Toxin–antitoxin regulation: bimodal interaction of YefM–YoeB with paired DNA palindromes exerts transcriptional autorepression

Toxin–antitoxin (TA) complexes function in programmed cell death or stress response mechanisms in bacteria. The YefM–YoeB TA complex of Escherichia coli consists of YoeB toxin that is counteracted by YefM antitoxin. When liberated from the complex, YoeB acts as an endoribonuclease, preferentially cleaving 3′ of purine nucleotides. Here we demonstrate that yefM-yoeB is transcriptionally autoregulated. YefM, a dimeric protein with extensive secondary structure revealed by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, is the primary repressor, whereas YoeB is a repression enhancer. The operator site 5′ of yefM-yoeB comprises adjacent long and short palindromes with core 5′-TGTACA-3′ motifs. YefM binds the long palindrome, followed sequentially by short palindrome recognition. In contrast, the repressor–corepressor complex recognizes both motifs more avidly, impyling that YefM within the complex has an enhanced DNA-binding affinity compared to free YefM. Operator interaction by YefM and YefM–YoeB is accompanied by structural transitions in the proteins. Paired 5′-TGTACA-3′ motifs are common in yefM-yoeB regulatory regions in diverse genomes suggesting that interaction of YefM–YoeB with these motifs is a conserved mechanism of operon autoregulation. Artificial perturbation of transcriptional autorepression could elicit inappropriate YoeB toxin production and induction of bacterial cell suicide, a potentially novel antibacterial strategy

High resolution 13C and 1H NMR studies of proteins and peptides

At the magnetic field used in this research (9.4T) many individual ¹³C resonances of protonated carbon atoms are resolved. To test the applicability of the high-resolution natural abundance ¹³C NMR method, lysozyme and ribonuclease A are used. The research includes the assignment of many methyl resonances and attempts to answer questions on the behaviour of these side-chains when the proteins are subjected to varying conditions of pD and temperature, and also in the presence of ligands, inhibitors and urea. Ribonuclease A is observed to undergo conformational changes on variation of pD. The region(s) of this protein which are involved consist of many hydrophobic residues such as ile, val and met. Many denaturation processes are effectively in slow exchange at the high magnetic field used. The individual resonances of ribonuclease A which are involved in the predenaturation transitions are identified. They are found to be of distinct types for the two denaturants. In contrast, lysozyme does not show such distinct conformational behaviour below its denaturation temperature. At higher temperatures the main transition from the 'native' to the denatured state of both proteins is two-state. None of the unfolded states of ribonuclease A and lysozyme are random coil. Rather, the unfolded states have definable structures which have hydrophobic bondings and significant, but still restrained, internal flexibility. The high-resolution spectra of ribonuclease A-inhibitor complexes have revealed unprecedented details of the structures of these complexes. Extensive structural changes, including the closure of the active site cleft, together with movements in the hydrophobic regions bordering the cleft, are deduced from the inhibitor-induced shift perturbations of the ¹³C and ¹H NMR resonances of the protein residues. In the assignment work, and the urea denaturation and ligand binding studies, high-resolution ¹H NMR is used to complement the ¹³C NMR technique. A theory relating ¹³C NMR relaxation parameters to molecular motion at three levels (3—τ) is tested with gramicidin-S and glutathione dimer. The results obtained give information about the conformations and rates of internal librational motions of the two peptides. The '3-τ' model is also applied to some more-limited data on proteins. The low nuclear Overhauser effects are partially explained by this theory

Interaction of the Oncofetal Thomsen-Friedenreich Antigen with Galectins in Cancer Progression and Metastasis

Aberrant glycosylation of cell membrane proteins is a universal feature of cancer cells. One of the most common glycosylation changes in epithelial cancer is the increased occurrence of the oncofetal Thomsen-Friedenreich disaccharide Galβ1-3GalNAc (T or TF antigen), which appears in about 90% of cancers but is rarely seen in normal epithelium. Over the past few years, increasing evidence has revealed that the increased appearance of TF antigen on cancer cell surface plays an active role in promoting cancer progression and metastasis by interaction with the β-galactoside-binding proteins, galectins, which themselves are also frequently overexpressed in cancer and pre-cancerous conditions. This review summarizes the current understanding of the molecular mechanism of the increased TF occurrence in cancer, the structural nature and biological impact of TF interaction with galectins, in particular galectin-1 and -3, on cancer progression and metastasis

A New Race (X12) of Soybean Cyst Nematode in China

The soybean cyst nematode (SCN), Heterodera glycines, is a serious economic threat to soybean-producing regions worldwide. A new SCN population (called race X12) was detected in Shanxi province, China. Race X12 could reproduce on all the indicator lines of both race and Heterodera glycines (HG) type tests. The average number of females on Lee68 (susceptible control) was 171.40 with the lowest Female Index (FI) 61.31 on PI88788 and the highest FI 117.32 on Pickett in the race test. The average number of females on Lee68 was 323.17 with the lowest FI 44.18 on PI88788 and the highest FI 97.83 on PI548316 in the HG type test. ZDD2315 and ZDD24656 are elite resistant germplasms in China. ZDD2315 is highly resistant to race 4, the strongest infection race in the 16 races with FI 1.51 while being highly sensitive to race X12 with FI 64.32. ZDD24656, a variety derived from PI437654 and ZDD2315, is highly resistant to race 1 and race 2. ZDD24656 is highly sensitive to race X12 with FI 99.12. Morphological and molecular studies of J2 and cysts confirmed the population as the SCN H. glycines. This is a new SCN race with stronger virulence than that of race 4 and is a potential threat to soybean production in China

Characterisation of the Interaction of the C-Terminus of the Dopamine D2 Receptor with Neuronal Calcium Sensor-1

NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca2+ binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca2+ channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca2+ and D2 peptide. The data show that NCS-1 binds D2 peptide with a Kd of ∼14.3 µM and stoichiometry of peptide binding to NCS-1 of 2∶1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer

Glycerol: An unexpected major metabolite of energy metabolism by the human malaria parasite

<p>Abstract</p> <p>Background</p> <p>Malaria is a global health emergency, and yet our understanding of the energy metabolism of the principle causative agent of this devastating disease, <it>Plasmodium falciparum</it>, remains rather basic. Glucose was shown to be an essential nutritional requirement nearly 100 years ago and since this original observation, much of the current knowledge of <it>Plasmodium </it>energy metabolism is based on early biochemical work, performed using basic analytical techniques (e.g. paper chromatography), carried out almost exclusively on avian and rodent malaria. Data derived from malaria parasite genome and transcriptome studies suggest that the energy metabolism of the parasite may be more complex than hitherto anticipated. This study was undertaken in order to further characterize the fate of glucose catabolism in the human malaria parasite, <it>P. falciparum</it>.</p> <p>Methods</p> <p>Products of glucose catabolism were determined by incubating erythrocyte-freed parasites with D-[1-¹³C] glucose under controlled conditions and metabolites were identified using ¹³C-NMR spectroscopy.</p> <p>Results</p> <p>Following a 2 h incubation of freed-<it>P. falciparum </it>parasites with 25 mM D-[1-¹³C] glucose (<it>n </it>= 4), the major metabolites identified included; [3-¹³C] lactate, [1,3-¹³C] glycerol, [3-¹³C] pyruvate, [3-¹³C] alanine and [3-¹³C] glycerol-3-phosphate. Control experiments performed with uninfected erythrocytes incubated under identical conditions did not show any metabolism of D-[1-¹³C] glucose to glycerol or glycerol-3-phosphate.</p> <p>Discussion</p> <p>The identification of glycerol as a major glucose metabolite confirms the view that energy metabolism in this parasite is more complex than previously proposed. It is hypothesized here that glycerol production by the malaria parasite is the result of a metabolic adaptation to growth in O₂-limited (and CO₂elevated) conditions by the operation of a glycerol-3-phosphate shuttle for the re-oxidation of assimilatory NADH. Similar metabolic adaptations have been reported previously for other microaerobic/anaerobic organisms, such as yeast, rumen protozoa and human parasitic protozoa.</p> <p>Conclusion</p> <p>These data highlight the need to re-evaluate the carbon and redox balance of this important human pathogen, ultimately leading to a better understanding of how the parasite is able to adapt to the variable environments encountered during parasite development and disease progression.</p

Intrinsic tryptophan fluorescence spectroscopy reliably determines galectin-ligand interactions

Galectins are involved in the regulation of divergent physiological and pathological processes and are increasingly recognized to play important roles in a number of diseases. However, a simple and effective way in assessing galectin-ligand interactions is lacking. Our examination of the sequence of all 12 human galectin members reveals the presence of one or more tryptophan residues in the carbohydrate-recognition domains of each galectin. This led us to investigate the possibility that alteration of the galectin intrinsic tryptophan fluorescence could be used in determining the strength of galectin-ligand interactions. One representative member from each of the three subtype galectins, galectin-2 (proto-), galectin-3 (chimera-) and galectin-4 (tandem repeat-type), was selected and analysed for galectin interaction with three ligands of different affinities: galactose, lactose and N-acetyl-lactosamine using tryptophan fluorescence spectroscopy (TFS) and, as a comparison, isothermal titration calorimetry (ITC). Good agreement between TFS and ITC measurements were revealed in ligand bindings of all galectin members. Moreover, TFS detected very weak galectin binding where ITC could not reliably do so. The reliability of TFS in determining galectin-ligand interactions was further validated by analysis of galectin-3 interaction with a semisynthetic ligand, F3. Thus, TFS can be used as a simple, sensitive and reliable way to determine galectin-ligand interactions and also as a drug-discovery platform in developing galectin-targeted therapeutic drugs