Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets.

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BSTRACTFactor inhibiting HIF (FIH, also known as HIF1AN) is an oxygendependent asparaginyl hydroxylase that regulates the hypoxiainducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.

Background: Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Results: Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Conclusion: Matrilin-4, collagen XII, thrombospondin-4, fibronectin, ␤ig-h3, and epiphycan are components of the in vivo collagen IX interactome. Significance: We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage.

Biomarkers of the age of mosquitoes are required to determine the risk of transmission of various pathogens as each pathogen undergoes a period of extrinsic incubation in the mosquito host. Using the 2-D Difference Gel Electrophoresis (2-D DIGE) procedure, we investigated the abundance of up to 898 proteins from the Yellow Fever and dengue virus vector, Aedes aegypti, during ageing. By applying a mixed-effects model of protein expression, we identified five common patterns of abundance change during ageing and demonstrated an age-related decrease in variance for four of these. This supported a search for specific proteins with abundance changes that remain tightly associated with ageing for use as ageing biomarkers. Using MALDI-TOF/TOF mass spectrometry we identified ten candidate proteins that satisfied strict biomarker discovery criteria (identified in two out of three multivariate analysis procedures and in two cohorts of mosquitoes). We validated the abundances of the four most suitable candidates (Actin depolymerising factor; ADF, Eukaryotic initiation factor 5A; eIF5A, insect cuticle protein Q17LN8, and Anterior fat body protein; AFP) using semi-quantitative Western analysis of individual mosquitoes of six ages. The redox-response protein Manganese superoxide dismutase (SOD2) and electron shuttling protein Electron transfer oxidoreductase (ETO) were subject to post-translational modifications affecting their charge states with potential effects on function. For the four candidates we show remarkably consistent decreases in abundance during ageing, validating initial selections. In particular, the abundance of AFP is an ideal biomarker candidate for whether a female mosquito has lived long enough to be capable of dengue virus transmission. We have demonstrated proteins to be a suitable class of ageing biomarkers in mosquitoes and have identified candidates for epidemiological studies of dengue and the evaluation of new disease reduction projects targeting mosquito longevity.