
IntroductionThe gene quiescin/sulfhydryl oxidase 1, QSOX1, encodes an enzyme directed to the secretory pathway and excreted into the extracellular space. QSOX1 participates in the folding and stability of proteins and thus could regulate the biological activity of its substrates in the secretory pathway and/or outside the cell. The involvement of QSOX1 in oncogenesis has been studied primarily in terms of its differential expression in systemic studies. QSOX1 is overexpressed in prostate cancers and in pancreatic adenocarcinoma. In contrast, QSOX1 gene expression is repressed in endothelial tumors. In the present study, we investigated the role of QSOX1 in breast cancer.MethodsWe analyzed QSOX1 mRNA expression in a cohort of 217 invasive ductal carcinomas of the breast. Moreover, we investigated QSOX1's potential role in regulating tumor growth and metastasis using cellular models in which we overexpressed or extinguished QSOX1 and xenograft experiments.ResultsWe showed that the QSOX1 expression level is inversely correlated to the aggressiveness of breast tumors. Our results show that QSOX1 leads to a decrease in cell proliferation, clonogenic capacities and promotes adhesion to the extracellular matrix. QSOX1 also reduces the invasive potential of cells by reducing cell migration and decreases the activity of the matrix metalloproteinase, MMP-2, involved in these mechanisms. Moreover, in vivo experiments show that QSOX1 drastically reduces the tumor development.ConclusionsTogether, these results suggest that QSOX1 could be posited as a new biomarker of good prognosis in breast cancer and demonstrate that QSOX1 inhibits human breast cancer tumorogenesis.
Cystic fibrosis (CF) is an inherited disease associated with chronic severe lung inflammation, leading to premature death. To develop innovative anti-inflammatory treatments, we need to characterize new cellular and molecular components contributing to the mechanisms of lung inflammation. Here, we focused on the potential role of "transient receptor potential vanilloid-4" (TRPV4), a nonselective calcium channel. We used both in vitro and in vivo approaches to demonstrate that TRPV4 expressed in airway epithelial cells triggers the secretion of major proinflammatory mediators such as chemokines and biologically active lipids, as well as a neutrophil recruitment in lung tissues. We characterized the contribution of cytosolic phospholipase A2, MAPKs, and NF-κB in TRPV4-dependent signaling. We also showed that 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, i.e., four natural lipid-based TRPV4 agonists, are present in expectorations of CF patients. Also, TRPV4-induced calcium mobilization and inflammatory responses were enhanced in cystic fibrosis transmembrane conductance regulator-deficient cellular and animal models, suggesting that TRPV4 is a promising target for the development of new anti-inflammatory treatments for diseases such as CF.
Two immunochemically related forms of cystatin C‐like inhibitors which differ in their M r app and isoelectric point have been found both in urine and seminal vesicles of rats. Amino‐terminal sequences of these two cystatins are identical within the same fluid and exhibit a high degree of homology with that of human cystatin C. However, cystatins C purified from urine lack eight residues at their amino‐terminal end when compared to those of seminal vesicles. The occurrence of two cystatin C‐like components in rat fluids has been found to be due to the presence of a glycosylated form reported here as cystatin Cg which specifically binds concanavalin A and is susceptible to endo‐β‐N‐acetylglucosaminidase treatment.
The overall mechanism of interaction with proteinases of cr,-inhibitor,, a plasma proteinase inhibitor so far specific to the rat, has been shown to be closely similar to that described for a-macroglobulins. This mechanism includes: (i) the cleavage of at least one susceptible peptidic bond which leads to structural changes in the molecule. (ii) The cleavage of a putative thiol ester bond in another site of the molecule which permits the covalent linkage of the enzyme. Moreover, fragmentation of a,-inhibitor, upon heating as observed for cr-macroglobulin quarter subunits has been demonstrated. The question is raised of the presence of such a molecule in rat plasma in addition to two a-macroglobulin species, all of these proteinase inhibitors being antigenically unrelated. Evidence is given here that the mechanism of ingiving enzymatically active complexes when they teraction of ~13 with proteinases closely resembles interact with proteinases (recent review [l]). In rat that described for CYM. In particular, the applasma, however, in addition to cul-macroglobulin pearance of a free thiol group presumably issuing and ~2 acute phase macroglobulin, a third protein, from the cleavage of a thiol ester bond upon innamely (~~13, is also able to give such active comteraction with proteinases or methylamine has plexes when binding serine or cysteine proteinases been demonstrated as well as a selective heat [2,3]. Circulating alI3 has an M, next to that of the fragmentation of the molecule which is prevented quarter subunit of either human CYZM or rat by prior incorporation of methylamine. This struca2APM as well as the higher subunit of rat (YIM tural relationship between arlI3 and the quarter [4,5]. It stands in normal rat plasma at a relatively subunit of cuM raises the question of the high concentration (about 30 PM) but behaves as a simultaneous presence of these proteins in rat negative acute phase reactant [2]. As found for plasma and that of the phylogeny of these uM, a conformational change of the tul13 molecule molecules.
Rat FAD-dependent sulfhydryl oxidase was purified; partial sequencing indicated that it was homologous to human quiescin Q6. A cDNA (GenBank™ accession no. AF285078) was cloned from rat seminal vesicles, and active recombinant sulfhydryl oxidase was expressed in Chinese hamster ovary epithelial cells. This 2472-nucleotide cDNA has an open reading frame of 1710 base pairs, encoding a protein of 570 amino acids including a 32-amino acid leader sequence and two potential sites for N-glycosylation. One of them is used and the 64,000 M r purified protein was transformed to 61,000 by the action of endoglycosidase F. Northern blotting and reverse transcription-polymerase chain reaction analyses showed that there were small amounts of sulfhydryl oxidase in the rat testis, prostate, lung, heart, kidney, spleen, and liver, and that the gene was highly expressed in seminal vesicles and epididymis. Rat sulfhydryl oxidase cDNA corresponds to the human cell growth inhibiting factor cDNA, which could be a differently spliced form of quiescin Q6. Comparing sulfhydryl oxidase sequences with those of human quiescin Q6 and mammalian and Caenorhabditis elegans quiescin Q6-related genes established the existence of a new family of FAD-dependent sulfhydryl oxidase/quiescin Q6-related genes containing protein-disulfide isomerase-type thioredoxin and yeast ERV1 domains.
Previous attempts to liberate T kinin from T kininogen Eur. J. Biochem. 15Y, 341 -346;Gutman et al. (1988) Eur. J. Biochem. 171, have shown that complete fragmentation of the precursor molecule into inhibitory peptides was achieved before any vasoactive peptide was released, suggesting a possible physiological significance for this phenomenon. In this study, cysteine-proteinase-inhibiting properties of rat T kininogen and of its proteolytic fragments issuing from trypsin and submaxillary gland endopeptidase k hydrolysis, have been investigated using rat lysosomal cathepsins B, H and L, papain and bovine calpains I and 11. All three lysosomal cathepsins were inhibited by T kininogen but tighter interactions were observed with cathepsin L and papain. Though higher Ki values were obtained for cathepsins B and H, rate constants for association were found to have high and almost similar values (in the lo6 M-' s-l range) whathever the enzyme used. Proteolytic fragments also inhibited cathepsin L and papain very strongly and even better than the entire molecule for some of them, but no significant inhibition of cathepsins B and H was observed. Bovine calpains were not inhibited by T kininogen nor by its proteolytic fragments. From the results of this kinetic analysis, which indicates that both the association and the dissociation of lysosomal cysteine proteinases with T kininogen may occur rapidly, an hypothesis has been put forward on the possible in vivo functioning of T kininogen as a proteinase inhibitor.Kininogens of high and low M , are able to inhibit to a variable extent a number of cysteine proteinases including lysosomal cathepsins B, H and L as well as calpains (for a review see [l]). It has been also demonstrated that kinin-free H kininogen (high-M, kininogen) retains strong inhibitory activity [2,3] just as proteolytic fragments issuing from human low-M, kininogen [4]. In addition to L kininogen (low-M, kininogen) and H kininogen, rat plasma possesses a third related molecule, reported as T kininogen [5], present as two molecular varieties [6]. T kininogen also releases inhibitory fragments after proteolysis [7] but at variance with other kininogens it behaves as a major acute-phase reactant [8, 91 and is not susceptible to tissue and plasma kallikrein to release a kinin [lo, 111. However several proteinases have been reported to be able to liberate immunoreactive kinin from T kininogen but those which have been clearly identified so far should react stoichiometrically with the precursor to accomplish this function [7, 121. Their role as kininogenases remains therefore questionable, especially as most of these enzymes induce large proteolytic cleavage of T kininogen before releasing immunoreactive kinin [7, 12, 131. This cleavage gives rise to peptides retaining strong inhibitory activity when trypsin or submaxillary gland endopeptidase k are used as T kininogenases [7, 121 so that the cysteine-proteinase-inhibiting function could well be the prevailing function of this peculiar kininogen.This has prom...
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