Perspectives La thiorédoxine est actuellement une cible thérapeutique émergente en cancérologie. Les preuves croissantes de l’implication de la thiorédoxine et des sulfhydryl oxydases dans la physio-pathologie tumorale pulmonaire permettent d’envisager l’application d’une thérapie anti-tumorale pulmonaire du NSCLC ciblée sur le métabolisme des ponts disulfure.

Experimental destruction of olfactory neurons stimulates proliferation and differentiation of local neural precursors and is used as a model to study in vivo mechanisms for degeneration and regeneration of the nervous system. Quiescin-sulfhydryl oxidases (QSOX) have a potential role in the control of the cell cycle or growth regulation and have recently been described in the central nervous system. In mice, we show an expression of QSOX in olfactory mucosa. Northern- and western-blot analysis show that the destruction of olfactory epithelium is associated with a reversible reduction in QSOX expression. Interestingly, QSOX is not localized in olfactory neurons (ON) but in cells of the lamina propria, suggesting that olfactory epithelium destruction may act as a signal of down-regulation of QSOX expression.

L'environnement redox joue un rôle capital dans les processus de progression tumorale au sein des tumeurs pulmonaires. La thioredoxine (TRX1) est la molécule de référence d'une superfamille de protéines impliquées dans la formation et le remaniement des ponts disulfures et à laquelle se rattachent également les Protéine Disulfure Isomérases (PDI), et les Sulfhydryl Oxydases (SOx) identifiées dans notre laboratoire, composées de SOx/Q6 et SOxN. La surexpression de TRX1, molécule permettant l'ouverture des ponts disulfures, confère aux cellules une capacité proliférative accrue ainsi qu'une résistance à l'apotose et aux anticancéreux. À l'inverse, SOxN, catalysant la fermeture des ponts disulfures, est impliquée dans l'exécution de l'apoptose dans le neuroblastome. SOx/Q6 est exprimée lors de la sortie de prolifération des cellules. L'importance de cette superfamille thioredoxine pour la prolifération cellulaire, l'apoptose, et l'impact que pourraient avoir des modifications de l'expression de ces gènes sur ces fonctions cellulaires nous ont conduits à développer une thématique portant sur la superfamille thioredoxine et les cancers broncho-pulmonaires. Après sélection des amorces spécifiques, obtention des standards externes et détermination des conditions optimales d'amplification, nous avons analysé l'expression des gènes de la superfamille thioredoxine (PDI, SOx/Q6, SOxN, TRX1 et TXNRD1) et 18S par PCR quantitative en temps réel sur 41 échantillons (tissu tumoral et tissu non tumoral correspondant) de cancers non à petites cellules (adénocarcinome et carcinome epi-dermoïde). Des tests non paramétriques (Stat Xact software) et une analyse statistique multivariée (SPAD software) nous ont permis d'associer les résultats avec les données cliniques et histologiques des patients. La comparaison du profil d'expression des gènes de la superfamille thioredoxine, dans les tissus tumoraux et non tumoraux, met en évidence une surexpression de TRX1, TXNRD1 et PDI dans les tumeurs. Par ailleurs, nous avons observé pour SOx/Q6, SOxN et PDI, une spécificité selon la nature histologique et le stade du cancer. Nous mettons ainsi en évidence un déséquilibre d'expression des membres réducteurs et oxydants de la superfamille Thioredoxine qui conditionne le statut apoptotique de la cellule tumorale et l'évolution de la tumeur.

Rat FAD-dependent sulfhydryl oxidase was purified; partial sequencing indicated that it was homologous to human quiescin Q6. A cDNA (GenBank(TM) 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.

An inflammatory reaction was induced in grass-cutters (Thryonomys swinderianus) by injecting turpentine. The changes in the plasma haptoglobin, fibrinogen, α2 macroglobulin and immunoglobulin G was followed for 23 days by immunonephelometry. The results were compared to rat and rabbit. The showed that (a) the inflammatory reaction is delayed in the grass-cutter compared to rats and rabbits; (b) the concentration of haptoglobin increases less than in rat and rabbit; (c) the fibrinogen concentration is very low in the grass-cutter, despite hypercoagulability of blood; (d) the changes in the plasma α2-macroglobulin in the grass-cutter seems to be comparable to that of rabbit α1 macroglobulin in amplitude and in its slow return to the initial concentration; and (e) fibrinogen and haptoglobin are suitable markers for grass-cutter inflammation monitoring.

The specific inhibitor of cysteine proteinases, cystatin C, was purified from ram rete testis fluid and the conditioned medium of Sertoli cells. This molecule associated with sheep liver cathepsin L at one of the fastest rates ever described for a proteinase/inhibitor interaction (1.75 +/- 0.20 x 10(8) M-1.s-1). But the association rate constant for the interaction of cathepsin L with alpha 2-macroglobulin, a non-specific inhibitor of proteinases, was also extremely high (8.8 +/- 0.75 x 10(6) M-1.s-1). Cathepsin L complexed with alpha 2-macroglobulin was protected from inhibition by type 2 and type 3 cystatins. The data indicate that cystatin C is the most potent inhibitor of cathepsin L in mammalian male genital tract fluids, whereas alpha 2-macroglobulin may act as a terminal acceptor of this enzyme. These inhibitors could therefore inhibit the activated form of procathepsin L which may appear during the complex process of spermatozoa production and maturation in the testis and epididymis.

alpha(1)-Inhibitor 3 (alpha(1)I(3)) is a rodent-specific proteinase inhibitor of about 190 kDa belonging to the alpha(2)-macroglobulin family. It consists of five globular domains, three of which are connected by disulfide bridges, and contains an intramolecular thiol ester which can react with attaching proteinases. To explore the folding of newly synthesized alpha(1)I(3), We have used rat hepatocytes and pulse-chase experiments. In one of the analyses, the radiolabeled protein was isolated from cell lysates by immunoprecipitation and its Asp-Pro bonds cleaved by treatment with formic acid. The size of the major fragment, as assessed by electrophoresis under nonreducing conditions, was found to increase from 100 to 150 kDa upon the chasing. This result, together with knowledge of the positions of the cleavage sites and the disulfide arrangement, indicates that one of the interdomain disulfide bonds is formed after the synthesis of the polypeptide. Analysis of the same material by limited proteolysis and by velocity centrifugation showed that the folded regions became larger and that the protein became more compact; the thiol ester was found to be formed after these conformational changes. These results suggest that the domains of alpha(1)I(3) are only partially developed directly after the synthesis of the polypeptide and that they acquire their final structure as the protein condenses and the domains interact with one another.

A mixture of crude collagenase and non-specific proteases has been used to isolate guinea pig ventricular heart cells. Measurements of collagenase activity with Wünsch's substrate and protein content with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggest that collagenase enzymes do not play a major role in heart cell isolation. On the other hand, an important factor in heart digestion seems to consist of some fractions of the proteases present in crude collagenase. It is also noted that crude collagenases do not present any sensitivity to added calcium but because this ion is important to obtain isolated cells its role is discussed. According to our results, the SDS-PAGE method can be used to determine the appropriate enzyme concentrations to obtain calcium-tolerant myocytes. These myocytes have electrophysiological properties as reported in the literature.

The Sertoli cells of the rat testis produce cystatin C, a cysteine proteinase inhibitor. Primary culture of Sertoli cells secreted both unglycosylated and glycosylated forms of rat cystatin C. Despite the low concentration of cystatin C in rete testis fluid, equilibrium dissociation constants ( K 1 ) for the interaction between cystatin C and lysosomal cathepsins indicate that this molecule could be involved in the local regulation of testicular cysteine proteinase activity which may be necessary for spermatogenesis and spermiogenesis.

alpha 1 Inhibitor3 (alpha 1I3) is a monomeric protease inhibitor of about 190 kDa which is secreted by rodent hepatocytes. We have studied intracellular modifications of this protein in [35]methionine-labelled rat hepatocytes by pulse/chase experiments followed by immunoprecipitation and gel electrophoresis under reducing and nonreducing conditions. Directly after the pulse, most of the unreduced alpha 1I3 migrated faster than the reduced form, indicating that disulphide bridges are formed during or shortly after synthesis yielding a compact structure. With increasing chase time however, an increasing portion of the unreduced alpha 1I3 migrated with a mobility lower than that of the reduced protein, half-maximal conversion occurring after about 10 min. This finding suggests that alpha 1I3 undergoes a conformational change in the endoplasmic reticulum, possibly becoming more elongated. During 10-30 min of chase, the protein acquired the capacity to undergo autolytic cleavage upon heating, a property due to the existence of an internal thiolester bond [Howard, J. B., Vermeulen, M. & Swenson, R. P. (1980) J. Biol. Chem. 255, 3820-3823; Esnard, F., Gutman, N., El Moujahed, A. & Gauthier, F. (1985) FEBS Lett. 182, 125-129]. Analysis by subcellular fractionation indicated that this bond is formed in the endoplasmic reticulum. Finally, we show that secreted alpha 1I3 is sulphated, presumably at Tyr618.

Tissue patterns of gene expression were analyzed by measuring mRNA levels and incorporation of radioactive amino acids for cystatin C and β2‐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 N‐linked 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 β2‐microglobulin cDNA was cloned and identified by nucleotide sequencing and comparison of the obtained sequence with that of mouse and human β2‐microglobulin cDNA. Tissue levels of β2‐microglobulin mRNA in the rat were measured by hybridization to rat β2‐microglobulin cDNA. The highest levels of β2‐microglobulin mRNA were observed in liver and choroid plexus. Other parts of the brain and testis contained lower levels of β2‐microglobulin mRNA.

Two immunochemically related forms of cystatin C-like inhibitors which differ in their Mr 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-beta-N-acetylglucosaminidase treatment.

Previous attempts to liberate T kinin from T kininogen [Moreau et al. (1986) Eur. J. Biochem. 159, 341-346; Gutman et al. (1988) Eur. J. Biochem. 171, 577-582] 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 II. 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 10(6) M-1 s-1 range) whatever 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.

Two cysteine proteinase inhibitors of the cystatin C type have been purified from urine of sodium chromate-treated rats. Both strongly inhibit papain as well as rat liver cathepsin L (Ki less than 10(-11) M) whereas rat liver cathepsins B and H are inhibited to a lesser extent. They differ by their apparent molecular mass of 17 kDa and 22 kDa and by their isoelectric point greater than or equal to 9.5 and 7.7 respectively. These two molecules share complete immunochemical identity and are precipitated by antibodies directed against human cystatin C but not by anti rat thiostatin and anti rat H-kininogen antibodies. They are also found in large amounts in seminal vesicles where they represent most of the cysteine proteinase inhibitory capacity.

The potential kininogenic function of rat T kininogen has been studied in parallel with the cysteine-proteinase-inhibitory function also carried by this molecule. Proteolytic cleavage of the molecule was observed upon incubation with catalytic amounts of trypsin. These conditions do not permit any significant release of immunoreactive kinin and do not modify the total papain-inhibiting capacity of T kininogen. As trypsin concentration increases in the reaction mixture, immunoreactive kinin is liberated and the total papain-inhibiting capacity decreases accordingly, as indicated by titration studies. This decrease, however, does not exceed 50% of the initial value even at a trypsin concentration as high as 75 microM, indicating that only one of the two inhibitory sites has been inactivated. The remaining inhibitory fragment corresponds to a peptide of apparent Mr 24 000, which binds papain at least as well as native T kininogen. T kininogen, therefore, appears as a potent proteinase inhibitor and/or a proteinase inhibitor precursor, whereas its kininogenic function remains questionable since no specific kininogenase able to release T kinin or another kinin under physiologically compatible conditions has been found so far.

The overall mechanism of interaction with proteinases of alpha 1-inhibitor3, a plasma proteinase inhibitor so far specific to the rat, has been shown to be closely similar to that described for alpha-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 alpha 1-inhibitor3 upon heating as observed for alpha-macroglobulin quarter subunits has been demonstrated. The question is raised of the presence of such a molecule in rat plasma in addition to two alpha-macroglobulin species, all of these proteinase inhibitors being antigenically unrelated.