Background: Dengue protease is a two-component protease that is important for viral replication. Results: An unlinked protease complex containing the NS2B regulatory region and the NS3 protease domain was obtained. Conclusion:The unlinked protease complex produces dispersed cross-peaks in NMR spectra and exists predominantly in a closed conformation in solution.Significance: This new construct will be a useful tool for drug discovery against the dengue virus.

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.

Knowledge of the cellular targets of ROS (reactive oxygen species) and their regulation is an essential prerequisite for understanding ROS-mediated signalling. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is known as a major target protein in oxidative stresses and becomes thiolated in its active site. However, the molecular and functional changes of oxidized GAPDH, the inactive form, have not yet been characterized. To examine the modifications of GAPDH under oxidative stress, we separated the oxidation products by two-dimensional gel electrophoresis and identified them using nanoLC-ESI-q-TOF MS/MS (nano column liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem MS). Intracellular GAPDH subjected to oxidative stress separated into multiple acidic spots on two-dimensional gel electrophoresis and were identified as cysteine disulfide and cysteic acids on Cys152 in the active site. We identified the interacting proteins of oxidized inactive GAPDH as p54nrb (54 kDa nuclear RNA-binding protein) and PSF (polypyrimidine tract-binding protein-associated splicing factor), both of which are known to exist as heterodimers and bind to RNA and DNA. Interaction between oxidized GAPDH and p54nrb was abolished upon expression of the GAPDH active site mutant C152S. The C-terminal of p54nrb binds to GAPDH in the cytosol in a manner dependent on the dose of hydrogen peroxide. The GAPDH-p54nrb complex enhances the intrinsic topoisomerase I activation by p54nrb-PSF binding. These results suggest that GAPDH exerts other functions beyond glycolysis, and that oxidatively modified GAPDH regulates its cellular functions by changing its interacting proteins, i.e. the RNA splicing by interacting with the p54nrb-PSF complex.

We previously reported that the four-transmembrane L6 family member 5 (TM4SF5) was highly expressed in hepatocarcinoma, induced morphological elongation and epithelialmesenchymal transition, and caused abnormal cell growth in multilayers in vitro and tumor formation in vivo. In this study, we identified a synthetic compound, 4 -(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC) that antagonized both the TM4SF5-mediated multilayer growth and TM4SF5-enhanced migration/invasion. TSAHC treatment induced multilayer-growing cells to grow in monolayers, recovering contact inhibition without accompanying apoptosis, and inhibited chemotactic migration and invasion. E pithelial monolayer integrity is maintained by integrin-mediated cell adhesion between the cell and the extracellular matrix (ECM) and by E-cadherin-mediated contact between adjacent cells. 1 Epithelial-mesenchymal transition (EMT) through loss of cellcell contacts disrupts monolayer integrity and alters cell-ECM interactions. 2 Tumor cells disseminated from primary tumors via loss of cell adhesion and contact can migrate to and invade distal tissues. 3 We recently reported that TM4SF5-mediated EMT results in a loss of contact inhibition and multilayer growth. 4 Therefore, altered cell adhesion and contact may lead to both a loss of contact inhibition and a dissemination of metastatic cells from the primary tumor. 5 Integrin-mediated cell adhesion reorganizes actin filaments 6 through activation of diverse intracellular signaling molecules, including focal adhesion kinase (FAK), Rho guanosine triphosphatases (RhoA, Rac1, and CDC42), and others, 7 which are critical for cellular morphology and migration. 8 TM4SF proteins (i.e., tetraspanins or tetraspans) are a group of membrane proteins with four transmembrane

Transforming growth factor ␣ (TGF-␣) is biosynthesized as a membrane-bound precursor protein, pro-TGF-␣, that undergoes sequential endoproteolytic cleavages to release a soluble form of the factor. In the present study, we have analyzed the biosynthesis and regulation of TGF-␣ production in human tumor-derived cell lines that endogenously express pro-TGF-␣ and the epidermal growth factor (EGF) receptor. These cells biosynthesized membrane-anchored forms of the TGF-␣ that accumulated on the cell surface. Membranebound pro-TGF-␣ interacted with the EGF receptor, and complexes of receptor and pro-TGF-␣ contained tyrosine-phosphorylated receptor. Activation of the EGF receptor by soluble EGF or TGF-␣ had a dual effect on TGF-␣ production: an increase in pro-TGF-␣ mRNA levels and an increase in pro-TGF-␣ cleavage. These effects were largely prevented by preincubation with an anti-EGF receptor monoclonal antibody that blocked ligand binding. Growth factor autoinduction of cleavage could be stimulated by several second messenger pathways that are activated by the EGF receptor, including protein kinase C and intracellular calcium, and by other alternative mechanisms. EGF-stimulated cleavage of pro-TGF-␣ could be partially blocked by inhibition of these second messenger pathways. These results suggest that juxtacrine stimulation takes place in human tumor cells that coexpress both the EGF receptor and membrane-anchored TGF-␣ and that TGF-␣ is able to induce its own endoproteolytic cleavage by activating the EGF receptor.

There has already been a great deal of attention devoted to the preparation of compounds containing TCNE (tetracyanoethylene) combined with transition-metal complexes,l and a great variety of product types have been described. It would seem reasonable to expect that the strong electron-acceptor capacity of TCNE, coupled with its ability to serve as a ligand through two or more of its CN groups, should allow the preparation of polymeric materials, [ML,,(TCNE)]-, that stand a g d chance of displaying interesting electronic properties. To date, however, the literature records but three examples of such materials, namely, ([Mn-(TPP)] [TCNE]),Z (TPP = tetraphenylphorphinato dianion) and the ([M(hfacac)z] [TCNE]), compounds3 (hfacac = F3CC(O)-CHC(O)CF3-with M = Co and Cu). All of these have infinite chain structures in which cl2,92-(trans-C2(CN),) units are coordinated to the planar Mn(TPP) or M(hfacac)2 units above and below the plane. It is clear that the size and shape of the Mn-(TPP) and M(hfacac)z moieties preclude the use of the remaining two CN groups on each TCNE unit.There have also been a few reported examples of TCNE employing one? two: or four4 of its CN groups as points of ligation to form discrete molecular species. These include the [LMn-(CO)2] 1 4 (TCNE) compounds4 (L = MeCp or MeSCp), which have not been structurally characterized and [(Ph3P)2Ir(CO)]z-[TCNE],S which has been structurally characterized.We report here the preparation of the first compound in which TCNE employs all four of its CN groups to function as a ~4 -9~ bridging group in such a way as to form infinite sheets, [(M,L,)z-(TCNE)],. The M,L,group is Rh2(02CCF3)4, which is already well known to be a good acceptor at both of its axial positions, whereby numerous Rh2(02CCF3)4L2 compounds may be formed, many of which have been structurally characterized.6 The compound reported here is one in which TCNE is attached to a dimetal core. The Rh2(02CCF& was chosen for two reasons:(1) it is extremely soluble in a variety of solvents and (2) it is a powerful acceptor with a preference for hard ligands.' Both of these properties arise because of the strong electron-withdrawing effect of the four CFS groups: molecular association is feeble becauseof the poor donor power of the carboxylateoxygen atoms, while the rhodium atoms have enhanced acceptor ability.The Rh2(02CCF3)4* (130 mg, 0.20 mmol) was dissolved in 15 mL of dichloromethane, and 25 mg of TCNE (0.20 mmol) was added to this solution. The green color of the starting material immediately changed, and a blue-black precipitate appeared. The precipitate was collected by filtration and dried under reduced pressure. Crystals suitable for X-ray crystallography were (1) A useful reference list can be found in ref 3 and need not be repeated here.Figure 1. Drawing of one T C N E unit surrounded by four Rh2(02CCF& units and portions of the neighboring TCNE units. For clarity, all fluorine atoms are omitted, and the sizes of the atoms are arbitrary.obtained by a diffusion technique in which a layer of ben...

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Snail family proteins regulate transcription of molecules for cell-cell adhesion during epithelial-mesenchymal transition (EMT). Based on putative glycogen synthase kinase 3β (GSK-3β) phosphorylation sites within the Slug/Snail2, we explored the significance of GSK-3β-mediated phosphorylation in Slug/Snail2 expression during EMT. Mutation of the putative GSK-3β phosphorylation sites (S92/96A or S100/104A) enhanced the Slug/Snail2-mediated EMT properties of E-cadherin repression and vimentin induction, compared with wild-type Slug/Snail2. S92/96A mutation inhibited degradation of Slug/Snail2 and S100/104A mutation extended nuclear stabilization. Inhibition of GSK-3β activity caused similar effects, as did the phosphorylation mutations. Thus, our study suggests that GSK-3β-mediated phosphorylation of Slug/Snail2 controls its turnover and localization during EMT.