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Two biflavonoids, ginkgetin (1) and sciadopitysin (2), were isolated from the MeOH extract of the young branches of Taxus cuspidata, which inhibited phosphatase of regenerating liver-3 (PRL-3) with IC50 values of 25.8 and 46.2 microM, respectively. This is the first report on PRL-3 inhibitors, isolated from natural sources.

In Vietnam, highly pathogenic avian influenza (HPAI), such as that caused by H5N1 viruses, is the most highly contagious infectious disease that has been affecting domestic poultry in recent years. Vietnam might be an evolutionary hotspot and a potential source of globally pandemic strains. However, few studies have reported viruses circulating in the south-central region of Vietnam. In the present study, 47 H5N1-positive samples were collected from both vaccinated and unvaccinated poultry farms in the South Central Coast region of Vietnam during 2013-2014, and their genetic diversity was analyzed. A common sequence motif for HPAI virus was identified at HA-cleavage sites in all samples: either RERRRKR/G (clades 2.3.2.1c and 2.3.2.1a) or REGRRKKR/G (clade 1.1.2). Phylogenetic analysis of HA genes identified three clades of HPAI H5N1: 1.1.2 (n=1), 2.3.2.1a (n=1), and 2.3.2.1c (n=45). The phylogenetic analysis indicated that these Vietnamese clades may have evolved from Chinese and Cambodian virus clades isolated in 2012-2013 but are less closely related to the clades detected from the Tyva Republic, Bulgaria, Mongolia, Japan, and Korea in 2009-2011. Detection of the coexistence of virus clades 2.3.2.1 and the very virulent 1.1.2 in the south-central regions suggests their local importance and highlights concerns regarding their spread, both northwards and southwards, as well as the potential for reassortment. The obtained data highlight the importance of regular identification of viral evolution and the development and use of region-specific vaccines.

H3N8 equine influenza virus (EIV) causes respiratory diseases in the horse population, and it has been demonstrated that EIV can transmit into dogs owing to its availability on receptors of canine respiratory epithelial cells. Recently, we isolated H3N8 EIV from an EIV-vaccinated horse that showed symptoms of respiratory disease, and which has a partially truncated nonstructural gene (NS). However, it is not clear that the NS-truncated EIV has an ability to cross the host species barrier from horses to dogs as well. Here, we experimentally infected the NS-truncated H3N8 EIV into dogs, and monitored their clinical signs and viral load in respiratory organs to determine the virus``s transmissibility.

We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009) and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naive dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection occurring in dogs since 2008. Furthermore, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses.

The Escherichia coli transmembrane protein DsbD transfers electrons from the cytoplasm to the periplasm through a cascade of thiol-disulfide exchange reactions. In this process, the C-terminal periplasmic domain of DsbD (DsbDgamma) shuttles the reducing potential from the membrane domain (DsbDbeta) to the N-terminal periplasmic domain (DsbDalpha). The crystal structure of DsbDgamma determined at 1.9 A resolution reveals that the domain has a thioredoxin fold with an extended N-terminal stretch. In comparison to thioredoxin, the DsbDgamma structure exhibits the stabilized active site conformation and the extended active site alpha2 helix that explain the domain's substrate specificity and the redox potential shift, respectively. The hypothetical model of the DsbDgamma:DsbDalpha complex based on the DsbDgamma structure and previous structural studies indicates that the conserved hydrophobic residue in the C-X-X-C motif of DsbDgamma may be important in the specific recognition of DsbDalpha.

Outbreaks of H5 highly pathogenic avian influenza viruses (HPAIVs) have caused economic loss for the poultry industry and posed a threat to public health. In South Korea, novel reassortants of HPAIVs such as H5N6 and H5N8 had been circulating in poultry. Here, we will discuss the identity of recent novel reassortants of Korean H5 HPAIVs and the recent advances in vaccine development, which will be useful for controlling HPAIV transmission in poultry and for effectively preventing future epidemics and pandemics.

Heat shock protein 33 (Hsp33) inhibits aggregation of partially denatured proteins during oxidative stress. The chaperone activity of Hsp33 is unique among heat shock proteins because the activity is reversibly regulated by cellular redox status. We report here the crystal structure of the N-terminal region of Hsp33 fragments with constitutive chaperone activity. The structure reveals that the N-terminal portion of Hsp33 forms a tightly associated dimer formed by a domain crossover. A concave groove on the dimeric surface contains an elongated hydrophobic patch that could potentially bind denatured protein substrates. The termini of the subunits are located near the hydrophobic patch, indicating that the cleaved C-terminal domain may shield the hydrophobic patch in an inactive state. Two of the four conserved zinc-coordinating cysteines are in the end of the N-terminal domain, and the other two are in the cleaved C-terminal domain. The structural information and subsequent biochemical characterizations suggest that the redox switch of Hsp33 occurrs by a reversible dissociation of the C-terminal regulatory domain through oxidation of zinc-coordinating cysteines and zinc release.

Dual-specificity protein phosphatases (DUSPs), which dephosphorylate both phosphoserine/threonine and phosphotyrosine, play vital roles in immune activation, brain function and cell-growth signalling. A family-wide structural library of human DUSPs was constructed based on experimental structure determination supplemented with homology modelling. The catalytic domain of each individual DUSP has characteristic features in the active site and in surface-charge distribution, indicating substrate-interaction specificity. The active-site loop-to-strand switch occurs in a subtype-specific manner, indicating that the switch process is necessary for characteristic substrate interactions in the corresponding DUSPs. A comprehensive analysis of the activity–inhibition profile and active-site geometry of DUSPs revealed a novel role of the active-pocket structure in the substrate specificity of DUSPs. A structure-based analysis of redox responses indicated that the additional cysteine residues are important for the protection of enzyme activity. The family-wide structures of DUSPs form a basis for the understanding of phosphorylation-mediated signal transduction and the development of therapeutics.

Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are primary virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications, such as hemolytic uremic syndrome and central nervous system abnormalities. Current therapeutic options to treat patients infected with toxin-producing bacteria are limited. The structures of Stxs, toxin-receptor binding, intracellular transport and the mode of action of the toxins have been well defined. However, in the last decade, numerous studies have demonstrated that in addition to being potent protein synthesis inhibitors, Stxs are also multifunctional proteins capable of activating multiple cell stress signaling pathways, which may result in apoptosis, autophagy or activation of the innate immune response. Here, we briefly present the current understanding of Stx-activated signaling pathways and provide a concise review of therapeutic applications to target tumors by engineering the toxins.

Anthraquinones have been reported as phosphatase inhibitors. Therefore, anthraquinone derivatives were screened to identify a potent phosphatase inhibitor against the phosphatase of regenerating liver-3 (PRL-3). Emodin strongly inhibited phosphatase activity of PRL-3 with IC(50) values of 3.5μM and blocked PRL-3-induced tumor cell migration and invasion in a dose-dependent manner. Emodin rescued the phosphorylation of ezrin, which is a known PRL-3 substrate. The results of this study reveal that emodin is a PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.

Dual-specificity protein tyrosine phosphatase localized to mitochondrion 1 (PTPMT1) has recently proved to be a promising therapeutic target for the treatment of type II diabetes. Herein we report the first example for a successful application of the structure-based virtual screening to identify the novel inhibitors of human PTPMT1. These inhibitors were computationally screened for having desirable physicochemical properties as a drug candidate and reveal a high potency with IC50 values ranging from 0.7 to 17.3 μM. Therefore, they deserve consideration for further development by structure–activity relationship studies to optimize the antidiabetic activities. Structural features relevant to the stabilization of the newly identified inhibitors in the active site of PTPMT1 are addressed in detail.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) has proved to be an attractive target for the development of therapeutics for the treatment of cancer. We report the first example for a successful application of the structure-based virtual screening to identify the novel inhibitors of MKP-1. It is shown that the efficiency of virtual screening can be enhanced significantly by the incorporation of a new solvation energy term in the scoring function. The newly found inhibitors have desirable physicochemical properties as a drug candidate and reveal a moderate potency with IC(50) values ranging from 20 to 50 μM. Therefore, they deserve a consideration for further development by structure-activity relationship studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the inhibitors in the active site of MKP-1 are discussed in detail.

Oxidative stress has been implicated in a number of neurological disorders, including cerebral ischemia and neuro-degenerative diseases. Comprehensive proteomic studies were carried out using an immortalized mouse hippocampal cell line, HT22, exhibiting oxidative stress-mediated cell death upon glutamate treatment. Two-dimensional fluorescence difference gel electrophoresis (2D DIGE) of subcellular organelle fractions revealed that significant numbers of proteins showed quantitative changes during HT22 cell death, among which a total of 51 proteins were identified by mass spectrometry. The identified proteins indicate that HT22 cell death occurs through perturbations in mitochondrial function, changes in translational elongation machinery, and translocation of proteins across subcellular organelles. This list of proteins may shed light on oxidative stress-mediated neuronal cell death.

Eya proteins are transcription factors that play pivotal roles in organ formation during development by mediating interactions between Sine Oculis (SO) and Dachshund (DAC). Remarkably, the transcriptional activity of Eya proteins is regulated by a dephosphorylating activity within its Eya domain (ED). However, the molecular basis for the link between catalytic and transcriptional activities remains unclear. Here we report the first description of the crystal structure of the ED of human Eya2 (ED-Eya2), determined at 2.4-A resolution. In stark contrast to other members of the haloacid dehalogenase (HAD) family to which ED-Eya2 belongs, the helix-bundle motif (HBM) is elongated along the back of the catalytic site. This not only results in a structure that accommodates large protein substrates but also positions the catalytic and the SO-interacting sites on opposite faces, which suggests that SO binding is not directly affected by catalytic function. Based on the observation that the DAC-binding site is located between the catalytic core and SO binding sites within ED-Eya2, we propose that catalytic activity can be translated to SO binding through DAC, which acts as a transcriptional switch. We also captured at two stages of reaction cycles-acyl-phosphate intermediate and transition state of hydrolysis step, which provided a detailed view of reaction mechanism. The ED-Eya2 structure defined here serves as a model for other members of the Eya family and provides a framework for understanding the role of Eya phosphatase mutations in disease.—Jung, S.–K., Jeong, D. G., Chung, S. J., Kim, J. H., Park, B. C., Tonks, N. K., Ryu, S. E., Kim, S. J.. Crystal structure of ED-Eya2: insight into dual roles as a protein tyrosine phosphatase and a transcription factor.

The receptor-type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPrho is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto-domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto-domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain-specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.

Decursinol, found in the roots of Angelica gigas Nakai, has been traditionally used to treat anemia and other various diseases. Recently, numerous biological activities such as cytotoxic effect on leukemia cells, and antitumor, neuroprotection, and antibacterial activities have been reported for this compound. Although a number of proteins including protein kinase C, androgen receptor, and acetylcholinesterase were proposed as molecular targets responsible for the activities of decursinol, they are not enough to explain such a diverse biological activity mentioned above. In this study, we employed a chemical proteomic approach, leading to identification of seven proteins as potential proteins interacting with decursinol. Most of the proteins contain a defined ATP or nucleic acid binding domain and have been implied to be involved in the pathogenesis and progression of various human diseases including cancer, autoimmune disorders, or neurodegenerative diseases. The present results may provide clues to understand the molecular mechanism of the biological activities shown by decursinol, an anticancer natural product.

Extracellular signal-regulated kinase 2 (ERK2) has become an attractive target for the development of therapeutics for the treatment of cancer. We have been able to identify eight new inhibitors of ERK2 by means of a drug design protocol involving the virtual screening with docking simulations and in vitro enzyme assay. The newly discovered inhibitors can be categorized into three structural classes and reveal a significant potency with IC50 values ranging from 1 to 30 μM. Therefore, all of the three inhibitor scaffolds deserve further development by structure–activity relationship or de novo design methods. Structural features relevant to the stabilizations of the newly identified inhibitors in the ATP-binding site of ERK2 are discussed in detail.

The inhibitors of phosphatase of regenerating liver-3 (PRL-3) have been shown to be useful as therapeutics for the treatment of cancer. We have been able to identify 12 novel PRL-3 inhibitors by means of the virtual screening with docking simulations under the consideration of the effects of ligand solvation in the scoring function. Because the newly identified inhibitors are structurally diverse and reveal a significant potency with IC50 values ranging from 10 to 50 μM, all of them can be considered for further development by structure–activity relationship or de novo design methods. Structural features relevant to the interactions of the newly identified inhibitors with the amino acid residues in the active site and the peripheral binding site of PRL-3 are discussed in detail.

The Rev-erb family is an orphan nuclear receptor acting as a negative regulator of transcription. Rev-erbalpha and Rev-erbbeta are crucial components of the circadian clock and involved in various lipid homeostasis. They are unique nuclear receptors that lack the activation function 2 helix (AF2-helix) required for ligand-dependent activation by other members of nuclear receptors. Here, we report the crystal structure of Rev-erbbeta (NR1D2) in a dimeric arrangement. The putative ligand-binding pocket (LBP) of Rev-erbbeta is filled with bulky hydrophobic residues resulting in a residual cavity size that is too small to allow binding of any known ligand molecules. However, an alternative conformation of the putative LBP observed in another crystal form suggests the flexibility of this region. The kinked conformation of helix H11 allows helix H11 to bend toward helix H3 over the putative ligand binding pocket by filling and closing the cavity with its side-chains. In the absence of the AF2-helix and a cognate ligand, Rev-erbbeta appears to stabilize the hydrophobic cluster in the putative ligand binding pocket and provide a structural platform for co-repressor binding by adopting the unique geometry of helix H11, a suitable conformation for the constitutive repression activity.

Insulinoma-associated protein-2 (IA-2) is a major autoantigen in type 1 diabetes that occurs through autoimmune-mediated β-cell destruction. We present here the crystal structure of the protein tyrosine phosphatase (PTP)-like domain of human IA-2. The structure reveals a canonical PTP domain with the closed WPD loop over the active site pocket, explaining the lack of enzyme activity in the native protein. The structural interpretation of previous mutagenesis studies indicates that the B-cell epitopes are concentrated on two distinctive regions on peripheral loops of the central β-sheet surrounding T-cell epitopes within the sheet. The detailed structural information on immune epitopes provides a framework for the future development of immune intervention strategies against diabetes.

The human dual-specificity protein phosphatase 18 (DSP18) gene and its protein product have recently been characterized. Like most DSPs, DSP18 displays dephosphorylating activity towards both phosphotyrosine and phosphothreonine residues. However, DSP18 is distinct from other known DSPs in terms of the existence of approximately 30 residues at the C-terminus of the catalytic domain and an unusual optimum activity profile at 328 K. The crystal structure of human DSP18 has been determined at 2.0 A resolution. The catalytic domain of DSP18 adopts a fold similar to that known for other DSP structures. Although good alignments are found with other DSPs, substantial differences are also found in the regions surrounding the active site, suggesting that DSP18 constitutes a unique structure with a distinct substrate specificity. Furthermore, the residues at the C-terminus fold into two antiparallel beta-strands and participate in extensive interactions with the catalytic domain, explaining the thermostability of DSP18.

Protein tyrosine phosphatase 1B (PTP1B) acts as a negative regulator of insulin signaling, and selective inhibition of PTP1B has served as a potential drug target for the treatment of type 2 diabetes. In the course of screening for PTP1B inhibitory natural products, the MeOH extract of the dried root of Salvia miltiorrhiza BUNGE (Labiatae) was found to exhibit significant inhibitory effect. Bioassay-guided fractionation and purification afforded three related abietane-type diterpene metabolites 1-3. Compounds 1-3 were identified as isotanshinone IIA (1), dihydroisotanshinone I (2), and isocryptotanshinone (3) mainly by analysis of NMR and MS data. Compounds 1-3 non-competitively inhibited PTP1B activity with 50% inhibitory concentration values of 11.4+/-0.6 microM, 22.4+/-0.6 microM and 56.1+/-6.3 microM, respectively.

An organic layer prepared from the Chinese crude drug 'Sang-Bai-Pi' (Morus root bark) was studied in order to identify the inhibitory compounds for protein tyrosine phosphatase 1B (PTP1B). Bioassay-guided fractionation resulted in the isolation of sanggenon C (1), sanggenon G (2), mulberrofuran C (3) and kuwanon L (4) as PTP1B inhibitors, along with moracin O (5) and moracin P (6). Compounds 1-4 inhibited PTP1B with IC(50) values ranging from 1.6+/-0.3 microM to 16.9+/-1.1 microM.

The PRL phosphatases, which constitute a subfamily of the protein tyrosine phosphatases (PTPs), are implicated in oncogenic and metastatic processes. Here, we report the crystal structure of human PRL-1 determined at 2.7 A resolution. The crystal structure reveals the shallow active-site pocket with highly hydrophobic character. A structural comparison with the previously determined NMR structure of PRL-3 exhibits significant differences in the active-site region. In the PRL-1 structure, a sulfate ion is bound to the active-site, providing stabilizing interactions to maintain the canonically found active conformation of PTPs, whereas the NMR structure exhibits an open conformation of the active-site. We also found that PRL-1 forms a trimer in the crystal and the trimer exists in the membrane fraction of cells, suggesting the possible biological regulation of PRL-1 activity by oligomerization. The detailed structural information on the active enzyme conformation and regulation of PRL-1 provides the structural basis for the development of potential inhibitors of PRL enzymes.

Cellular redox control is often mediated by oxidation and reduction of cysteine residues in the redox-sensitive proteins, where thioredoxin and glutaredoxin (Grx) play as electron donors for the oxidized proteins. Despite the importance of protein-protein interactions between the electron donor and acceptor proteins, there has been no structural information for the interaction of thioredoxin or Grx with natural target proteins. Here, we present the crystal structure of a novel Haemophilus influenza peroxiredoxin (Prx) hybrid Prx5 determined at 2.8-A resolution. The structure reveals that hybrid Prx5 forms a tightly associated tetramer where active sites of Prx and Grx domains of different monomers interact with each other. The Prx-Grx interface comprises specific charge interactions surrounded by weak interactions, providing insight into the target recognition mechanism of Grx. The tetrameric structure also exhibits a flexible active site and alternative Prx-Grx interactions, which appear to facilitate the electron transfer from Grx to Prx domain. Differences of electron donor binding surfaces in Prx proteins revealed by an analysis based on the structural information explain the electron donor specificities of various Prx proteins.

The master switch of cellular hypoxia responses, hypoxia-inducible factor 1 (HIF-1), is hydroxylated by factor inhibiting HIF-1 (FIH-1) at a conserved asparagine residue under normoxia, which suppresses transcriptional activity of HIF-1 by abrogating its interaction with transcription coactivators. Here we report the crystal structure of human FIH-1 at 2.8-A resolution. The structural core of FIH-1 consists of a jellyroll-like beta-barrel containing the conserved ferrous-binding triad residues, confirming that FIH-1 is a member of the 2-oxoglutarate-dependent dioxygenase family. Except for the core structure and triad residues, FIH-1 has many structural deviations from other family members including N- and C-terminal insertions and various deletions in the middle of the structure. The ferrous-binding triad region is highly exposed to the solvent, which is connected to a prominent groove that may bind to a helix near the hydroxylation site of HIF-1. The structure, which is in a dimeric state, also reveals the putative von Hippel-Lindau-binding site that is distinctive to the putative HIF-1-binding site, supporting the formation of the ternary complex by FIH-1, HIF-1, and von Hippel-Lindau. The unique environment of the active site and cofactor-binding region revealed in the structure should allow design of selective drugs that can be used in ischemic diseases to promote hypoxia responses.

The gene for Escherichia coli ribonuclease P (RNase P) protein (also known as C5 protein) and its mutant C5-C113A have been expressed as GST fusion proteins in E. coli at a high level. After cleavage of the fusion protein, highly purified functional C5 protein is obtained that can be crystallized with 2.5-2.6 M (NH(4))(2)HPO(4)/(NH(4))H(2)PO(4) pH 7.0 at room temperature. These crystals are suitable for X-ray analysis, belong to the space group P3(1)21 or P3(2)21 (unit-cell parameters a = b = 66.67, c = 142.09 A) and diffract to 2.9 A at 100 K using sorbitol and glycerol as cryoprotectants. For three molecules in the asymmetric unit a V(M) of 2.17 A(3) Da(-1) was calculated.

Heat shock protein 33 (Hsp33) from Escherichia coli is a redox-regulated molecular chaperone that protects cells from oxidative stress. To understand the molecular basis for the monomer–dimer switch in the functional regulation of E. coli Hsp33, we generated a constitutively monomeric Hsp33 by introducing the Q151E mutation in the dimeric interface and determined its crystal structure. The overall scaffold of the monomeric Hsp331–235 (Q151E) mutant is virtually the same as that of the dimeric form, except that there is no domain swapping. The measurement of chaperone activity to thermally denatured luciferase showed that the constitutively monomeric Hsp33 mutant still retains chaperone activity similar to that of wild-type Hsp331–235, suggesting that a Hsp33 monomer is sufficient to interact with slowly unfolded substrate.