S. M. Elizarov

Search for inhibitors of bacterial and human protein kinases among derivatives of diazepines[1,4] annelated with maleimide and indole cycles.

Aminomethylation of 9b,10-dihydro-1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino-[1,7-a]indole-1,3(2H)-diones or 1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)-diones resulted in dialkylaminomethyl derivatives. Alkylation of the nitrogen atom of maleimide moiety of polyannelated diazepines with 1,3-dibromopropane and subsequent reaction with thiourea or its N-alkyl derivatives gave isothiourea-carrying compounds. The compounds containing isothiourea moiety were active against individual human serine/threonine and tyrosine kinases at low micromolar concentrations. Dialkylaminomethyl derivatives of diazepines sensitized Streptomyces lividans with overexpressed aminoglycoside phosphotransferase type VIII (aphVIII) to kanamycin by inhibiting serine/threonine kinase(s) mediated aphVIII phosphorylation.

Ca2+-dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca2+ ions. The inhibitors of Ca2+/calmodulin and Ca2+/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca2+-dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca2+/calmodulin and Ca2+/phospholipid-dependent STPK reduced the Ca2+-induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.

[Calcium as a regulator of intracellular processes in actinomycetes: a review].

Data on the effects of calcium ions (Ca2+) on processes of morphological and physiological differentiation in cultures of actinomycetes have been reviewed, with emphasis on representatives of the genus Strepomyces. Evidence accumulated thus far regarding the regulatory role of serine-threonine protein kinases in the differentiation and the possible involvement of Ca2+-dependent protein kinases in secondary metabolism (including antibiotic biosynthesis) are analyzed. The possibility that regulatory elements of apoptosis (including Ca2+-dependent) function in actinomycetes is discussed. A hypothesis is advanced, according to which determinants of antibiotic resistance play a key role in the network of signal transduction systems of actinomycetes.

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F0F1 ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F0F1 complex is Mg2+-dependent and moderately stimulated by high concentrations of Ca2+ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F0F1 ATPase inhibitors that react with the membrane-bound F0 complex. The assay of biochemical properties of the F0F1 ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F0F1 complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F0F1 ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.

Calcium‐Induced Alterations in the Functioning of Protein Serine/Threonine and Tyrosine Kinases in Streptomyces fradiae Cells

In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate sporulation. Using in vivo labeling, we demonstrate that in S. fradiae phosphorylation of some proteins are also influenced by Ca2+ added exogenously. Calcium ions at physiological concentration increase phosphorylation of multiple proteins on serine/threonine residues and suppress modification of a 140‐kDa protein on tyrosine residues. Assay of protein kinases in situ demonstrated that Ca2+‐induced differences in the pattern of protein phosphorylation in vivo are accompanied by Ca2+‐dependent cessation of autophosphorylation of 140‐kDa tyrosine kinase and by increased autophosphorylation of three serine/threonine kinases with molecular masses of 127, 65, and 31.5 kDa.

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 β- 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, β, γ, δ, ɛ, 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-, β-, α- and ɛ-subunits. The β-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.

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca2+-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.

Dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain and its chloramphenicol-resistant mutant

The dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain 964 and its chloramphenicol-resistant (Cmlr) pleiotropic mutant with an enhanced production of avermectins was studied by measuring the transfer of radiolabeled phosphate from [y-32P]ATP to the serine and threonine residues of proteins in cell-free extracts. In both of the strains studied, radiolabeled phosphate was found to incorporate into polypeptides with molecular masses of 32, 35, 41, 68, 75, 79, 83, and 137 kDa; however, the degree and the dynamics of phosphorylation of particular peptides were different in these strains. The differences revealed could not be accounted for by the interference of ATPases or phosphoprotein phosphatases. The data obtained may be interpreted as evidence that Cmlr mutation activates the protein kinase signalling system ofS.avermitilis cells in the early stationary growth phase and thus enhances the production of avermectins and leads to some other physiological changes in the mutant strain.