Species-specific serine-threonine protein kinase Pkb2 of Bifidobacterium longum subsp. longum: Genetic environment and substrate specificity.

The objective of this study was to determine for phosphorylated substrates of the species-specific serine-threonine protein kinase (STPK) Pkb2 from Bifidobacterium longum subsp. longum GT15. Two approaches were employed: analyses of phosphorylated membrane vesicles protein spectra following kinase reactions and analyses of the genes surrounding pkb2. A bioinformatics analysis of the genes surrounding pkb2 found a species-specific gene cluster PFNA in the genomes of 34 different bifidobacterial species. The identified cluster consisted of 5-8 genes depending on the species. The first five genes are characteristic for all considered species. These are the following genes encoding serine-threonine protein kinase (pkb2), fibronectin type III domain-containing protein (fn3), AAA-ATPase (aaa-atp), hypothetical protein with DUF58 domain (duf58) and transglutaminase (tgm). The sixth (protein phosphatase, prpC), seventh (hypothetical protein, BLGT_RS02790), and eighth (FHA domain-containing protein, fha) genes are included in this cluster, but they are not found in all species. The operon organization of the PFNA gene cluster was confirmed with transcriptional analysis. AAA-ATPase, which is encoded by a gene of the PFNA gene cluster, was found to be a substrate of the STPK Pkb2. Fourteen AAA-ATPase sites (seven serine, six threonine, and one tyrosine) phosphorylated by STPK Pkb2 were revealed. Analysis of the spectra of phosphorylated membrane vesicles proteins allowed us to identify eleven proteins that were considered as possible Pkb2 substrates. They belong to several functional classes: proteins involved in transcription and translation; proteins of the F1-domain of the FoF1-ATPase; ABC-transporters; molecular chaperone GroEL; and glutamine synthase, GlnA1. All identified proteins were considered moonlighting proteins. Three out of 11 proteins (glutamine synthetase GlnA1 and FoF1-ATPase alpha and beta subunits) were selected for further in vitro phosphorylation assays and were shown to be phosphorylated by Pkb2. Four phosphorylated substrates of the species-specific STPK Pkb2 from B. longum subsp. longum GT15 were identified for the first time. They included the moonlighting protein glutamine synthase GlnA, FoF1-ATPase alpha and beta subunits, and the chaperone MoxR family of AAA-ATPase. The ability of bifidobacterial STPK to phosphorylate the substrate on serine, threonine, and tyrosine residues was shown for the first time.

FoF1-ATP synthase of Streptomyces fradiae ATCC 19609: structural, biochemical, and functional characterization.

The patterns of protein phosphorylation in inverted membrane vesicles from the strain Streptomyces fradiae ATCC 19609 were investigated to elucidate the mechanisms of regulation of bacterial membrane bound FoF1-ATP synthase. We found for the first time by two-dimensional gel electrophoresis and mass spectrometry that the ß- and b-subunits of the FoF1-ATP synthase complex undergo phosphorylation; 20 proteins with known functions were identified. All eight subunits of FoF1-ATP synthase, i.e. α, ß, γ, δ, ε, a, b, and c, were cloned into Escherichia coli and expressed as recombinant proteins. Using a crude preparation of serine/threonine protein kinases, we demonstrated the phosphorylation of recombinant γ-, ß-, α- and ε-subunits. The ß-subunit was phosphorylated both as a recombinant protein and in vesicles. Differential phosphorylation of membrane-bound and recombinant proteins can be attributed to different pools of protein kinases in each preparation; in addition, certain steps of FoF1-ATP synthase assembly and function might be accompanied by individual phosphorylation patterns. The structure of the operon containing all subunits and regulatory protein I was identified. The phylogenetic similarity of FoF1-ATP synthase from Streptomyces fradiae ATCC 19609 with the respective proteins in saprophytic and pathogenic (including Mycobacterium tuberculosis) bacteria was investigated. Thus, bacterial serine/threonine protein kinases are important for the regulation of FoF1-ATP synthase. From the practical standpoint, our results provide a basis for designing targeted antibacterial drugs.

Isolation and Purification of Recombinant Serine/Threonine Protein Kinases of the Strain Bifidobacterium longum B379M and Investigation of Their Activity.

Previously, we identified six serine/threonine protein kinases (STPK) of Bifidobacterium and named them Pkb1-Pkb6. In the present study, we optimized methods for isolation of the six STPK catalytic domains proteins of B. longum B379M: a method for isolation of Pkb3 and Pkb4 in native conditions, a method for isolation of Pkb5 in denaturing conditions, and a method for isolation of Pkb1, Pkb2, and Pkb6 from inclusion bodies. The dialysis conditions for the renaturation of the proteins were optimized. All of the enzymes were isolated in quantities sufficient for study of the protein activity. The proteins were homogeneous according to SDS-PAGE. The autophosphorylation ability of Pkb1, Pkb3, Pkb4, and Pkb6 was investigated for the first time. Autophosphorylation was detected only for the Pkb3 catalytic domain.

[Protection of radiosensitive human cells against the action of heavy metals by antimutagens and adapting factors: association with genetic and protein polymorphisms].

Cells of a diploid line obtained from embryos with the Down's syndrome, known to be unable to repair gamma-induced DNA damage, were treated with natural (garlic extract, retinol) and synthetic (crown compound) antimutagens and with adapting factors (heat shock, low CdCl2 concentrations, 10(-8) M). The protective effect was evaluated by registering DNA breaks and cell survival, and the protection coefficients were calculated. The most effective results were obtained with the use of the garlic extract and retinol. No protection of the DNA structure was observed when cells were treated with low concentrations of cadmium chloride and then with high concentrations, i. e., no adaptive response (AR) was formed under these conditions. The spectrum of proteins in treated and control cells as well as detoxication genes (GSTM1, GSTT1 , CYPIA1) were determined.

[Gene expression in normal and virally transformed erythroid cells and the effect of stress factors]. / Ekspressiia genov v normal'nykh i transformirovannykh virusom éritroidnykh kletkakh i deistvie faktorov stressa.

The globin gene expression in normal and Rauscher virus-transformed murine erythroid cells and the effect of heatshock and cycloheximide (CHI) were investigated. The hybridization analysis of RNA transcripts synthesized using erythroid cell chromatin as a template with globin cDNA demonstrated the disturbance of globin gene transcription in transformed erythroblasts. Using the Northern hybridization analysis of poly(A) +RNA with pCR1 beta MG9 as a probe, the presence of a rather broad RNA set (from approximately 1500 to 450 nucleotides) in these cells was discovered, while hemoglobin was not synthesized. This indicated that the processing of globin mRNA was probably also disturbed. As a result of heatshock, the RNA set approached that in normal erythroid cells (500-650 nucleotides), and the proteins corresponding to globins were synthesized. The stable appearance of low molecular weight proteins and the irregular synthesis of HSP-90 and HSP-70 in response to heat shock were the characteristic features of transformed erythroblasts. After CHI treatment, the content of globin sequences in RNA of transformed erythroblasts increased and beta-globin protein chains appeared.

[Effect of non-histone chromatin proteins on the transcription of globin genes]. / Vliianie negistonovykh belkov khromatina na transkriptsiiu globinovykh genov.

Globin gene transcription was studied in vitro using chromatin of normal and Rauscher virus-transformed erythroid cells. For this purpose the reconstituted chromatin was obtained and used as a template for RNA synthesis. The RNA-transcript was then analysed for the presence of globin mRNA sequences. When chromatin was reconstituted using DNA from Rauscher-transformed erythroblasts and reticulocyte nonhistone proteins they enhanced transcription. Most of the globin gene activation was due to the nonhistone protein fraction eluted from hydroxylapatite in 0.05 M Na-phosphate and the RNA-transcript in this case contained the largest quantity of globin sequences.

[Effect of chemotherapy on cytosol proteins of ovarian tumors]. / Vliianie khimioterapii na belki tsitozolia opukholei iaichnika.

Cytosol proteins of malignant ovarian tumors were studied with one- and two-dimensional gel electrophoresis, prior to and after effective chemotherapy with cisplatin and taxotere. Effective chemotherapy caused the bands and spots of 29, 36 and 50-55 kDa to fade significantly or disappear completely. It is suggested that protein 36 kDa is the proliferating nuclear cell antigen while one of the proteins 50-55 kDa--betatubulin. These proteins may serve as additional markers of ovarian tumor sensitivity to chemotherapy.

[Heat-shock gene expression in murine erythroid cells]. / Ekspressiia genov teplovogo shoka v éritroidnykh kletkakh myshi.

The effect of the heat-shock stress factor on protein synthesis in Rauscher virus-transformed murine erythroblasts with blocked hemoglobin synthesis was studied. The most pronounced heat-shock protein (HSP) synthesis was observed at 43 and 45 degrees C. Under conditions of heat shock the cells examined demonstrated enhanced (or new) synthesis of HSP with relative mol. mass of approximately 70-80 kD, approximately 50 kD and approximately 15-25 kD. No stable induction of 90 kD heat-shock protein was observed. The characteristic feature of transformed erythroblasts was a sufficiently stable appearance of low-molecular HSP with mol. mass from 14 kD to 25 kD, among which there were proteins with relative mol. mass corresponding to that of globin chains.

[Effect of cycloheximide on transcription in eukaryotic cells]. / Vliianie tsiklogeksimida na transkriptsiiu v éukarioticheskikh kletkakh.

The effects of the translation inhibitor cycloheximide on transcription in Rauscher virus transformed mouse erythroblasts and in barley rootlets were studied. In transformed erythroblasts, the globin gene transcription appeared to be blocked. Consecutive treatment of cells with sublethal doses of cycloheximide (7 micrograms/ml) and actinomycin D resulted in the induction of synthesis of the protein fraction corresponding to beta-globin chains. An exposure of barley rootlets to cycloheximide caused changes in total RNA synthesis.