Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Structure and function of a silicic acid channel Lsi1.

Silicon is a beneficial element for plant growth and production, especially in rice. Plant roots take up silicon in the form of silicic acid. Silicic acid channels, which belong to the NIP subfamily of aquaporins, are responsible for silicic acid uptake. Accumulated experimental results have deepened our understanding of the silicic acid channel for its uptake mechanism, physiological function, localization, and other aspects. However, how the silicic acid channel efficiently and selectively permeates silicic acid remains to be elucidated. Recently reported crystal structures of the silicic acid channel enabled us to discuss the mechanism of silicic acid uptake by plant roots at an atomic level. In this mini-review, we focus on the crystal structures of the silicic acid channel and provide a detailed description of the structural determinants of silicic acid permeation and its transport mechanism, which are crucial for the rational creation of secure and sustainable crops.

Structural basis for high selectivity of a rice silicon channel Lsi1.

Silicon (Si), the most abundant mineral element in the earth's crust, is taken up by plant roots in the form of silicic acid through Low silicon rice 1 (Lsi1). Lsi1 belongs to the Nodulin 26-like intrinsic protein subfamily in aquaporin and shows high selectivity for silicic acid. To uncover the structural basis for this high selectivity, here we show the crystal structure of the rice Lsi1 at a resolution of 1.8 Å. The structure reveals transmembrane helical orientations different from other aquaporins, characterized by a unique, widely opened, and hydrophilic selectivity filter (SF) composed of five residues. Our structural, functional, and theoretical investigations provide a solid structural basis for the Si uptake mechanism in plants, which will contribute to secure and sustainable rice production by manipulating Lsi1 selectivity for different metalloids.

Morphologic determinant of tight junctions revealed by claudin-3 structures.

Tight junction is a cell adhesion apparatus functioning as barrier and/or channel in the paracellular spaces of epithelia. Claudin is the major component of tight junction and polymerizes to form tight junction strands with various morphologies that may correlate with their functions. Here we present the crystal structure of mammalian claudin-3 at 3.6 Å resolution. The third transmembrane helix of claudin-3 is clearly bent compared with that of other subtypes. Structural analysis of additional two mutants with a single mutation representing other subtypes in the third helix indicates that this helix takes a bent or straight structure depending on the residue. The presence or absence of the helix bending changes the positions of residues related to claudin-claudin interactions and affects the morphology and adhesiveness of the tight junction strands. These results evoke a model for tight junction strand formation with different morphologies - straight or curvy strands - observed in native epithelia.

Correction: EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

[This corrects the article DOI: 10.1371/journal.pone.0174136.].

EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

Epstein-Barr virus (EBV) has been detected in several T- and NK-cell neoplasms such as extranodal NK/T-cell lymphoma nasal type, aggressive NK-cell leukemia, EBV-positive peripheral T-cell lymphoma, systemic EBV-positive T-cell lymphoma of childhood, and chronic active EBV infection (CAEBV). However, how this virus contributes to lymphomagenesis in T or NK cells remains largely unknown. Here, we examined NF-κB activation in EBV-positive T or NK cell lines, SNT8, SNT15, SNT16, SNK6, and primary EBV-positive and clonally proliferating T/NK cells obtained from the peripheral blood of patients with CAEBV. Western blotting, electrophoretic mobility shift assays, and immunofluorescent staining revealed persistent NF-κB activation in EBV-infected cell lines and primary cells from patients. Furthermore, we investigated the role of EBV in infected T cells. We performed an in vitro infection assay using MOLT4 cells infected with EBV. The infection directly induced NF-κB activation, promoted survival, and inhibited etoposide-induced apoptosis in MOLT4 cells. The luciferase assay suggested that LMP1 mediated NF-κB activation in MOLT4 cells. IMD-0354, a specific inhibitor of NF-κB that suppresses NF-κB activation in cell lines, inhibited cell survival and induced apoptosis. These results indicate that EBV induces NF-κB-mediated survival signals in T and NK cells, and therefore, may contribute to the lymphomagenesis of these cells.

Tight junctions. Structural insight into tight junction disassembly by Clostridium perfringens enterotoxin.

The C-terminal region of Clostridium perfringens enterotoxin (C-CPE) can bind to specific claudins, resulting in the disintegration of tight junctions (TJs) and an increase in the paracellular permeability across epithelial cell sheets. Here we present the structure of mammalian claudin-19 in complex with C-CPE at 3.7 Å resolution. The structure shows that C-CPE forms extensive hydrophobic and hydrophilic interactions with the two extracellular segments of claudin-19. The claudin-19/C-CPE complex shows no density of a short extracellular helix that is critical for claudins to assemble into TJ strands. The helix displacement may thus underlie C-CPE-mediated disassembly of TJs.

CD137 expression is induced by Epstein-Barr virus infection through LMP1 in T or NK cells and mediates survival promoting signals.

To clarify the mechanism for development of Epstein-Barr virus (EBV)-positive T- or NK-cell neoplasms, we focused on the costimulatory receptor CD137. We detected high expression of CD137 gene and its protein on EBV-positive T- or NK-cell lines as compared with EBV-negative cell lines. EBV-positive cells from EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs) patients also had significantly higher CD137 gene expression than control cells from healthy donors. In the presence of IL-2, whose concentration in the serum of EBV-T/NK-LPDs was higher than that of healthy donors, CD137 protein expression was upregulated in the patients' cells whereas not in control cells from healthy donors. In vitro EBV infection of MOLT4 cells resulted in induction of endogenous CD137 expression. Transient expression of LMP1, which was enhanced by IL-2 in EBV-T/NK-LPDs cells, induced endogenous CD137 gene expression in T and NK-cell lines. In order to examine in vivo CD137 expression, we used EBV-T/NK-LPDs xenograft models generated by intravenous injection of patients' cells. We identified EBV-positive and CD8-positive T cells, as well as CD137 ligand-positive cells, in their tissue lesions. In addition, we detected CD137 expression on the EBV infected cells from the lesions of the models by immune-fluorescent staining. Finally, CD137 stimulation suppressed etoposide-induced cell death not only in the EBV-positive T- or NK-cell lines, but also in the patients' cells. These results indicate that upregulation of CD137 expression through LMP1 by EBV promotes cell survival in T or NK cells leading to development of EBV-positive T/NK-cell neoplasms.

Novel vasotocin-regulated aquaporins expressed in the ventral skin of semiaquatic anuran amphibians: evolution of cutaneous water-absorbing mechanisms.

Until now, it was believed that only one form of arginine vasotocin (AVT)-regulated aquaporin (AQP) existed to control water absorption from the ventral skin of semiaquatic anuran amphibians, eg, AQP-rj3(a) in Rana japonica. In the present study, we have identified a novel form of ventral skin-type AQP, AQP-rj3b, in R. japonica by cDNA cloning. The oocyte swelling assay confirmed that AQP-rj3b can facilitate water permeability. Both AQP-rj3a and AQP-rj3b were expressed abundantly in the ventral hindlimb skin and weakly in the ventral pelvic skin. For the hindlimb skin, water permeability was increased in response to AVT, although the hydroosmotic response was not statistically significant in the pelvic skin. Isoproterenol augmented water permeability of the hindlimb skin, and the response was inhibited by propranolol. These events were well correlated with the intracellular trafficking of the AQPs. Immunohistochemistry showed that both AQP-rj3 proteins were translocated from the cytoplasmic pool to the apical membrane of principal cells in the first-reacting cell layer of the hindlimb skin after stimulation with AVT and/or isoproterenol. The type-b AQP was also found in R. (Lithobates) catesbeiana and R. (Pelophylax) nigromaculata. Molecular phylogenetic analysis indicated that the type-a is closely related to ventral skin-type AQPs from aquatic Xenopus, whereas the type-b is closer to the AQPs from terrestrial Bufo and Hyla, suggesting that the AQPs from terrestrial species are not the orthologue of the AQPs from aquatic species. Based on these results, we propose a model for the evolution of cutaneous water-absorbing mechanisms in association with AQPs.

NF-κB inducing kinase, a central signaling component of the non-canonical pathway of NF-κB, contributes to ovarian cancer progression.

Ovarian cancer is one of the leading causes of female death and the development of novel therapeutic approaches is urgently required. Nuclear factor-κB (NF-κB) is constitutively activated in several types of cancer including ovarian cancer and is known to support the survival of cancer cells. However, molecular mechanisms of persistent activation of NF-κB in ovarian cancer remain largely unknown. We report here that, in addition to the previously reported canonical activation, NF-κB is activated through the noncanonical pathway in ovarian cancer cells. RNA interference-mediated silencing of NF-κB inducing kinase (NIK), a central regulator of the noncanonical pathway, reduced the NF-κB2/p52 DNA binding activity and NF-κB-dependent reporter gene expression as well as NF-κB target gene expression. Notably, anchorage-dependent and -independent cell growth was impaired in NIK-depleted cells. Depletion of NIK also suppressed tumor formation in the nude mouse xenograft assay. These results indicate that NIK plays a key role in constitutive NF-κB activation and the progression of ovarian cancer cells and suggest that NIK represents an attractive therapeutic target for ovarian cancer.

Zinc-finger antiviral protein mediates retinoic acid inducible gene I-like receptor-independent antiviral response to murine leukemia virus.

When host cells are infected by an RNA virus, pattern-recognition receptors (PRRs) recognize the viral RNA and induce the antiviral innate immunity. Toll-like receptor 7 (TLR7) detects the genomic RNA of incoming murine leukemia virus (MLV) in endosomes and mediates the antiviral response. However, the RNA-sensing PRR that recognizes the MLV in the cytosol is not fully understood. Here, we definitively demonstrate that zinc-finger antiviral protein (ZAP) acts as a cytosolic RNA sensor, inducing the degradation of the MLV transcripts by the exosome, an RNA degradation system, on RNA granules. Although the retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 detect various RNA viruses in the cytosol and induce the type I IFN-dependent antiviral response, RLR loss does not alter the replication efficiency of MLV. In sharp contrast, the loss of ZAP greatly enhances the replication efficiency of MLV. ZAP localizes to RNA granules, where the processing-body and stress-granule proteins assemble. ZAP induces the recruitment of the MLV transcripts and exosome components to the RNA granules. The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to RNA granules and MLV transcripts degradation by the exosome. Although ZAP was known as a regulator of RIG-I signaling in a human cell line, ZAP deficiency does not affect the RIG-I-dependent production of type I IFN in mouse cells. Thus, ZAP is a unique member of the cytosolic RNA-sensing PRR family that targets and eliminates intracellular RNA viruses independently of TLR and RLR family members.

Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1.

Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps (NETs), which are genomic DNA-based net-like structures that capture bacteria and fungi. Although NETs also express antiviral factors, such as myeloperoxidase and α-defensin, the involvement of NETs in antiviral responses remains unclear. We show that NETs capture human immunodeficiency virus (HIV)-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin. Neutrophils detect HIV-1 by Toll-like receptors (TLRs) TLR7 and TLR8, which recognize viral nucleic acids. Engagement of TLR7 and TLR8 induces the generation of reactive oxygen species that trigger NET formation, leading to NET-dependent HIV-1 elimination. However, HIV-1 counteracts this response by inducing C-type lectin CD209-dependent production of interleukin (IL)-10 by dendritic cells to inhibit NET formation. IL-10 suppresses the reactive oxygen species-dependent generation of NETs induced upon TLR7 and TLR8 engagement, resulting in disrupted NET-dependent HIV-1 elimination. Therefore, NET formation is an antiviral response that is counteracted by HIV-1.

An IκB kinase 2 inhibitor IMD-0354 suppresses the survival of adult T-cell leukemia cells.

Adult T-cell leukemia (ATL) is a fatal T-cell malignancy associated with human T-cell leukemia virus type I infection. The aberrant expression of nuclear factor-κB (NF-κB) is considered to contribute to the malignant phenotype and chemo-resistance of ATL cells. Because of the poor prognosis of ATL, the development of new therapeutic strategies is direly needed. In the present study, we show that an IκB kinase 2 (IKK2) inhibitor, IMD-0354, efficiently inhibits the survival of CD4(+) CD25(+) primary ATL cells and prevents the growth of or induces apoptosis of patient-derived ATL cell lines. Assays of transcription with integrated forms of reporter genes revealed that IMD-0354 suppresses NF-κB-dependent transcriptional activity. Moreover, the daily administration of IMD-0354 prevents the growth of tumors in mice inoculated with ATL cells. Our results suggest that targeting IKK2 with a small molecule inhibitor, such as IMD-0354, is an attractive strategy for the treatment of ATL.

Overexpression of NF-κB inducing kinase underlies constitutive NF-κB activation in lung cancer cells.

The present study investigates roles for NF-κB inducing kinase (NIK) in constitutive NF-κB activation in lung cancer cells. A wealth of evidence showed that NF-κB is often constitutively activated in human cancer cells, including non-small cell lung cancer tissue specimens and cell lines, which may lead to deregulated apoptosis and enhanced resistance of tumor cells to chemotherapy. However, the mechanisms of NF-κB activation in lung cancer cells remain largely unknown. We report here that NF-κB inducing kinase (NIK) is aberrantly expressed at the pre-translational level in non-small cell lung cancer (NSCLC) cell lines. Depletion of NIK by RNA interference remarkably diminished nuclear NF-κB DNA binding activity and reporter gene expression. NIK depletion induced apoptosis in A549 cells, reduced the matrix metalloproteinase 9 (MMP-9) and survivin mRNA expression and affected efficiency of anchorage-independent H1299 cell growth, suggesting a role for NIK in the manifestation of oncogenic phenotype. These results indicate that NIK plays a key role in constitutive NF-κB activation in NSCLC cells and implicate NIK as a molecular target for lung cancer therapy.

Role for protein geranylgeranylation in adult T-cell leukemia cell survival.

Adult T-cell leukemia (ATL) is a fatal lymphoproliferative disease that develops in human T-cell leukemia virus type I (HTLV-I)-infected individuals. Despite the accumulating knowledge of the molecular biology of HTLV-I-infected cells, effective therapeutic strategies remain to be established. Recent reports showed that the hydroxyl-3-methylglutaryl (HMG)-CoA reductase inhibitor statins have anti-proliferative and apoptotic effects on certain tumor cells through inhibition of protein prenylation. Here, we report that statins hinder the survival of ATL cells and induce apoptotic cell death. Inhibition of protein geranylgeranylation is responsible for these effects, since simultaneous treatment with isoprenoid precursors, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, but not a cholesterol precursor squalene, restored the viability of ATL cells. Simvastatin inhibited geranylgeranylation of small GTPases Rab5B and Rac1 in ATL cells, and a geranylgeranyl transferase inhibitor GGTI-298 reduced ATL cell viability more efficiently than a farnesyl transferase inhibitor FTI-277. These results not only unveil an important role for protein geranylgeranylation in ATL cell survival, but also implicate therapeutic potentials of statins in the treatment of ATL.

Pycnogenol, a procyanidin-rich extract from French maritime pine, inhibits intracellular replication of HIV-1 as well as its binding to host cells.

A procyanidin-rich extract from French maritime pine, Pycnogenol(R) (PYC), is known as an antioxidant that exerts a variety of physiological activities and is widely used in human beings. We report here that PYC inhibits not only human immunodeficiency virus type-1 (HIV-1) binding to host cells, but also its replication after entry in susceptible cells in vitro. Prominent biochemical alterations induced by PYC were the elevated expression of an intracellular antioxidant protein, manganese superoxide dismutase (Mn-SOD), and the inhibition of phosphorylation of the ribosomal S6 protein. Interestingly, ectopic expression of Mn-SOD inhibited HIV-1 replication as well. Inhibition of HIV-1 replication associated with induced expression of Mn-SOD in cells treated with PYC suggests the potential of this natural antioxidant inducer as a new anti-HIV-1 agent.

Statin-induced inhibition of HIV-1 release from latently infected U1 cells reveals a critical role for protein prenylation in HIV-1 replication.

Latent infection of human immunodeficiency virus type 1 (HIV-1) represents a major hurdle in the treatment of acquired immunodeficiency syndrome (AIDS) patients. Statins were recently reported to suppress acute HIV-1 infection and reduce infectious virion production, but the precise mechanism of inhibition has remained elusive. Here we demonstrate that lypophilic statins suppress HIV-1 virion release from tumor necrosis factor alpha-stimulated latently infected U1 cells through inhibition of protein geranylgeranylation, but not by cholesterol depletion. Indeed, this suppression was reversed by the addition of geranylgeranylpyrophosphate, and a geranylgeranyltransferase-1 inhibitor reduced HIV-1 production. Notably, silencing of the endogenous Rab11a GTPase expression in U1 cells by RNA interference destabilized Gag and reduced virion production both in vitro and in NOD/SCID/gammac null mice. Our findings thus suggest that small GTPase proteins play an important role in HIV-1 replication, and therefore could be attractive molecular targets for anti-HIV-1 therapy.

Overexpressed NF-kappaB-inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and Hodgkin Reed-Sternberg cells.

The nuclear factor-kappaB (NF-kappaB) transcription factors play important roles in cancer development by preventing apoptosis and facilitating the tumor cell growth. However, the precise mechanisms by which NF-kappaB is constitutively activated in specific cancer cells remain largely unknown. In our current study, we now report that NF-kappaB-inducing kinase (NIK) is overexpressed at the pretranslational level in adult T-cell leukemia (ATL) and Hodgkin Reed-Sternberg cells (H-RS) that do not express viral regulatory proteins. The overexpression of NIK causes cell transformation in rat fibroblasts, which is abolished by a super-repressor form of IkappaBalpha. Notably, depletion of NIK in ATL cells by RNA interference reduces the DNA-binding activity of NF-kappaB and NF-kappaB-dependent transcriptional activity, and efficiently suppresses tumor growth in NOD/SCID/gammac(null) mice. These results indicate that the deregulated expression of NIK plays a critical role in constitutive NF-kappaB activation in ATL and H-RS cells, and suggest also that NIK is an attractive molecular target for cancer therapy.

Efficient induction of HIV-1 replication in latently infected cells through contact with CD4+ T cells: involvement of NF-kappaB activation.

Reservoir cells latently infected with HIV-1 pose one of the major obstacles that hamper ultimate eradication of HIV-1 from infected patients. In this report, we showed that direct contact with MOLT-4 T cells induced HIV-1 replication in J(22)-HL-60 latently infected cells without any additional stimulus. Neutralization experiments revealed that pro-inflammatory cytokines, whose production was increased following cell-cell contact, were unlikely to be primarily involved in the induced HIV-1 replication. Cell-cell contact, but not soluble components in the culture supernatant, caused a rapid phosphorylation and degradation of IkappaBalpha, which led to elevated NF-kappaB DNA binding activity in J(22)-HL-60 cells. Furthermore, forced expression of a super-repressor form of IkappaBalpha or pretreatment with ritonavir efficiently blocked the activation of NF-kappaB and HIV-1 replication in J(22)-HL-60 cells co-cultured with MOLT-4 T cells. Moreover, either resting or PHA stimulated primary CD4(+) T cells induced HIV-1 replication in J(22)-HL-60 cells in a similar way with that of MOLT-4 cells. These results indicated that direct contact with CD4(+) T cells induced HIV-1 replication in latently infected cells and provide insight into the molecular mechanism of virus release from myeloid progenitor cells latently infected with HIV-1.