Proteomic changes occurring in the malaria mosquitoes Anopheles gambiae and Anopheles stephensi during aging.

The age of mosquitoes is a crucial determinant of their ability to transmit pathogens and their resistance to insecticides. We investigated changes to the abundance of proteins found in heads and thoraces of the malaria mosquitoes Anopheles gambiae and Anopheles stephensi as they aged. Protein expression changes were assessed using two-dimensional difference gel electrophoresis and the identity of differentially expressed proteins was determined by using either matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry or capillary high-pressure liquid chromatography coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometer. Protein biomarkers were validated by semi quantitative Western blot analysis. Nineteen and nine age dependent protein spots were identified for A. stephensi and A. gambiae, respectively. Among the proteins down-regulated with age were homologs of ADF/Cofilin, cytochome c1, heat shock protein-70 and eukaryotic translation initiation factor 5A (eIF5a). Proteins up-regulated with age included probable methylmalonate-semialdehyde dehydrogenase, voltage-dependent anion-selective channel and fructose bisphosphate aldolase. Semi quantitative Western blot analysis confirmed expression patterns observed by 2-D DIGE for eIF5a and ADF/Cofilin. Further work is recommended to determine whether these biomarkers are robust to infection, blood feeding and insecticide resistance. Robust biomarkers could then be incorporated into rapid diagnostic assays for ecological and epidemiological studies. BIOLOGICAL SIGNIFICANCE: In this study, we have identified several proteins with characteristic changes in abundance in both A. gambiae and A. stephensi during their aging process. These changes may highlight underlying mechanisms beneath the relationship between mosquito age and factors affecting Plasmodium transmission and mosquito control. The similarity of changes in protein abundance between these species and the primary dengue vector Aedes aegypti, has revealed conserved patterns of aging-specific protein regulation.

Proteomic biomarkers for ageing the mosquito Aedes aegypti to determine risk of pathogen transmission.

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.

Mutation of the elongin C binding domain of human respiratory syncytial virus non-structural protein 1 (NS1) results in degradation of NS1 and attenuation of the virus.

BACKGROUND: Human respiratory syncytial virus (RSV) is an important cause of lower respiratory tract disease in the paediatic population, immunocompromised individuals and the elderly worldwide. However, despite global efforts over the past several decades there are no commercially available vaccines. RSV encodes 2 non-structural proteins, NS1 and NS2, that are type I interferon antagonists. RSV restricts type I interferon signaling and the expression of antiviral genes by degrading STAT2. It has been proposed that NS1 binds to elongin C to form a ubiquitin ligase (E3) complex that targets STAT2 for ubiquitination and proteosomal degradation. RESULTS: Here, we have engineered a live recombinant RSV in which the 3 consensus amino acids of the NS1 elongin C binding domain have been replaced with alanine (NS1F-ELCmut). Mutation of this region of NS1 resulted in attenuation of RSV replication in A549 cells to levels similar to that observed when the NS1 gene is completely deleted (ΔNS1). This mutation also resulted in moderate attenuation in Vero cells. Attenuation was correlated to intracellular degradation of the mutated NS1 protein. Time course analysis showed that mutant NS1 protein accumulated in cytoplasmic bodies that contained the lysosomal marker LAMP1. However lack of cleavage of LC3 suggested that autophagy was not involved. Induction of IFN-ß mRNA expression also was observed in association with the degradation of NS1 protein and attenuation of viral growth. CONCLUSIONS: These results indicate that the elongin C binding region of NS1 is crucial for survival of the protein and that disruption of this region results in the degradation of NS1 and restriction of RSV replication.

Identification of a dominant self-ligand bound to three HLA B44 alleles and the preliminary crystallographic analysis of recombinant forms of each complex.

A naturally processed and presented ligand that is shared by human leukocyte antigen (HLA) B*4402, B*4403 and B*4405 molecules has been identified in peptides isolated from immunoaffinity purified HLA B44 complexes. This peptide derived from HLA DPalpha residues 46-54, an endogenous product of HLA DP expressed in the cell line Hmy2.C1R, is a prominent peptide in the mass spectra of species isolated as bound peptides from each allele when the three HLA B44 subtypes were introduced as transfected gene products. Recombinant truncated forms of HLA B*4405(1-276), HLA B*4403(1-276), HLA B*4402(1-276) and beta(2)-microglobulin have been prepared as inclusion bodies in Escherichia coli and refolded in the presence of the DPalpha(46-54) peptide and purified by a combination of size exclusion and anion exchange chromatography. This material was determined to be correctly folded based on detection of a conformational epitope recognized by the W6/32 monoclonal antibody. Large, plate-like crystals of the three complexes were produced using polyethylene glycol as the precipitant. All the crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of approximately a=51, b=82, c=110 A. The crystals of three B44/DPalpha complexes diffracted to a resolution of 1.9 A or better. For the first time, using this natural, high abundance ligand of the HLA B44 molecules we have successfully expressed and refolded the three HLA B44 molecules and produced crystals amenable to structural studies.