Band 398, Heft 12

Decades of chemical, biochemical and pathophysiological research have established the relevance of post-translational protein modifications induced by processes related to oxidative stress, with critical reflections on cellular signal transduction pathways. A great deal of the so-called ‘redox regulation’ of cell function is in fact mediated through reactions promoted by reactive oxygen and nitrogen species on more or less specific aminoacid residues in proteins, at various levels within the cell machinery. Modifications involving cysteine residues have received most attention, due to the critical roles they play in determining the structure/function correlates in proteins. The peculiar reactivity of these residues results in two major classes of modifications, with incorporation of NO moieties ( S -nitrosation, leading to formation of protein S -nitrosothiols) or binding of low molecular weight thiols ( S -thionylation, i.e. in particular S -glutathionylation, S -cysteinylglycinylation and S -cysteinylation). A wide array of proteins have been thus analyzed in detail as far as their susceptibility to either modification or both, and the resulting functional changes have been described in a number of experimental settings. The present review aims to provide an update of available knowledge in the field, with a special focus on the respective (sometimes competing and antagonistic) roles played by protein S -nitrosations and S -thionylations in biochemical and cellular processes specifically pertaining to pathogenesis of cardiovascular diseases.

The cystine knot disulfide pattern has been found to be widespread in nature, since it has been detected in proteins from plants, marine snails, spiders and mammals. Cystine knot proteins are secreted proteins. Their functions range from defense mechanisms as toxins, e.g. ion channel or enzyme inhibitors, to hormones, blood factors and growth factors. Cystine knot proteins can be divided into two superordinate groups. (i) The cystine knot peptides, also referred to – with other non-cystine knot proteins – as knottins, with linear and cyclic polypeptide chains. (ii) The cystine knot growth factor family, which is in the focus of this article. The disulfide ring structure of the cystine knot peptides is made up by the half-cystines 1-4 and 2-5, and the threading disulfide bond is formed by the half-cystines, 3-6. In the growth factor group, the disulfides of half-cystines 1 and 4 pass the ring structure formed by the half-cystines 2-5 and 3-6. In this review, special emphasis will be devoted to the growth factor cystine knot proteins and their proregions. The latter have shifted into the focus of scientific interest as their important biological roles are just to be unravelled.

Kallistatin, via its two structural elements – an active site and a heparin-binding domain – displays a double-edged function in angiogenesis, apoptosis and oxidative stress. First, kallistatin has both anti-angiogenic and pro-angiogenic effects. Kallistatin treatment attenuates angiogenesis and tumor growth in cancer-bearing mice. Kallistatin via its heparin-binding site inhibits angiogenesis by blocking vascular endothelial growth factor (VEGF)-induced growth, migration and adhesion of endothelial cells. Conversely, kallistatin via the active site promotes neovascularization by stimulating VEGF levels in endothelial progenitor cells. Second, kallistatin inhibits or induces apoptosis depending on cell types. Kallistatin attenuates organ injury and apoptosis in animal models, and its heparin-binding site is essential for blocking tumor necrosis factor (TNF)-α-induced apoptosis in endothelial cells. However, kallistatin via its active site induces apoptosis in breast cancer cells by up-regulating miR-34a and down-regulating miR-21 and miR-203 synthesis. Third, kallistatin can act as an antioxidant or pro-oxidant. Kallistatin treatment inhibits oxidative stress and tissue damage in animal models and cultured cells. Kallistatin via the heparin-binding domain antagonizes TNF-α-induced oxidative stress, whereas its active site is crucial for stimulating antioxidant enzyme expression. In contrast, kallistatin provokes oxidant formation, leading to blood pressure reduction and bacterial killing. Kallistatin-mediated vasodilation is partly mediated by H 2 O 2 , as the effect is abolished by the antioxidant enzyme catalase. Moreover, kallistatin exerts a bactericidal effect by stimulating superoxide production in neutrophils of mice with microbial infection as well as in cultured immune cells. Thus, kallistatin’s dual roles in angiogenesis, apoptosis and oxidative stress contribute to its beneficial effects in various diseases.

Sphingomyelin (SM) is among the most important biomolecules in eukaryotes and acts as both constructive components and signal carrier in physiological processes. SM is catalyzed by a membrane protein family, sphingomyelin synthases (SMSs), consisting of three members, SMS1, SMS2 and SMSr. SMSs modulate sphingomyelin and other sphingolipids levels, thereby regulating membrane mobility, ceramide-dependent apoptosis and DAG-dependent signaling pathways. SMSs was found associated with various diseases. Downregulation of SMS2 activity results in protective effects against obesity, atherosclerosis and diabetes and makes SMS2 inhibitors potential medicines. Structural guided specific drug design could be the next breakthrough, discriminating SMS2 from other homologs.

Cytochromes P450 (CYPs) are important for bone homeostasis, but only limited information is available on their expression in human bone cells. We analyzed the expression levels of eight CYPs in osteoblasts cultured in human bone pieces, in osteoblasts differentiated from human periosteum mesenchymal stem cells, in primary human osteoblasts and in the human osteoblast cell line MG63, respectively. Our results confirm previous reports about the presence of CYP11A1, CYP17A1, CYP24A1 and CYP27B1, while demonstrating expression of CYP2E1, CYP26A1, CYP39A1 and CYP51A1 for the first time. However, expression patterns in the four models were remarkably different from each other.

We have compared the effect of three legume lectins, wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA) and Lens culinaris agglutinin (LCA), on the function of human platelets. We have found that WGA is more active than PHA in stimulating platelet activation/aggregation, while LCA has no effect. Studies on the mechanisms involved show that WGA and PHA induce phosphorylation/activation of PLCγ2 and increase [Ca 2+ ] i . For the first time, it has been shown that Src/Syk pathway, the adapter protein SLP-76 and the exchange protein VAV, participate in the PLCγ2 activation by these lectins. Moreover WGA and PHA stimulate the PI3K/AKT pathway. PI3K, through its product phosphatidylinositol-3,4,5-trisphosphate activates Bruton’s tyrosine kinase (BTK) and contributes to PLCγ2 activation. In conclusion, our findings suggest that PLCγ2 activation induced by WGA and PHA is regulated by Src/Syk and by PI3K/BTK pathways through their concerted action.

Targeted inhibition of histone deacetylase (HDAC) is one of the potent anticancer therapy approaches. Our data showed that mRNA and protein levels of HDAC1 in breast cancer cells were greater than that in normal fibroblast 3T3 cells and normal epithelial breast MCF10A cells. The mRNA levels of HDAC1 in 75% of breast cancer tissues (18/24) were greater than that in their corresponding adjacent normal tissues. Knockdown of HDAC1 by specific siRNAs can suppress the proliferation and migration of breast cancer cells and inhibit the expression of interleukin-8 (IL-8), while not IL-6. While recombinant IL-8 (rIL-8) can attenuate the suppression effects of si-HDAC1 on the proliferation and migration of breast cancer cells. HDAC1 can positively regulate the transcription and promoter activities of IL-8. While NF-κB and MAPK, two important signals responsible for the transcription of IL-8, did not mediate HDAC1 regulated IL-8 expression. The expression and nuclear translocation of Snail were increased in HDAC1 over expressed breast cancer cells. Targeted inhibition of Snail can attenuate HDAC1 over expression induced cell proliferation and migration. Collectively, our data showed that HDAC1 can trigger the proliferation and migration of breast cancer cells via activation of Snail/IL-8 signals.

The study aimed to explore the regulation of heat shock protein 47 (HSP47) on expressions of receptors associated with hepatic stellate cell (HSC) in liver fibrosis mouse models induced by Schistosoma japonicum ( S. japonicum ). Mouse fibroblasts (NIH/3T3) were transfected with HSP47 shRNA plasmid by lipofectamine transfection, and experimental fibrosis in HSCs was studied in S. japonicum mouse models treated with HSP47 shRNA in vivo . HSP47 expression was assessed using Western blot and real-time PCR. Flow cytometry was adopted to determine the expression of cell membrane receptors. HSP47-shRNA could markedly down-regulate the expression of collagen (Col1a1 and Col3a1). The expressions of HSP47, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) significantly increased in the liver tissue of infected mice. However, the expressions of ETAR and HSP47 and ETBR remarkably decreased after the administration of HSP47 shRNA in vitro and in vivo . ETAR and ETBR levels were found to be positively correlated with HSP47 expression. HSP47 might exert influence on liver fibrosis via the regulation of ETAR and ETBR.