Identification and characterization of KvgAS,a two-component system in Klebsiella pneumoniae CG43

A two-component system encoding gene cluster kvgAS that is present only in virulent Klebsiella pneumoniae CG43 was isolated and its sequence determined. RT-PCR and Southern analysis demonstrated that kvgAS is organized as an operon. No apparent effect of a kvgS deletion on bacterial virulence was observed in a mouse peritonitis model. In the presence of paraquat or 2,2-dipyridyl, the activity of kvgAS promoter in the kvgS mutant was found to be reduced to half of the level in the wild-type strain. The data suggest that the KvgAS system is autoregulated and plays a role in countering free radical stresses and sensing iron-limiting conditions.     

Functional insights from the molecular modelling of a novel two-component system

Two-component systems (TCSs) are the major signalling pathway in bacteria and represent potential drug targets. Among the 11 paired TCS proteins present in Mycobacterium tuberculosis H37Rv, the histidine kinases (HKs) Rv0600c (HK1) and Rv0601c (HK2) are annotated to phosphorylate one response regulator (RR) Rv0602c (TcrA). We wanted to establish the sequence-structure-function relationship to elucidate the mechanism of phosphotransfer using in silico methods. Sequence alignments and codon usage analysis showed that the two domains encoded by a single gene in homologous HKs have been separated into individual open-reading frames in M. tuberculosis. This is the first example where two incomplete HKs are involved in phosphorylating a single RR. The model shows that HK2 is a unique histidine phosphotransfer (HPt)-mono-domain protein, not found as lone protein in other bacteria. The secondary structure of HKs was confirmed using "far-UV" circular dichroism study of purified proteins. We propose that HK1 phosphorylates HK2 at the conserved H131 and the phosphoryl group is then transferred to D73 of TcrA.     

Evolution of Gene Overlaps: Relative Reading Frame Bias in Prokaryotic Two-Component System Genes

During a survey of two-component system genes, a list of neighboring histidine kinase and response regulator genes, encoded on the same strand, was compiled from over 200 fully sequenced bacteria. It was observed that many gene pairs overlapped, and although such overlaps can potentially occur in two phases (relative reading frames), one phase predominated for overlaps of seven or more nucleotides. Preference for a particular phase cannot be explained by arguments of sequence restraint (mutations in one gene differentially affect an overlapping gene, depending on phase). We have therefore investigated a potential explanation of the observed phase bias. For phase +1 gene overlaps, simulated point mutations in the overlapping region result in more severe changes to the downstream gene product than to the upstream gene product; vice versa in phase +2. Additionally, codon usage frequencies in nonoverlapping regions are more similar to those at the end of the upstream gene than the beginning of the downstream gene in overlaps. Taking both observations together, we propose that new gene overlaps generally arise by N-terminal extension of a downstream gene, creating a novel sequence at the start of the downstream gene. Sequence changes in this newly coding sequence will alter the sequences of both the new and the original coding sequence (the C-terminal region of the upstream gene). However, these changes will be less detrimental to the original coding sequence if the two genes overlap in phase +1, leading to selective retention during evolution of phase +1 overlaps relative to phase +2 overlaps. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Supplementary Information: The gene list and overlap dataset can be downloaded from the journal’s web site (). [Reviewing Editor: Dr. Hector Musto]     

The two-component signal system in rice (Oryza sativa L.): a genome-wide study of cytokinin signal perception and transduction

In this report we define the genes of two-component regulatory systems in rice through a comprehensive computational analysis of rice (Oryza sativa L.) genome sequence databases. Thirty-seven genes were identified, including 5 HKs (cytokinin-response histidine protein kinase) (OsHK1–4, OsHKL1), 5 HPs (histidine phosphotransfer proteins) (OsHP1–5), 15 type-A RRs (response regulators) (OsRR1–15), 7 type B RR genes (OsRR16–22), and 5 predicted pseudo-response regulators (OsPRR1–5). Protein motif organization, gene structure, phylogenetic analysis, chromosomal location, and comparative analysis between rice, maize, and Arabidopsis are described. Full-length cDNA clones of each gene were isolated from rice. Heterologous expression of each of the OsHKs in yeast mutants conferred histidine kinase function in a cytokinin-dependent manner. Nonconserved regions of individual cDNAs were used as probes in expression profiling experiments. This work provides a foundation for future functional dissection of the rice cytokinin two-component signaling pathway.     

Mammalian diacylglycerol kinases: Molecular interactions and biological functions of selected isoforms

The mammalian diacylglycerol kinases (DGK) are a group of enzymes having important roles in regulating many biological processes. Both the product and the substrate of these enzymes, i.e. diacylglycerol and phosphatidic acid, are important lipid signalling molecules. Each DGK isoform appears to have a distinct biological function as a consequence of its location in the cell and/or the proteins with which it associates. This review discusses three of the more extensively studied forms of this enzyme, DGKα, DGK?, and DGKζ. DGKα has an important role in immune function and its activity is modulated by several mechanisms. DGK? has several unique features among which is its specificity for arachionoyl-containing substrates, suggesting its importance in phosphatidylinositol cycling. DGKζ is expressed in many tissues and also has several mechanisms to regulate its functions. It is localized in several subcellular organelles, including the nucleus. The current state of our understanding of the properties and functions of these proteins is reviewed.     

Distinct functions of Cdk5(Y15) phosphorylation and Cdk5 activity in stress fiber formation and organization

Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.     

Gene expression, cellular localization, and enzymatic activity of diacylglycerol kinase isozymes in rat ovary and placenta

Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGK, -, -, and - was detected in the ovary and placenta. Intense expression signals for DGK and - were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGK were seen more intensely in granulosa cells. In the placenta, signals for DGK and - were observed in the junctional zone, whereas those for DGK were detected in the labyrinthine zone. At higher magnification, the signals for DGK were mainly discerned in giant cytotrophoblasts, and those for DGK were found in small cytotrophoblasts of the junctional zone. DGK signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGK signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.This work was supported by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, the Uehara Memorial Foundation, the ONO Medical Research Foundation, the Ciba-Geigy Foundation (Japan) for the Promotion of Science, the Kato Memorial Bioscience Foundation, and the Yamagata Health Support Foundation (to K.G.).     

Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21(WAF1/Cip1), p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.     

AtoSC two-component system is involved in cPHB biosynthesis through fatty acid metabolism in E. coli

Background

We have shown previously that AtoSC two-component system regulates the biosynthesis of E. coli cPHB [complexed poly-(R)-3-hydroxybutyrate].

Methods

The AtoSC involvement on fatty acids metabolism, towards cPHB synthesis, was studied using cPHB determination, gene expression, and fatty acid metabolic pathways inhibitors.

Results

Deletion of the atoDAEB operon from the E. coli genome resulted in a consistent reduction of cPHB accumulation. When in ΔatoDAEB cells, the atoDAEB operon and the AtoSC system were introduced extrachromosomally, a significant enhancement of cPHB levels was observed. Moreover, the introduction of a plasmid with atoSC genes regulated positively cPHB biosynthesis. A lesser cPHB enhancement was triggered when plasmids carrying either atoS or atoC were introduced. The intracellular distribution of cPHB was regulated by AtoSC or AtoC according to the inducer (acetoacetate or spermidine). Blockage of β-oxidation by acrylic acid reduced cPHB levels, suggesting the involvement of this pathway in cPHB synthesis; however, the overproduction of AtoSC or its constituents separately resulted in cPHB enhancement. Inhibition of fatty acid biosynthesis by cerulenin resulted to a major cPHB reduction, indicating the contribution of this pathway in cPHB production. Inhibition of both β-oxidation and fatty acid biosynthesis reduced dramatically cPHB, suggesting the contribution of both pathways in cPHB biosynthesis.

Conclusions

Short fatty acid catabolism (atoDAEB operon) and fatty acids metabolic pathways participate in cPHB synthesis through the involvement of AtoSC system.

General significance

The involvement of the AtoSC system in the fatty acids metabolic pathways interplay towards cPHB biosynthesis provides additional perceptions of AtoSC role on E. coli regulatory biochemical processes.     

Ca2+-activated, phospholipid-dependent protein kinase catalyzes the phosphorylation of actin-binding proteins

Chicken gizzard vinculin and filamin were found to be phosphorylated by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). These two actin-binding proteins serve as substrates for protein kinase C specifically in the free form, whereas they are little phosphorylated by protein kinase C in the presence of F-actin. In contrast, alpha-actinin from chicken gizzard is less susceptible to phosphorylation by protein kinase C, either in the presence or in the absence of F-actin. In light of these data, the possibility that Ca2+ and phospholipid-dependent phosphorylation by protein kinase C may modulate the function of actin-binding proteins has to be considered.     

Endometrial stromal cells and decidualized stromal cells: Origins,transformation and functions

Decidualization of endometrium, which is characterized by endometrial stromal cell (ESC) decidualization, vascular reconstruction, immune cell recruitment, and plentiful molecule production, is a crucial step for uterus to become receptive for embryo. When implantation takes place, ESCs surround and directly interact with embryo. Decidualized stromal cells (DSCs) are of great importance in endometrial decidualization, having a broad function in regulating immune activity and vascular remodeling of uterus. DSCs are shown to have a higher metabolic level and looser cytoskeleton than ESCs. What's the origin of ESCs and how ESCs successfully transform into DSCs had puzzled scientists in the last decades. Breakthrough had been achieved recently, and many studies had elucidated some of the characters and functions of DSCs. However, several questions still remain unclear. This paper reviews current understanding of where ESCs come from and how ESCs differentiate into DSCs, summarizes some characters and functions of DSCs, analyzes current studies and their limitations and points out research areas that need further investigation.     

Modulation of Na,K-ATPase by Associated Small Transmembrane Regulatory Proteins and by Lipids

The effects of phospholipid acyl chain length (n_c) and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal hydrophobic thickness of the lipid bilayer decreases from n_c = 22 to 18 in the presence of 40 mol% cholesterol. Hydrophobic matching as well as specific interactions of cholesterol with the phosphorylation/dephosphorylation reactions is found to be important. A novel regulatory protein has been identified in Na,K-ATPase membrane preparations from the shark (phospholemmanlike protein from shark, PLMS) with significant homology to phospholemman (PLM), the major protein kinase substrate in myocardium. Both are members of the FXYD gene family. Another member of this family is the Na,K-ATPase subunit indicating that these proteins may be specific regulators of the Na,K-ATPase. A regulatory mechanism is described in which association/dissociation of PLMS with the Na,K-ATPase is governed by its phosphorylation by protein kinases.     

Probing the nucleotide binding and phosphorylation by the histidine kinase of a novel three-protein two-component system from Mycobacterium tuberculosis

The two-component signal transduction system from Mycobacterium tuberculosis bears a unique three-protein system comprising of two putative histidine kinases (HK1 and HK2) and one response regulator TcrA. By sequence analysis, HK1 is found to be an adenosine 5'-triphosphate (ATP) binding protein, similar to the nucleotide-binding domain of homologous histidine kinases, and HK2 is a unique histidine containing phosphotransfer (HPt)-mono-domain protein. HK1 is expected to interact with and phosphorylate HK2. Here, we show that HK1 binds 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate monolithium trisodium salt and ATP with a 1:1 stoichiometric ratio. The ATPase activity of HK1 in the presence of HK2 was measured, and phosphorylation experiments suggested that HK1 acts as a functional kinase and phosphorylates HK2 by interacting with it. Further phosphorylation studies showed transfer of a phosphoryl group from HK2 to the response regulator TcrA. These results indicate a new mode of interaction for phosphotransfer between the two-component system proteins in bacteria.     

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): anatomical and cell differentiation

The ontogeny of the antennal glands was studied during the embryonic and post-embryonic development of Astacus leptodactylus. The future glands arising from undifferentiated columnar cells were detectable at the metanauplius stage EI 150 m (EI: eye index; approximately 440 m at hatching). The tubule and labyrinth differentiated in embryos at EI 190 m, and the bladder and coelomosac at EI 250 m. At EI 350 m, the tubule lengthened and divided into proximal and distal sub-regions. In later stages, the gland retained the same morpho-anatomy but the differentiation and size of each part increased. The cells of the coelomosac displayed the cytological features of podocytes in late embryonic development at EI 440 m. Only small apical microvilli and a few mitochondria were observable in the labyrinth cells at EI 250 m; by EI 440 m, these cells presented well-shaped apical microvilli, formed bodies, basal infoldings and mitochondria. In the cells of the tubules and bladder, mitochondria and basal infoldings occurred at EI 440 m and EI 250 m, respectively. The differentiation of the tubules and bladder cells suggested that they were involved in active transport at EI 440 m. Following hatching, the differentiation of the cells and the size of the glands increased. The ontogeny of the antennal glands thus starts in early embryos, the specific cellular functional features being differentiated in the various parts of the glands by EI 440 m. The antennal glands are probably functional just before hatching, i.e., before the juveniles are confronted with the low osmolality of freshwater.Thanks are due to the University of Tarbiat Modarres and Ministry of Science, Research and Technology, Islamic Republic of Iran, for financial aid and support. Special thanks are also extended to the Société Française dExportation des Ressources Educatives (SFERE) for a scholarship to S.K.     

New insights on glucosylated lipids: Metabolism and functions

Ceramide, cholesterol, and phosphatidic acid are major basic structures for cell membrane lipids. These lipids are modified with glucose to generate glucosylceramide (GlcCer), cholesterylglucoside (ChlGlc), and phosphatidylglucoside (PtdGlc), respectively. Glucosylation dramatically changes the functional properties of lipids. For instance, ceramide acts as a strong tumor suppressor that causes apoptosis and cell cycle arrest, while GlcCer has an opposite effect, downregulating ceramide activities. All glucosylated lipids are enriched in lipid rafts or microdomains and play fundamental roles in a variety of cellular processes. In this review, we discuss the biological functions and metabolism of these three glucosylated lipids.     

Targeting the X-linked inhibitor of apoptosis protein through 4-substituted azabicyclo[5.3.0]alkane smac mimetics. Structure, activity, and recognition principles

The X-linked inhibitor of apoptosis protein (XIAP) is overexpressed in several malignant cells where it prevents apoptosis by binding to, and blocking, the activation of caspase-3, -7, and -9. Human XIAP (479 residues) is composed of three tandem-repeated baculoviral IAP repeat (BIR) domains (BIR1-3), and by a C-terminal RING domain. Smac-DIABLO [second mitochondria-derived activator of caspases (Smac)-direct IAP binding protein with low pI (DIABLO)], the natural antagonist of XIAP, binds through its N-terminal sequence AVPI to the same surface groove, in the BIR domains, that binds caspases. Synthetic compounds mimicking such tetrapeptide motif effectively block the interaction between IAP and active caspases, thus triggering apoptosis. Peptidomimetics based on an azabicyclo[x.y.0]alkane scaffolds, have been shown to bind the BIR3 domain of XIAP with micromolar to nanomolar affinities, thus presenting attractive features for drug lead optimization. Here we report a study on three newly synthesized Smac mimetics, which have been characterized in their complexes with XIAP BIR3 domain through X-ray crystallography and molecular modelling/docking simulations. Based on analysis of the crystal structures, we show that specific substitutions at the 4-position of the azabicyclo[5.3.0]alkane scaffold results in sizeable effects on the peptidomimetic-BIR3 domain affinity. By means of functional, biophysical and simulative approaches we also propose that the same Smac mimetics can bind XIAP BIR2 domain at a location structurally related to the BIR3 domain AVPI binding groove. Details of the XIAP-Smac mimetic recognition principles highlighted by this study are discussed in light of the drug-like profile of the three (potentially proapoptotic) compounds developed that show improved performance in ADMET (adsorption, distribution, metabolism, excretion and toxicity) tests.