Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2 alpha kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2 alpha kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2 alpha kinases might play similar roles and that PoPKR is the predominant kinase. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

Molecular cloning and stress-induced expression of paralichthys olivaceus heme-regulated initiation factor 2 alpha kinase

The heme-regulated initiation factor 2 alpha kinase (HRI) is acknowledged to play an important role in translational shutoff in reticulocytes in response to various cellular stresses. In this study, we report its homologous cDNA cloning and characterization from cultured flounder embryonic cells (FEC) after treatment with UV-inactivated grass carp haemorrhagic virus (GCHV). The full-length cDNA of Paralichthys olivaceus HRI homologue (PoHRI) has 2391 bp and encodes a protein of 651 amino acids. The putative PoHRI protein exhibits high identity with all members of eIF2 alpha kinase family. It contains 12 catalytic subdomains located within the C-terminus of all Ser/Thr protein kinases, a unique kinase insertion of 136 amino acids between subdomains IV and V, and a relatively conserved N-terminal domain (NTD). Upon heat shock, virus infection or Poly PC treatment, PoHRI mRNA and protein are significantly upregulated in FEC cells but show different expression patterns in response to different stresses. In healthy flounders, PoHRI displays a wide tissue distribution at both the mRNA and protein levels. These results indicate that PoHRI is a ubiquitous eIF2a kinase and might play an important role in translational control over nonheme producing FEC cells under different stresses. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus

The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. Previously, some important IFN system genes had been identified from an IFN-producing CAB (crucian carp Carassius auratus blastulae embryonic) cells after treatment with UV-inactivated GCHV (grass carp haemorrhage virus). Here, a fish PKR-like gene, named CaPKR-like, is cloned and sequenced from the same virally infected CAB cells. It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein of 513 amino acid residues. BLAST search reveals that the putative CaPKR-like protein is most homologous to human PKR and also has a high-level homology with all members of a family of eIF2alpha kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2alpha kinase family and the most similarity to mammalian PKRs. Within its N-terminus, there are no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Zalpha). Like mammalian PKRs, CaPKR-like had a very low level of constitutive expression in normal CAB cells but was up-regulated in response to active GCHV, UV-inactivated GCHV and CAB IFN, implying that the transcriptional activation of CaPKR-like by viral infection is mediated possibly by newly produced CAB IFN, which was further supported by using cycloheximide, a potent inhibitor of protein synthesis. The results together suggested that CaPKR-like was the first identified fish gene most similar to mammalian PKRs. (C) 2004 Elsevier Ltd. All rights reserved.

alr0117, a two-component histidine kinase gene, is involved in heterocyst development in Anabaena sp PCC 7120

Anabaena sp. PCC; 7120 was mutagenized by transposon Tn5-1087b, generating a mutant whose heterocysts lack the envelope polysaccharide layer. The transposon was located between nucleotides 342 and 343 of alr0117, a 918 bp gene encoding a histidine kinase for a two-component regulatory system. Complementation of the mutant with a DNA fragment containing alr0117 and targeted inactivation of the gene confirmed that alr0117 is involved in heterocyst development. RT-PCR showed that alr0117 was constitutively expressed in the presence or absence of a combined-nitrogen source. hepA and patB, the two genes turned on during wild-type heterocyst development, were no longer activated in an alr0117-null mutant. The two-component signal transduction system involving alr0117 may control the formation of the envelope polysaccharide layer and certain late events essential to the function of heterocysts.

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43') but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

A SERINE KINASE REGULATES INTRACELLULAR-LOCALIZATION OF SPLICING FACTORS IN THE CELL CYCLE

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells(1). It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts(2). Splicing factors redistribute in response to transcription inhibition(3,4) or viral infection(5), and nuclear speckles break down and reform as cells progress through mitosis(6). We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 mag have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.

Molecular Dynamics Simulation of Methane Hydrate Dissociation Process in the Presence of Thermodynamic Inhibitor

The dissociation of methane hydrate in the presence of ethylene glycol (11.45 mol.L-1) at 277.0 K was studied using canonical ensemble (NVT) molecular dynamics simulations. Results show that hydrate dissociation starts from the surface layer of the solid hydrate and then gradually expands to the internal layer. Thus, the solid structure gradually shrinks until it disappears. A distortion of the hydrate lattice structure occurs first and then the hydrate evolves from a fractured frame to a fractional fragment. Finally, water molecules in the hydrate construction exist in the liquid state. The inner dissociating layer is, additionally, coated by a liquid film formed from outer dissociated water molecules outside. This film inhibits the mass transfer performance of the inner molecules during the hydrate dissociation process.

Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1IIIB entry and replication with EC50 values of 3.92±0.62 and 6.59±1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1KM018 with EC50 values of 44.44±10.20 nM, as well as suppressing HIV-1- induced cytopathic effect with an EC50 value of 3.04±1.20 nM. It also inhibited HIV-2ROD and HIV-2CBL-20 entry and replication in the μM range. Notably, HR212 was highly effective against T20-resistant strains with EC50 values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/ AIDS patients, particularly for those infected by T20-resistant variants.

Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

In this paper, we study the ability of DNA-PK-deficient (M059J) and -proficient (M059K) cells to undergo the rate of cellular proliferation, cell cycle distribution and apoptosis after 10 Gy X-ray irradiation, and the role of DNA-PK in radiosensitivity. The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells. A strong G2 phase arrest was observed in M059J cells post irradiation. Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h. Altogether, the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage, a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint, by which DNA-PK may affect cellular sensitivity to ionizing radiation. 地址: [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Chinese Acad Sci, Inst Modern Phys, Lanzhou 730000, Peoples R China; [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Key Lab Heavy Ion Radiat Med Gansu Prov, Lanzhou 730000, Peoples R China; [Li Ning; Wang Yanling] Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China; [Wang Xiaohu] Gansu Tumor Hosp, Dept Radiotherapy, Lanzhou 730050, Peoples R China

Expression, purification and spectral characterization of p21(Waf1/Cip1)

p21(Waf1/Cip1), best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, can interact with various target proteins, and this ability relies on its structural plasticity. Therefore, studies on the structural properties of p(21) are very important to understand its structure-function relationship. However, detailed studies on its secondary structure and biophysical properties have been comparatively sparse. A human p(21) gene was cloned into the temperature expression vector pBV220 and transformed into Escherichia coli strain JM109.