The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release

NADPH oxidase (Nox) family proteins produce superoxide (O2.-) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2.- intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2.- dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2.- leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2.- even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2.- in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2.- leakage; chimeric Duox1 possessing the A-loop of Duox2 released O2.-. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2.- release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2.- leakage. Although we identified Duox1 A-loop residues (His1071, His1072, and Gly1074) important for reducing O2.- release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2.- releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex. © 2015, American Society for Biochemistry and Molecular Biology Inc

P67-phox (NCF2) Lacking Exons 11 and 12 Is Functionally Active and Leads to an Extremely Late Diagnosis of Chronic Granulomatous Disease (CGD)

Two brothers in their fifties presented with a medical history of suspected fungal allergy, allergic bronchopulmonary aspergillosis, alveolitis, and invasive aspergillosis and pulmonary fistula, respectively. Eventually, after a delay of 50 years, chronic granulomatous disease (CGD) was diagnosed in the index patient. We found a new splice mutation in the NCF2 (p67-phox) gene, c.1000+2T→G, that led to several splice products one of which lacked exons 11 and 12. This deletion was in frame and allowed for remarkable residual NADPH oxidase activity as determined by transduction experiments using a retroviral vector. We conclude that p67-phox which lacks the 34 amino acids encoded by the two exons can still exert considerable functional activity. This activity can partially explain the long-term survival of the patients without adequate diagnosis and treatment, but could not prevent progressing lung damage

A change in the optical polarization associated with a gamma-ray flare in the blazar 3C 279

It is widely accepted that strong and variable radiation detected over all accessible energy bands in a number of active galaxies arises from a relativistic, Doppler-boosted jet pointing close to our line of sight. The size of the emitting zone and the location of this region relative to the central supermassive black hole are, however, poorly known, with estimates ranging from light-hours to a light-year or more. Here we report the coincidence of a gamma-ray flare with a dramatic change of optical polarization angle. This provides evidence for co-spatiality of optical and gamma-ray emission regions and indicates a highly ordered jet magnetic field. The results also require a non-axisymmetric structure of the emission zone, implying a curved trajectory for the emitting material within the jet, with the dissipation region located at a considerable distance from the black hole, at about 10^5 gravitational radii.Comment: Published in Nature issued on 18 February 2010. Corresponding authors: Masaaki Hayashida and Greg Madejsk

Comparative tissue transcriptomics reveal prompt inter-organ communication in response to local bacterial kidney infection

<p>Abstract</p> <p>Background</p> <p>Mucosal infections elicit inflammatory responses via regulated signaling pathways. Infection outcome depends strongly on early events occurring immediately when bacteria start interacting with cells in the mucosal membrane. Hitherto reported transcription profiles on host-pathogen interactions are strongly biased towards <it>in vitro </it>studies. To detail the local <it>in vivo </it>genetic response to infection, we here profiled host gene expression in a recent experimental model that assures high spatial and temporal control of uropathogenic <it>Escherichia coli </it>(UPEC) infection within the kidney of a live rat.</p> <p>Results</p> <p>Transcriptional profiling of tissue biopsies from UPEC-infected kidney tissue revealed 59 differentially expressed genes 8 h post-infection. Their relevance for the infection process was supported by a Gene Ontology (GO) analysis. Early differential expression at 3 h and 5 h post-infection was of low statistical significance, which correlated to the low degree of infection. Comparative transcriptomics analysis of the 8 h data set and online available studies of early local infection and inflammation defined a core of 80 genes constituting a "General tissue response to early local bacterial infections". Among these, 25% were annotated as interferon-γ (IFN-γ) regulated. Subsequent experimental analyses confirmed a systemic increase of IFN-γ in rats with an ongoing local kidney infection, correlating to splenic, rather than renal <it>Ifng </it>induction and suggested this inter-organ communication to be mediated by interleukin (IL)-23. The use of comparative transcriptomics allowed expansion of the statistical data handling, whereby relevant data could also be extracted from the 5 h data set. Out of the 31 differentially expressed core genes, some represented specific 5 h responses, illustrating the value of comparative transcriptomics when studying the dynamic nature of gene regulation in response to infections.</p> <p>Conclusion</p> <p>Our hypothesis-free approach identified components of infection-associated multi-cellular tissue responses and demonstrated how a comparative analysis allows retrieval of relevant information from lower-quality data sets. The data further define marked representation of IFN-γ responsive genes and a prompt inter-organ communication as a hallmark of an early local tissue response to infection.</p

The Type I NADH Dehydrogenase of Mycobacterium tuberculosis Counters Phagosomal NOX2 Activity to Inhibit TNF-α-Mediated Host Cell Apoptosis

The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb) to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbΔnuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-α-neutralizing antibodies, and also after infection of murine TNF−/− macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS) reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-α secretion. The MtbΔnuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbΔnuoG induced ROS and apoptosis was abolished in NOX-2 deficient (gp91−/−) macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-α-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-α signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense

Chronic Granulomatous Disease; fundamental stages in our understanding of CGD

It has been 50 years since chronic granulomatous disease was first reported as a disease which fatally affected the ability of children to survive infections. Various milestone discoveries from the insufficient ability of patients' leucocytes to destroy microbial particles to the underlying genetic predispositions through which the disease is inherited have had important consequences. Longterm antibiotic prophylaxis has helped to fight infections associated with chronic granulomatous disease while the steady progress in bone marrow transplantation and the prospect of gene therapy are hailed as long awaited permanent treatment options. This review unearths the important findings by scientists that have led to our current understanding of the disease

Oxidative protein labeling in mass-spectrometry-based proteomics

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade