Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity

Transient Receptor Potential Channel A1 (TRPA1) Regulates Sulfur Mustard-Induced Expression of Heat Shock 70 kDa Protein 6 (HSPA6) In Vitro

The chemosensory transient receptor potential ankyrin 1 (TRPA1) ion channel perceives different sensory stimuli. It also interacts with reactive exogenous compounds including the chemical warfare agent sulfur mustard (SM). Activation of TRPA1 by SM results in elevation of intracellular calcium levels but the cellular consequences are not understood so far. In the present study we analyzed SM-induced and TRPA1-mediated effects in human TRPA1-overexpressing HEK cells (HEKA1) and human lung epithelial cells (A549) that endogenously exhibit TRPA1. The specific TRPA1 inhibitor AP18 was used to distinguish between SM-induced and TRPA1-mediated or TRPA1-independent effects. Cells were exposed to 600 mu M SM and proteome changes were investigated 24 h afterwards by 2D gel electrophoresis. Protein spots with differential staining levels were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano liquid chromatography electrospray ionization tandem mass spectrometry. Results were verified by RT-qPCR experiments in both HEKA1 or A549 cells. Heat shock 70 kDa protein 6 (HSPA6) was identified as an SM-induced and TRPA1-mediated protein. AP18 pre-treatment diminished the up-regulation. RT-qPCR measurements verified these results and further revealed a time-dependent regulation. Our results demonstrate that SM-mediated activation of TRPA1 influences the protein expression and confirm the important role of TRPA1 ion channels in the molecular toxicology of SM

Concerted Actions of a Thermo-labile Regulator and a Unique Intergenic RNA Thermosensor Control Yersinia Virulence

Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein-and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25 degrees C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37 degrees C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency

Temperature-dependent binding of ribosomes to the <i>yscW-lcrF</i> intergenic region.

<p>Toeprinting analysis was performed with the wildtype, the repressed (UU-28/-27CC) and derepressed (GUU-30/-28AAA) variants as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002518#s4" target="_blank">material and methods</a>. The presence (+) and absence (−) of the 30S ribosomal subunits are indicated. The terminated primer extension products (toeprints) are marked. The sequencing ladder (ACGU) generated with the same <i>lcrF</i>-specific primer is loaded on the left. The positions of the fourU motif, the Shine-Dalgarno sequence and the start codon AUG are indicated.</p

<i>lcrF</i> RNA thermometer variants affect survival of <i>Y. pseudotuberculosis</i> infected mice.

<p>(<b>A</b>) 2·10⁹ CFU of <i>Y. pseudotuberculosis</i> YPIII (wildtype), the <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA), and YP66 (Δ<i>lcrF</i>) were used to orally infect BALB/c mice (n = 10/strain). (<b>B</b>) 1·10¹⁰ CFU of <i>Y. pseudotuberculosis</i> IP32953 (wildtype), the <i>yscW-lcrF</i> variants YPIP01 (UU-28/-27CC) and YPIP02 (GUU-30/-28AAA) were used to orally infect BALB/c mice (n = 10/strain). Survival of the mice was monitored up to 14 days.</p

<i>lcrF</i> thermosensor-dependent expression of the <i>yadA</i> and <i>yopE</i> genes.

<p>(<b>A</b>) Strains YPIII (wildtype), YP66 (Δ<i>lcrF</i>) and the repressed and derepressed <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) harboring the <i>yadA-lacZ</i> fusion plasmid pSF1 were grown in LB medium at 25°C or 37°C. β-Galactosidase activity from overnight cultures was determined and is given in µmol min⁻¹ mg⁻¹ for comparison. The data represent the average ± SD from at least three different experiments each done in duplicate. Data were analyzed by the Student's t test. Stars indicate the results that differed significantly from those of the wildtype at the same temperature with ** (P<0.01), and *** (P<0.001). Whole-cell extracts from overnight cultures of <i>Y. pseudotuberculosis</i> YPIII (wildtype) and the repressed and derepressed <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) grown at 25°C or 37°C were prepared, and analysed by Western blotting with a polyclonal antibody directed against LcrF and YadA. A higher molecular weight protein (c) was used as control the protein content of the cell extracts. (<b>B</b>) Strains YPIII (wildtype) and the repressed and derepressed <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) harboring the <i>yopE-luxCDABE</i> plasmid pWO34 were grown in LB medium at 25°C and 37°C. Bioluminescence emitted by the cultures was monitored and is given as relative luminescence units (RLU) and represents the mean of three independent experiments done in triplicate. Data were analyzed by the Student's t test. Stars indicate the results that differed significantly from those of the wildtype at the same temperature with ** (P<0.01), and *** (P<0.001). The panel below shows TCA-precipitated supernatants of YPIII (wildtype), the repressed and derepressed <i>yscW-lcrF</i> variants YP90 (UU-28/-27CC) and YP95 (GUU-30/-28AAA) grown at 25°C and 37°C in the presence (+) or absence (−) of Ca²⁺. The secreted Yop proteins are indicated.</p

The intergenic region of the <i>yscW-lcrF</i> operon is implicated in the temperature control of LcrF production.

<p>(<b>A</b>) Schematic presentation of the reporter gene fusion harboring the <i>yscW-lcrF</i> intergenic region and different portions of <i>yscW</i> under control of the P<i>_BAD</i> promoter. (<b>B</b>) <i>E. coli</i> K-12 harboring the different P<i>_BAD</i>::<i>yscW-lcrF-lacZ</i> reporter plasmids (pED10, pED11 and pKB14) or the P<i>_BAD</i>::<i>gnd-lacZ</i> control plasmid (pED05) were grown overnight in LB medium at 25°C or 37°C in the presence of 0.05% arabinose. β-Galactosidase activity from overnight cultures was determined and is given in mmol min⁻¹ mg⁻¹ for comparison. The data represent the average ± SD from at least three different experiments each done in duplicate. Data were analyzed by the Student's t test. Stars indicate the reporter activity that differed significantly between 25°C and 37°C with ** (P<0.01), and *** (P<0.001).</p

Expression of the <i>yscW-lcrF</i> operon in response to temperature.

<p>(<b>A</b>) Schematic presentation of the <i>yscW-lacZ</i> and <i>yscW-lcrF-lacZ</i> fusion plasmids. Numbers given in brackets represent the nucleotide positions of the 5′-end of the <i>yscW</i> regulatory region of the fusion constructs with respect to the start codon of <i>yscW</i>. The <i>yscW</i> gene is indicated in grey, the 5′-portion of the <i>lcrF</i> gene is given in black and the <i>lacZ</i> reporter gene is illustrated by a white arrow. (<b>B</b>) Strains YPIII and YP50 (Δ<i>ymoA</i>) harboring the <i>yscW-lacZ</i> (pKB10) or the <i>yscW-lcrF-lacZ</i> (pSF3 and pSF4) fusion plasmids ± pAKH71 (<i>ymoA</i>⁺) were grown overnight in LB medium at 25°C or 37°C. β-Galactosidase activity from overnight cultures was determined and is given in µmol min⁻¹ mg⁻¹ for comparison. The data represent the average ± SD from at least three different experiments each done in duplicate. Data were analyzed by the Student's t test. Stars indicate the results that differed significantly from those of YPIII at the same temperature with ** (P<0.01), and *** (P<0.001). The activity of all reporter constructs differed significantly between 25°C and 37°C with P<0.001 (not shown). (<b>C</b>) Whole-cell extracts from overnight cultures of <i>Y. pseudotuberculosis</i> wild type and the mutant strains YP66 (Δ<i>lcrF</i>) and YP50 (Δ<i>ymoA</i>) grown at 25°C or 37°C were prepared and analysed by Western blotting with a polyclonal antibody directed against LcrF. A molecular weight marker is loaded on the left. A higher molecular weight protein (c) that reacted with the polyclonal antiserum was used as loading control.</p

Analysis of the <i>yscW-lcrF</i> mRNA in wildtype and the <i>ymoA</i> mutant strain.

<p>(<b>A</b>) Schematic presentation of the <i>yscW-lcrF</i> operon, the <i>yscW-lcrF</i> mRNA and the <i>lcrF</i> probe used for Northern Blot analysis shown below. (<b>B</b>) Total RNA of YPIII, YP50 and YP66 was prepared, separated on a 1.2% agarose gel, transferred onto a Nylon membrane and probed with Digoxigenin-labelled PCR fragment encoding the <i>lcrF</i> gene. The 16S and 23S rRNAs are shown as RNA loading control. A RNA marker is loaded on the left.</p