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BBA - General Subjects (v.1780, #12)
A SARS-CoV protein, ORF-6, induces caspase-3 mediated, ER stress and JNK-dependent apoptosis by Zhongde Ye; Chung Kai Wong; Peng Li; Yong Xie (pp. 1383-1387).
Severe acute respiratory syndrome (SARS) coronavirus (CoV) spread from China to more than 30 countries, causing severe outbreaks of atypical pneumonia and over 800 deaths worldwide. CoV primarily infects the upper respiratory and gastrointestinal tract; however, SARS-CoV has a unique pathogenesis because it infects both the upper and lower respiratory tracts and leads to human respiratory diseases. SARS-CoV genome has shown containing 14 open reading frames (ORFs) and 8 of them encode novel proteins. Previous reports show that overexpression of ORF-3a, ORF-3b and ORF-7a induce apoptosis. In this report, we demonstrate that overexpression of ORF-6 also induces apoptosis and that Caspase-3 inhibitor and JNK inhibitor block ORF-6 induced apoptosis. Importantly, the protein level of ER chaperon protein, GRP94, was up-regulated when ORF-6 was overexpressed. All these data suggest that ORF-6 induces apoptosis via Caspase-3 mediated, ER stress and JNK-dependent pathways.
Keywords: ORF-6; Severe acute respiratory syndrome coronavirus; Endoplasmic reticulum; Caspase; Apoptosis; c-Jun N-terminal kinase; ER stress
The α-l-Rhamnose recognizing lectin site of human dermal fibroblasts functions as a signal transducer by Gilles Faury; E. Ruszova; J. Molinari; B. Mariko; S. Raveaud; V. Velebny; L. Robert (pp. 1388-1394).
An α-l-Rhamnose specific lectin site was described on human skin keratinocytes and fibrobasts. The addition of Rhamnose-rich oligo- and polysaccharides (RROPs) to fibroblasts has been shown to stimulate cell proliferation and increase extracellular matrix biosynthesis, suggesting that this lectin site functions as a “true” receptor transmitting messages to the cell interior. It was confirmed here that addition of the Rhamnose-rich polysaccharide, RROP-1, to normal human dermal fibroblasts (NHDFs) and human endothelial cells produced a dose-dependent stimulation of the calcium-signaling pathway, inducing fast and transient increases in Ca2+ influx and intracellular free Ca2+ level. The Rhamnose-rich oligosaccharide RROP-3 as well asl-Rhamnose alone were also able to trigger similar intracellular free Ca2+ concentration increases in NHDFs. Moreover, the recording of the RROP-1-induced modification of the gene-expression profile in fibroblasts showed that this polysaccharide triggered a down-regulation of the expression of several growth factors, adhesion molecules and extracellular matrix proteins involved in pro-tumoral activity and/or fibrotic processes. These results further support the hypothesis of a receptor function for the Rhamnose-recognizing lectin site in fibroblasts. Anti-fibrotic and anti-tumoral potential of RROP-1 remains to be further explored.
Keywords: Rhamnose; Lectin; Receptor; Calcium channel; Gene regulation; Human endothelial cell; Human dermal fibroblast
Ectopic expression of mouse Sry interferes with Wnt/β-catenin signaling in mouse embryonal carcinoma cell lines by Dana Ann A. Tamashiro; Vernadeth B. Alarcón; Yusuke Marikawa (pp. 1395-1402).
In mammals, Sry is the master regulator of male sex determination, although how it functions is still unclear. By contrast, female sex determination depends on the action of Rspo1 and Wnt4, the regulators of Wnt/β-catenin signaling. To seek a possible interaction between male and female sex determination mechanisms, we examined whether Sry affects Wnt/β-catenin signaling. Using the TOPFLASH reporter system to measure Lef/Tcf-dependent transcriptional activity, we showed that ectopic expression of mouse Sry strongly suppressed Wnt/β-catenin signaling in mouse embryonal carcinoma and human embryonic kidney cell lines. This inhibition occurred downstream of β-catenin but upstream of Lef/Tcf, and depended on both the HMG-box and the C-terminal transcriptional activation domain. By contrast, TOPFLASH was not inhibited by human SRY, which apparently lacks a transcriptional activation domain. However, a fusion construct consisting of human SRY attached to the C-terminal domain of mouse Sry was able to inhibit TOPFLASH effectively. Furthermore, Sry constructs carrying point mutations equivalent to those in human sex reversal mutations were less effective in inhibiting Wnt/β-catenin signaling. Also, we showed that the action of Sry as a transcriptional activator was both necessary and sufficient to inhibit Wnt/β-catenin signaling, suggesting that the transcriptional targets of Sry are responsible for the inhibition of signaling. Sox9 is a potential transcriptional target of Sry, although quantitative RT-PCR analysis indicates that the expression of Sox9 was not up-regulated by the ectopic expression of mouse Sry in mouse embryonal carcinoma cells. While the present study demonstrates an impact of mouse Sry on Wnt/β-catenin signaling at an in vitro level, it requires further investigations to assess whether such action also takes place in vivo to regulate male sex determination.
Keywords: Sox; HMG-box; Transcription factor; Rspo1; Sex reversal; Clinical mutation
Real-time imaging of NF-AT nucleocytoplasmic shuttling with a photoswitchable fluorescence protein in live cells by Oh Yeun Kwon; Ick Chan Kwon; Hyun Kyu Song; Hyesung Jeon (pp. 1403-1407).
The transcription factor NF-AT plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-AT translocates from the cytoplasm to the nucleus then returns in response to the intracellular calcium level.We have investigated NF-AT nucleocytoplasmic shuttling in real-time in living cells using NF-ATc1 tagged with the reversibly photoswitchable fluorescence protein, Dronpa. We monitored both nuclear import and export rate of Dronpa-tagged NF-AT in live cells upon stimulation with ionomycin plus calcium (I+Ca2+) or cyclosporin A (CsA).The results show that NF-AT moved into the nucleus within 3–9 min after stimulation and moved back out into the cytoplasm within 15–50 min after CsA addition. In the absence of stimulation, NF-AT stayed in the cytoplasm as in the cells overexpressing GSK-3β, a calcineurin-opposing regulator.This semi-quantitative imaging with constant fluorescence provides the basis to detect the real-time effect by several regulators on NF-AT family proteins.
Keywords: Abbreviations; NF-AT; nuclear factor of activated T cells; GSK-3; glycogen synthase kinase-3; CsA; cyclosporin A; I; +; Ca; 2+; ionomycin plus calciumReal-time imaging; Nucleocytoplasmic shuttling; NF-AT; Dronpa; Calcineurin; GSK-3β
The role of trehalose and its transporter in protection against reactive oxygen species by Débora da Costa Morato Nery; Carmelita Gomes da Silva; Diana Mariani; Patrícia Neves Fernandes; Marcos Dias Pereira; Anita Dolly Panek; Elis Cristina Araújo Eleutherio (pp. 1408-1411).
During menadione stress, trehalose was necessary intracellularly, but under H2O2, the sugar was required on the outside of the plasma membrane. The mechanism of protection involves minimizing the oxidative damage caused to both proteins and lipids, which would require the presence of trehalose on both sides of the lipid bilayer.
Keywords: Trehalose; Agt1; Oxidative stress; Saccharomyces cerevisiae
Salicylaldehyde derivatives as new protein kinase CK2 inhibitors by Renaud Prudent; Lopez-Ramos Miriam López-Ramos; Virginie Moucadel; Caroline Barette; David Grierson; Liliane Mouawad; Jean-Claude Florent; Lafanechere Laurence Lafanechère; Frédéric Schmidt; Claude Cochet (pp. 1412-1420).
Protein kinase CK2 is a Ser/Thr kinase, with a constitutive activity, that is considered as a promising target for cancer therapy. The currently available CK2 inhibitors lack the potency and the pharmacological properties necessary to be suitable and successful in clinical settings. We report the development of new potent CK2 inhibitors from salicylaldehyde derivatives identified by automated screening of a proprietary small-molecule library. Docking simulations and analysis of the structure–activity relationship for the hits allowed to determine their binding modes on CK2, and to carry out the optimization of their structures. This strategy led to the discovery of potent CK2 inhibitors with novel structures, one of which was able to inhibit CK2 activity in living cells and promote tumor cell death. The essential features required for potent CK2 inhibitory activity of this class of compounds are discussed.
Keywords: Protein kinase CK2; Inhibitor; Salicylaldehyde; Docking; Molecular modeling
Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines by Marcelina E. Kremser; Przybylo Małgorzata Przybyło; Hoja-Lukowicz Dorota Hoja-Łukowicz; Pochec Ewa Pocheć; Angela Amoresano; Andrea Carpentieri; Monika Bubka; Litynska Anna Lityńska (pp. 1421-1431).
It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched complex type N-glycans, the presence of poly- N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, α3β1 and αvβ3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with β1,6-branches and short polylactosamine chains. In WM9 cells, α3β1 integrin was more variously glycosylated than αvβ3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and α3β1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of αvβ3 integrin glycans in melanoma or in any cancer cells.
Keywords: α; 3; β; 1; integrin; α; V; β; 3; integrin; N; -oligosaccharide; MALDI MS; Lectin
An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection by Kaori Motomura; Akiko Hagiwara; Akiko Komi-Kuramochi; Yoshiro Hanyu; Emi Honda; Masashi Suzuki; Miho Kimura; Junko Oki; Masahiro Asada; Nagako Sakaguchi; Fumiaki Nakayama; Makoto Akashi; Toru Imamura (pp. 1432-1440).
Structural instability of wild-type fibroblast growth factor (FGF)-1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. Here we evaluated FGFC, an FGF1:FGF2 chimeric protein, for its receptor affinity, absolute heparin-dependence, stability and potential clinical applicability. Using BaF3 transfectants overexpressing each FGF receptor (FGFR) subtype, we found that, like FGF1, FGFC activates all of the FGFR subtypes (i.e., FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b and FGFR4) in the presence of heparin. Moreover, FGFC activates FGFRs even in the absence of heparin. FGFC stimulated keratinocytes proliferation much more strongly than FGF2, as would be expected from its ability to activate FGFR2b. FGFC showed greater structural stability, biological activity and resistance to trypsinization, and less loss in solution than FGF1 or FGF2. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body γ-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. These results suggest that FGFC could be useful in a variety of clinical applications, including promotion of wound healing and protection against radiation-induced damage.
Keywords: Fibroblast growth factor; Receptor specificity; Stability; Wound healing; Radiation-induced damage
Molecular mechanism for neuro-protective effect of prosaposin against oxidative stress: Its regulation of dimeric transcription factor formation by Takashi Ochiai; Yuka Takenaka; Yukako Kuramoto; Masakazu Kasuya; Kanemasa Fukuda; Masahiko Kimura; Hiroshi Shimeno; Roberta Misasi; Masao Hiraiwa; Shinji Soeda (pp. 1441-1447).
Prosaposin triggers G-protein-coupled receptor (GPCR)-mediated protein kinase B (Akt)/extracellular signal-regulated kinase (ERK) phosphorylation cascades to exert its neurotrophic and myelinotrophic activity capable of preventing neural cell death and promoting neural proliferation and glial differentiation. In the present study, we investigated the down-stream neurotrophic signaling mechanism of prosaposin by which rat pheochromocytoma (PC-12) cells are protected from cell death induced by oxidative stress. When PC-12 cells were exposed to H2O2, the cells underwent abrupt shrinkage followed by apoptosis. Prosaposin treatment at as low as 1 nM protected PC-12 cells from cell death by the oxidative stress with the activation of an ERK phosphorylation cascade. Simultaneously, prosaposin blocked the oxidative stress induced-Akt phosphorylation that acts on the down-stream of caspase-3 activation. A MEK inhibitor, PD98059, or a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, abolished the survival effect of prosaposin on the oxidative stress-induced cell death. Furthermore, prosaposin blocked the oxidative stress-induced phosphorylations of c-Jun N-terminal kinase (JNK) and p38 stress-activated protein kinase. We further investigated the effect of prosaposin treatment on the phosphorylation of activating protein-1 (AP-1) complex components, c-Jun and activating transcription factor (ATF)-3. Western blot analysis demonstrated that prosaposin treatment at 100 ng/ml decreased the levels of c-Jun and ATF-3 induced by H2O2 stimulation. Our results suggest that prosaposin aids survival of PC-12 cells from oxidative stress not only by reducing the phosphorylation levels of JNK and p38, but also by regulating the c-Jun/AP-1 pathway.
Keywords: Prosaposin; Oxidative stress; c-Jun; ATF-3; PC12 cell
Hyperoside prevents oxidative damage induced by hydrogen peroxide in lung fibroblast cells via an antioxidant effect by Mei Jing Piao; Kyoung Ah Kang; Rui Zhang; Dong Ok Ko; Zhi Hong Wang; Ho Jin You; Hee Sun Kim; Ju Sun Kim; Sam Sik Kang; Jin Won Hyun (pp. 1448-1457).
We elucidated the cytoprotective effects of hyperoside (quercetin-3- O-galactoside) against hydrogen peroxide (H2O2)-induced cell damage. We found that hyperoside scavenged the intracellular reactive oxygen species (ROS) detected by fluorescence spectrometry, flow cytometry, and confocal microscopy. In addition, we found that hyperoside scavenged the hydroxyl radicals generated by the Fenton reaction (FeSO4+H2O2) in a cell-free system, which was detected by electron spin resonance (ESR) spectrometry. Hyperoside was found to inhibit H2O2-induced apoptosis in Chinese hamster lung fibroblast (V79-4) cells, as shown by decreased apoptotic nuclear fragmentation, decreased sub-G1 cell population, and decreased DNA fragmentation. In addition, hyperoside pretreatment inhibited the H2O2-induced activation of caspase-3 measured in terms of levels of cleaved caspase-3. Hyperoside prevented H2O2-induced lipid peroxidation as well as protein carbonyl. In addition, hyperoside prevented the H2O2-induced cellular DNA damage, which was established by comet tail, and phospho histone H2A.X expression. Furthermore, hyperoside increased the catalase and glutathione peroxidase activities. Conversely, the catalase inhibitor abolished the cytoprotective effect of hyperoside from H2O2-induced cell damage. In conclusion, hyperoside was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing antioxidant enzyme activity.
Keywords: Hyperoside; Reactive oxygen species; Apoptosis; Lipid peroxidation; Protein carbonyl; DNA damage
Emergence of a novel highly specific and catalytically efficient enzyme from a naturally promiscuous glutathione transferase by Cecilia Blikstad; Abeer Shokeer; Sanela Kurtovic; Bengt Mannervik (pp. 1458-1463).
Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A4-4 mimic, called GIMFhelix, with >300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A4-4 by determining catalytic efficiencies with nine alternative substrates. The enzymes can be represented by vectors in multidimensional substrate-activity space, and the vectors of GIMFhelix and GST A1-1, expressed in kcat/ Km values for the alternative substrates, are essentially orthogonal. By contrast, the vectors of GIMFhelix and GST A4-4 have approximately similar lengths and directions. The broad substrate acceptance of GST A1-1 contrasts with the high selectivity of GST A4-4 and GIMFhelix for alkenal substrates. Multivariate analysis demonstrated that among the diverse substrates used, nonenal, cumene hydroperoxide, and androstenedione are major determinants in the portrayal of the three enzyme variants. These GST substrates represent diverse chemistries of naturally occurring substrates undergoing Michael addition, hydroperoxide reduction, and steroid double-bond isomerization, respectively. In terms of function, GIMFhelix is a novel enzyme compared to its progenitor GST A1-1 in spite of 94% amino-acid sequence identity between the enzymes. The redesign of GST A1-1 into GIMFhelix therefore serves as an illustration of divergent evolution leading to novel enzymes by minor structural modifications in the active site. Notwithstanding low sequence identity (60%), GIMFhelix is functionally an isoenzyme of GST A4-4.
Keywords: Abbreviations; PEITC; phenethyl isothiocyanate; EA; ethacrynic acid; Diiodobutane; 1,4-diiodobutane; pNPA; para; -nitrophenylacetate; CuOOH; cumene hydroperoxide; NPTI; 1-methyl-4-nitro-5-[(4-nitrophenyl)thio]-1H-imidazole; AD; Δ; 5; -androstene-3,17-dione; CDNB; 1-chloro-2,4-dinitrobenzene; GST; glutathione transferaseRational design; Protein evolution; Substrate selectivity; Multivariate data analysis