HIV-1 Tat Activates Akt/mTORC1 Pathway and AICDA Expression by Downregulating Its Transcriptional Inhibitors in B Cells.

HIV-1 infects T cells, but the most frequent AIDS-related lymphomas are of B-cell origin. Molecular mechanisms of HIV-1-induced oncogenic transformation of B cells remain largely unknown. HIV-1 Tat protein may participate in this process by penetrating and regulating gene expression in B cells. Both immune and cancer cells can reprogram communications between extracellular signals and intracellular signaling pathways via the Akt/mTORC1 pathway, which plays a key role in the cellular response to various stimuli including viral infection. Here, we investigated the role of HIV-1 Tat on the modulation of the Akt/mTORC1 pathway in B cells. We found that HIV-1 Tat activated the Akt/mTORC1 signaling pathway; this leads to aberrant activation of activation-induced cytidine deaminase (AICDA) due to inhibition of the AICDA transcriptional repressors c-Myb and E2F8. These perturbations may ultimately lead to an increased genomic instability and proliferation that might cause B cell malignancies.

Characterization of atypical pathogenic Aeromonas salmonicida isolated from a diseased Siberian sturgeon (Acipenser baerii).

Acipenser baerii (Siberian sturgeon) is native to Kazakhstan and is currently endangered and is listed within the first class of protected animals. Sturgeon aquaculture is becoming an important tool for the recovery of this endangered species. Nonetheless, diseases involving typical symptoms of skin ulceration and systemic bacterial hemorrhagic septicemia have occurred in cultured A. baerii on a fish farm located in Western Kazakhstan. In this study, an infectious strain of bacteria isolated from an ulcer of diseased A. baerii was identified as Aeromonas salmonicida (strain AB001). This identification involved analyses of 16S rRNA, gyrB, rpoD, and flaA genes' sequences. Even though strain AB001 belongs to A. salmonicida, it exhibited noticeable mobility and growth at temperatures of ≥37 °C. Profiling of virulence genes uncovered the presence of seven such genes related to pathogenicity. Antibiotic sensitivity testing showed that the strain is sensitive to aminoglycosides, amphenicols, nitrofurans, quinolones, and tetracyclines. Half-lethal doses (LD50) of strain AB001 for Oreochromis mossambicus and A. baerii were determined: respectively 1.7 × 108 and 7.2 × 107 colony-forming units per mL. The experimentally induced infection revealed that strain AB001 causes considerable histological lesions in O. mossambicus, including tissue degeneration, necrosis, and hemorrhages of varied severity.

A polyclonal antibody against a recombinantly expressed Triticum aestivum RHT-D1A protein.

BACKGROUND: Reduced height-1 dwarfing alleles affect DELLA proteins belonging to a family of putative transcriptional regulators that modulate plant growth and development. The Arabidopsis thaliana genome encodes five DELLA proteins, whereas monocot plants, such as rice, barley, and wheat, each have a single DELLA protein. In wheat, wild-type Rht-B1a and Rht-D1a genes encode DELLA proteins and have many alleles that contain lesions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key part in the creation of high-yielding wheat varieties. Despite their fundamental roles in plant biology, until now, DELLA proteins in wheat have been mainly researched regarding the phenotypic effect of defective Rht mutants on yield-related traits, without studies on the underlying mechanisms. The RHT-1 protein has yet to be detected in wheat tissues, owing to a lack of appropriate molecular tools for characterization of RHT function and protein interactions in signal transduction. This study is focused on the production of a polyclonal antibody to the wheat RHT-D1A protein. RESULTS: To generate the anti-RHT-D1A antibody, we expressed and purified soluble 6xHis-tagged RHT-D1A. The purified recombinant RHT-D1A was injected into New Zealand white rabbits to generate polyclonal antiserum. The polyclonal anti-RHT-D1A antibody was purified by ammonium sulfate precipitation, followed by affinity chromatography on protein A-agarose beads. The purified polyclonal antibody was demonstrated to be effective in immunoblotting, western blot hybridization, and immunoprecipitation. In wheat seedling extracts, the polyclonal antibody recognized a protein with a molecular mass close to the predicted molecular weight of the endogenous RHT-D1A protein. We also demonstrated that RHT-D1A disappears in response to exogenous and endogenous gibberellic acid. CONCLUSION: The purified polyclonal antibody raised against the recombinant RHT-D1A protein is sufficiently specific and sensitive and could be a useful tool for future insights into upstream and downstream components of DELLA-regulatory mechanisms in wheat plants.

Wheat Germination Is Dependent on Plant Target of Rapamycin Signaling.

Germination is a process of seed sprouting that facilitates embryo growth. The breakdown of reserved starch in the endosperm into simple sugars is essential for seed germination and subsequent seedling growth. At the early stage of germination, gibberellic acid (GA) activates transcription factor GAMYB to promote de novo synthesis of isoforms of α-amylase in the aleurone layer and scutellar epithelium of the embryo. Here, we demonstrate that wheat germination is regulated by plant target of rapamycin (TOR) signaling. TOR is a central component of the essential-nutrient-dependent pathway controlling cell growth in all eukaryotes. It is known that rapamycin, a highly specific allosteric inhibitor of TOR, is effective in yeast and animal cells but ineffective in most of higher plants likely owing to structural differences in ubiquitous rapamycin receptor FKBP12. The action of rapamycin on wheat growth has not been studied. Our data show that rapamycin inhibits germination of wheat seeds and of their isolated embryos in a dose-dependent manner. The involvement of Triticum aestivum TOR (TaTOR) in wheat germination was consistent with the suppression of wheat embryo growth by specific inhibitors of the TOR kinase: pp242 or torin1. Rapamycin or torin1 interfered with GA function in germination because of a potent inhibitory effect on α-amylase and GAMYB gene expression. The TOR inhibitors selectively targeted the GA-dependent gene expression, whereas expression of the abscisic acid-dependent ABI5 gene was not affected by either rapamycin or torin1. To determine whether the TaTOR kinase activation takes place during wheat germination, we examined phosphorylation of a ribosomal protein, T. aestivum S6 kinase 1 (TaS6K1; a substrate of TOR). The phosphorylation of serine 467 (S467) in a hydrophobic motif on TaS6K1 was induced in a process of germination triggered by GA. Moreover, the germination-induced phosphorylation of TaS6K1 on S467 was dependent on TaTOR and was inhibited by rapamycin or torin1. Besides, a gibberellin biosynthesis inhibitor (paclobutrazol; PBZ) blocked not only α-amylase gene expression but also TaS6K1 phosphorylation in wheat embryos. Thus, a hormonal action of GA turns on the synthesis of α-amylase in wheat germination via activation of the TaTOR-S6K1 signaling pathway.

Heterologous secretory expression of ß-glucosidase from Thermoascus aurantiacus in industrial Saccharomyces cerevisiae strains.

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer ß-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a ß-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.

The Arabidopsis thaliana Poly(ADP-Ribose) Polymerases 1 and 2 Modify DNA by ADP-Ribosylating Terminal Phosphate Residues.

Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyze the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are viewed as DNA damage sensors that, upon binding to strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. The flowering plant Arabidopsis thaliana contains three genes encoding homologs of mammalian PARPs: atPARP1, atPARP2, and atPARP3. Both atPARP1 and atPARP2 contain poly(ADP-ribosyl)ating activity; however, it is unknown whether they could covalently modify DNA by ADP-ribosylating the strand break termini. Here, we report that similar to their mammalian counterparts, the plant atPARP1 and atPARP2 proteins ADP-ribosylate 5'-terminal phosphate residues in duplex DNA oligonucleotides and plasmid containing at least two closely spaced DNA strand breaks. AtPARP1 preferentially catalyzes covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 5'-phosphate, whereas atPARP2 preferentially ADP-ribosylates the nicked and gapped DNA duplexes containing the terminal 5'-phosphate. Similar to their mammalian counterparts, the plant PARP-catalyzed DNA ADP-ribosylation is particularly sensitive to the distance that separates two strand breaks in the same DNA molecule, 1.5 and 1 or 2 turns of helix for atPARP1 and atPARP2, respectively. PAR glycohydrolase (PARG) restored native DNA structure by hydrolyzing the PAR-DNA adducts generated by atPARPs. Biochemical and mass spectrometry analyses of the PAR-DNA adducts showed that atPARPs utilize phosphorylated DNA termini as an alternative to protein acceptor residues to catalyze PAR chain synthesis via phosphodiester bond formation between C1' of ADP-ribose and a phosphate residue of the terminal nucleotide in DNA fragment. Taken together, these data establish the presence of a new type of DNA-modifying activity in Arabidopsis PARPs, suggesting a possible role of DNA ADP-ribosylation in DNA damage signaling and repair of terrestrial plants.

Mechanism of stimulation of DNA binding of the transcription factors by human apurinic/apyrimidinic endonuclease 1, APE1.

Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB. Also, APE1 participates in the base excision repair (BER) and nucleotide incision repair (NIR) pathways to remove oxidative DNA base damage. At present, the molecular mechanism underlying the TF-stimulating/redox function of APE1 and its biological role remains disputed. Here, we provide evidence that, instead of direct cysteine reduction in TFs by APE1, APE1-catalyzed NIR and TF-stimulating activities may be based on transient cooperative binding of APE1 to DNA and induction of conformational changes in the helix. The structure of DNA duplex strongly influences NIR and TF-stimulating activities. Homologous plant AP endonucleases lacking conserved cysteine residues stimulate DNA binding of the p50 subunit of NF-κB. APE1 acts synergistically with low-molecular-weight reducing agents on TFs. Finally, APE1 stimulates DNA binding of the redox-insensitive p50-C62S mutant protein. Electron microscopy imaging of APE1 complexes with DNA revealed preferential polymerization of APE1 on the gapped and intrinsically curved DNA duplexes. Molecular modeling offers a structural explanation how full-length APE1 can oligomerize on DNA. In conclusion, we propose that DNA-directed APE1 oligomerization can be regarded as a substitute for diffusion of APE1 along the DNA contour to probe for anisotropic flexibility. APE1 oligomers exacerbate pre-existing distortions in DNA and enable both NIR activity and DNA binding by TFs regardless of their oxidation state.

Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli.

BACKGROUND: DNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2'-deoxyguanosine-5'-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. METHODS: Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproduct (6-4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured. RESULTS: We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6-4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains. DISCUSSION: These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

The major Arabidopsis thaliana apurinic/apyrimidinic endonuclease, ARP is involved in the plant nucleotide incision repair pathway.

Apurinic/apyrimidinic (AP) endonucleases are important DNA repair enzymes involved in two overlapping pathways: DNA glycosylase-initiated base excision (BER) and AP endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, AP endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in NIR, the same AP endonucleases incise DNA 5' to a wide variety of oxidized bases. The flowering plant Arabidopsis thaliana contains three genes encoding homologues of major human AP endonuclease 1 (APE1): Arp, Ape1L and Ape2. It has been shown that all three proteins contain AP site cleavage and 3'-repair phosphodiesterase activities; however, it was not known whether the plant AP endonucleases contain the NIR activity. Here, we report that ARP proteins from Arabidopsis and common wheat (Triticum aestivum) contain NIR and 3'→5' exonuclease activities in addition to their AP endonuclease and 3'-repair phosphodiesterase functions. The steady-state kinetic parameters of reactions indicate that Arabidopsis ARP cleaves oligonucleotide duplexes containing α-anomeric 2'-deoxyadenosine (αdA) and 5,6-dihydrouridine (DHU) with efficiencies (kcat/KM=134 and 7.3 µM-1·min-1, respectively) comparable to those of the human counterpart. However, the ARP-catalyzed 3'-repair phosphodiesterase and 3'→5' exonuclease activities (kcat/KM=314 and 34 µM-1·min-1, respectively) were about 10-fold less efficient as compared to those of APE1. Interestingly, homozygous A. thaliana arp-/- mutant exhibited high sensitivity to methyl methanesulfonate and tert-butyl hydroperoxide, but not to H2O2, suggesting that ARP is a major plant AP endonuclease that removes abasic sites and specific types of oxidative DNA base damage. Taken together, these data establish the presence of the NIR pathway in plants and suggest its possible role in the repair of DNA damage generated by oxidative stress.

Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA.

Oxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichia coli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg(2+) dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases.

Cloning and characterization of a wheat homologue of apurinic/apyrimidinic endonuclease Ape1L.

BACKGROUND: Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized. METHODOLOGY/PRINCIPAL FINDINGS: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' → 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+) and Ca(2+) (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+), Co(2+) and Fe(2+) cations (0.1-1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6-7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 µM(-1) · min(-1), respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1. CONCLUSIONS/SIGNIFICANCE: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.

Presence of base excision repair enzymes in the wheat aleurone and their activation in cells undergoing programmed cell death.

Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the ß-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.

ER stress inhibits mTORC2 and Akt signaling through GSK-3ß-mediated phosphorylation of rictor.

In response to environmental cues, cells coordinate a balance between anabolic and catabolic pathways. In eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival through activation of the phosphoinositide 3-kinase (PI3K)-Akt pathway. Akt-mediated phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) inhibits its enzymatic activity, thereby stimulating glycogen synthesis. We show that GSK-3ß itself inhibits Akt by controlling the mammalian target of rapamycin complex 2 (mTORC2), a key activating kinase for Akt. We found that during cellular stress, GSK-3ß phosphorylated the mTORC2 component rictor at serine-1235, a modification that interfered with the binding of Akt to mTORC2. The inhibitory effect of GSK-3ß on mTORC2-Akt signaling and cell proliferation was eliminated by blocking phosphorylation of rictor at serine-1235. Thus, in response to cellular stress, GSK-3ß restrains mTORC2-Akt signaling by specifically phosphorylating rictor, thereby balancing the activities of GSK-3ß and Akt, two opposing players in glucose metabolism.