Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

Abstract Background All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. Results The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. Conclusion Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss-vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of,3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells. Citation: Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier. PLoS ONE 7(11): e48913. doi:10.1371/journal.pone.0048913

RefZ Facilitates the Switch from Medial to Polar Division during Spore Formation in Bacillus subtilis

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.

Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins

Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.

Mutations in SRCAP, Encoding SNF2-Related CREBBP Activator Protein, Cause Floating-Harbor Syndrome

Floating-Harbor syndrome (FHS) is a rare condition characterized by short stature, delayed osseous maturation, expressive-language deficits, and a distinctive facial appearance. Occurrence is generally sporadic, although parent-to-child transmission has been reported on occasion. Employing whole-exome sequencing, we identified heterozygous truncating mutations in SRCAP in five unrelated individuals with sporadic MS. Sanger sequencing identified mutations in SRCAP in eight more affected persons. Mutations were de novo in all six instances in which parental DNA was available. SRCAP is an SNF2-related chromatin-remodeling factor that serves as a coactivator for CREB-binding protein (CREBBP, better known as CBP, the major cause of Rubinstein-Taybi syndrome [RTS]). Five SRCAP mutations, two of which are recurrent, were identified; all are tightly clustered within a small (111 codon) region of the final exon. These mutations are predicted to abolish three C-terminal AT-hook DNA-binding motifs while leaving the CBP-binding and ATPase domains intact. Our findings show that SRCAP mutations are the major cause of FHS and offer an explanation for the clinical overlap between FHS and RTS.

Structural effect of cationic amphiphiles in diacetylenic photopolymerizable membranes

Liposomes have been an excellent option as drug delivery systems, since they are able of incorporating lipophobic and/or lipophilic drugs, reduce drug side effects, increase drug targeting, and control delivery. Also, in the last years, their use reached the field of gene therapy, as non-viral vectors for DNA delivery. As a strategy to increase system stability, the use of polymerizable phospholipids has been proposed in liposomal formulations. In this work, through differential scanning calorimetry (DSC) and electron spin resonance (ESR) of spin labels incorporated into the bilayers, we structurally characterize liposomes formed by a mixture of the polymerizable lipid diacetylenic phosphatidylcholine 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in a 1:1 molar ratio. It is shown here that the polymerization efficiency of the mixture (c.a. 60%) is much higher than that of pure DC8,9PC bilayers (c.a. 20%). Cationic amphiphiles (CA) were added, in a final molar ratio of 1:1:0.2 (DC8,9PC:DMPC:CA), to make the liposomes possible carriers for genetic material, due to their electrostatic interaction with negatively charged DNA. Three amphiphiles were tested, 1,2-dioleoyl-3-trimetylammonium-propane (DOTAP), stearylamine (SA) and trimetyl (2-miristoyloxietyl) ammonium chloride (MCL), and the systems were studied before and after UV irradiation. Interestingly, the presence of the cationic amphiphiles increased liposomes polymerization. MCL displaying the strongest effect. Considering the different structural effects the three cationic amphiphiles cause in DC8,9PC bilayers, there seem to be a correlation between the degree of DC8,9PC polymerization and the packing of the membrane at the temperature it is irradiated (gel phase). Moreover, at higher temperatures, in the bilayer fluid phase, more polymerized membranes are significantly more rigid. Considering that the structure and stability of liposomes at different temperatures can be crucial for DNA binding and delivery, we expect the study presented here contributes to the production of new carrier systems with potential applications in gene therapy. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-kappa B) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream kappa B binding sites in RAW 264.7 macrophage cell lines was repressed when NF-kappa B activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-kappa B subunits. Therefore, transcription of aa-nat driven by NF-kappa B dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-kappa B in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Low-Resolution Molecular Models Reveal the Oligomeric State of the PPAR and the Conformational Organization of Its Domains in Solution

The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPAR gamma and RXR alpha is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPAR gamma alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPAR gamma remains in the monomeric form by itself but forms heterodimers with hRXR alpha. The low-resolution models of hPPAR gamma/RXR alpha complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the full-length receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex.

Neutral Gold Complexes with Tridentate SNS Thiosemicarbazide Ligands

Na[AuCl4].2H(2)O reacts with tridentate thiosemicarbazide ligands, H(2)L1, derived from N-[N',N'-dialkylamino(thiocarbonyl)]benzimidoyl chloride and thiosemicarbazides under formation of air-stable, green [AuCl(L1)] complexes. The organic ligands coordinate in a planar SNS coordination mode. Small amounts of gold(I) complexes of the composition [AuCl(L3)] are formed as side-products, where L3 is an S-bonded 5-diethylamino-3-phenyl-1-thiocarbamoyl-1,2,4-triazole. The formation of the triazole L3 can be explained by the oxidation of H(2)L1 to an intermediate thiatriazine L2 by Au3+, followed by a desulfurization reaction with ring contraction. The chloro ligands in the [AuCl(L1)] complexes can readily be replaced by other monoanionic ligands such as SCN- or CN- giving [Au(SCN)(L1)] or [Au(CN)(L1)] complexes. The complexes described in this paper represent the first examples of fully characterized neutral Gold(III) thiosemicarbazone complexes. All the [AuCl(L1)] compounds present a remarkable cell growth inhibition against human MCF-7 breast cancer cells. However, systematic variation of the alkyl groups in the N(4)-position of the thiosemicarbazone building blocks as well as the replacement of the chloride by thiocyanate ligands do not considerably influence the biological activity. On the other hand, the reduction of Au-III to Au-I leads to a considerable decrease of the cytotoxicity.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-kappa B pathway in 3T3-L1 adipocytes

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-kappa B) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-kappa B DNA binding activity (NF-kappa Bp50 and NF-kappa Bp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-kappa B pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-kappa B (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-kappa B (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-kappa B signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals. (C) 2011 Elsevier Ltd. All rights reserved.

Analysis of Schistosoma mansoni genes shared with Deuterostomia and with possible roles in host interactions

Abstract Background: Schistosoma mansoni is a blood helminth parasite that causes schistosomiasis, a disease that affects 200 million people in the world. Many orthologs of known mammalian genes have been discovered in this parasite and evidence is accumulating that some of these genes encode proteins linked to signaling pathways in the parasite that appear to be involved with growth or development, suggesting a complex co-evolutionary process. Results: In this work we found 427 genes conserved in the Deuterostomia group that have orthologs in S. mansoni and no members in any nematodes and insects so far sequenced. Among these genes we have identified Insulin Induced Gene (INSIG), Interferon Regulatory Factor (IRF) and vasohibin orthologs, known to be involved in mammals in mevalonate metabolism, immune response and angiogenesis control, respectively. We have chosen these three genes for a more detailed characterization, which included extension of their cloned messages to obtain full-length sequences. Interestingly, SmINSIG showed a 10-fold higher expression in adult females as opposed to males, in accordance with its possible role in regulating egg production. SmIRF has a DNA binding domain, a tryptophan-rich N-terminal region and several predicted phosphorylation sites, usually important for IRF activity. Fourteen different alternatively spliced forms of the S. mansoni vasohibin (SmVASL) gene were detected that encode seven different protein isoforms including one with a complete C-terminal end, and other isoforms with shorter C-terminal portions. Using S. mansoni homologs, we have employed a parsimonious rationale to compute the total gene losses/gains in nematodes, arthropods and deuterostomes under either the Coelomata or the Ecdysozoa evolutionary hypotheses; our results show a lower losses/gains number under the latter hypothesis. Conclusion: The genes discussed which are conserved between S. mansoni and deuterostomes, probably have an ancient origin and were lost in Ecdysozoa, being still present in Lophotrochozoa. Given their known functions in Deuterostomia, it is possible that some of them have been co-opted to perform functions related (directly or indirectly) to host adaptation or interaction with host signaling processes.

Cloning and characterization of a novel alternatively spliced transcript of the human CHD7 putative helicase

Abstract Background The CHD7 (Chromodomain Helicase DNA binding protein 7) gene encodes a member of the chromodomain family of ATP-dependent chromatin remodeling enzymes. Mutations in the CHD7 gene are found in individuals with CHARGE, a syndrome characterized by multiple birth malformations in several tissues. CHD7 was identified as a binding partner of PBAF complex (Polybromo and BRG Associated Factor containing complex) playing a central role in the transcriptional reprogramming process associated to the formation of multipotent migratory neural crest, a transient cell population associated with the genesis of various tissues. CHD7 is a large gene containing 38 annotated exons and spanning 200 kb of genomic sequence. Although genes containing such number of exons are expected to have several alternative transcripts, there are very few evidences of alternative transcripts associated to CHD7 to date indicating that alternative splicing associated to this gene is poorly characterized. Findings Here, we report the cloning and characterization by experimental and computational studies of a novel alternative transcript of the human CHD7 (named CHD7 CRA_e), which lacks most of its coding exons. We confirmed by overexpression of CHD7 CRA_e alternative transcript that it is translated into a protein isoform lacking most of the domains displayed by the canonical isoform. Expression of the CHD7 CRA_e transcript was detected in normal liver, in addition to the DU145 human prostate carcinoma cell line from which it was originally isolated. Conclusions Our findings indicate that the splicing event associated to the CHD7 CRA_e alternative transcript is functional. The characterization of the CHD7 CRA_e novel isoform presented here not only sets the basis for more detailed functional studies of this isoform, but, also, contributes to the alternative splicing annotation of the CHD7 gene and the design of future functional studies aimed at the elucidation of the molecular functions of its gene products.

Changes in CREB activation in the prefrontal cortex and hippocampus blunt ethanol-induced behavioral sensitization in adolescent mice

Drug dependence is a major health problem in adults and has been recognized as a significant problem in adolescents. We previously demonstrated that repeated treatment with a behaviorally sensitizing dose of ethanol in adult mice induced tolerance or no sensitization in adolescents and that repeated ethanol-treated adolescents expressed lower Fos and Egr-1 expression than adult mice in the prefrontal cortex (PFC). In the present work, we investigated the effects of acute and repeated ethanol administration on cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) DNA-binding activity using the electrophoretic mobility shift assay (EMSA) and the phosphorylated CREB (pCREB)/CREB ratio using immunoblotting in both the PFC and hippocampus in adolescent and adult mice. Adult mice exhibited typical locomotor sensitization after 15 days of daily treatment with 2.0 g/kg ethanol, whereas adolescent mice did not exhibit sensitization. Overall, adolescent mice displayed lower CREB binding activity in the PFC compared with adult mice, whereas opposite effects were observed in the hippocampus. The present results indicate that ethanol exposure induces significant and differential neuroadaptive changes in CREB DNA-binding activity in the PFC and hippocampus in adolescent mice compared with adult mice. These differential molecular changes may contribute to the blunted ethanol-induced behavioral sensitization observed in adolescent mice.