Crosstalk between reactive oxygen species and pro-inflammatory markers in developing various chronic diseases: a review

The inflammation process in the human body plays a central role in the pathogenesis of many chronic diseases. In addition, reactive oxygen species (ROS) exert potentially a decisive role in human body, particularly in physiological and pathological process. The chronic inflammation state could generate several types of diseases such as cancer, atherosclerosis, diabetes mellitus and arthritis, especially if it is concomitant with high levels of pro-inflammatory markers and ROS. The respiratory burst of inflammatory cells during inflammation increases the production and accumulation of ROS. However, ROS regulate various types of kinases and transcription factors such nuclear factor-kappa B which is related to the activation of pro-inflammatory genes. The exact crosstalk between pro-inflammatory markers and ROS in terms of pathogenesis and development of serious diseases is still ambitious. Many studies have been attempting to determine the mechanistic mutual relationship between ROS and pro-inflammatory markers. Therefore hereby, we review the hypothetical relationship between ROS and pro-inflammatory markers in which they have been proposed to initiate cancer, atherosclerosis, diabetes mellitus and arthritis

The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σ^V Activation

<div><p>σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF) σ factor, σ^V, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP). In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of σ^V. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and σ^V activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for σ^V activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits σ^V activity in the absence of lysozyme, RsiV binds lysozyme triggering σ^V activation and RsiV inhibits the enzymatic activity of lysozyme.</p></div

Plasmid list.

<p>Plasmid list.</p

RsiV mutants that cannot bind lysozyme are less efficient at inhibiting lysozyme muramidase activity.

<p>Peptidoglycan from <i>M</i>. <i>lysodekticus</i> was combined with lysozyme (20 μg/ ml) and purified RsiV or RsiV mutants at a molar ratio of 1:1 with lysozyme. The OD₄₅₀ was monitored every minute for 30 minutes to determine lysozyme specific activity as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006287#pgen.1006287.ref041" target="_blank">41</a>].</p

Strain list.

<p>Strain list.</p

RsiV binding to lysozyme is required for σ^V activation.

<p><b>A.</b> The effect of RsiV mutants on σ^V activation using a P_<i>sigV</i><i>-lacZ</i> reporter assay. <i>B</i>. <i>subtilis</i> strains CDE1546 (WT), JLH1473 (S169W, P259A, Y261A), JLH1474 (S169W), JLH1477 (P259A, Y261A), JLH1527 (P259A), JLH1536 (S169W, P259A), JLH1476 (Y261A) and JLH1538 (S169W, Y261A) were grown to mid log and then 20 μl were spotted on LB plates with various concentrations of lysozyme (0, 2, 5, 10, 20 μg/ml). Plates were incubated 37°C for 6 hours and then β-galactosidase assays were performed. <b>B.</b> Immunoblot analysis of RsiV mutant protein levels. An aliquot of 1 ml was taken from each strain before spotting. The aliquot was pelleted and resuspended in 50 μl sample buffer. Samples were immunoblotted with anti-RsiV^59-285 and expression levels were compared using the Li-Cor software ImageStudio.</p

RsiV provides lysozyme resistance <i>in vivo</i>.

<p>RsiV provides lysozyme resistance <i>in vivo</i>.</p

RsiV binding to lysozyme is required for site-1 cleavage of RsiV.

<p>Site-1 cleavage of RsiV mutants in response to increasing concentration of lysozyme. Overnight <i>B</i>. <i>subtilis</i> strains were grown to OD₆₀₀ of 1 in LB IPTG. Strains were divided into 1.5 ml aliquots and incubated with increasing concentrations of lysozyme (0, 0.01, 0.1, 1, and 2 μg/mL) for 10 minutes. Following lysozyme exposure, cells were pelleted, resuspended in 100 μl sample buffer, and immunoblotted with anti-RsiV and anti-σ^A. The blots are labelled on the right with either α-RsiV^59-285 or α-σ^A. The FL arrow denotes full length RsiV. The C arrow denotes cleaved extracellular domain of RsiV. WT = JLH402; S169W P259A Y261A refers to JLH1312; S169W refers to JLH1271; P259A Y261A refers to JLH1343; P259A refers to JLH1481; S169W P259A refers to JLH1326; Y261A refers to JLH1342; S169W Y261A refers to JLH1504.</p

Crystallography statistics.

<p>Crystallography statistics.</p