The complement system, by virtue of its dual effector and priming functions, is a major host defense against pathogens. Flavivirus nonstructural protein (NS)-1 has been speculated to have immune evasion activity, because it is a secreted glycoprotein, binds back to cell surfaces, and accumulates to high levels in the serum of infected patients. Herein, we demonstrate an immunomodulatory function of West Nile virus NS1. Soluble and cell-surface-associated NS1 binds to and recruits the complement regulatory protein factor H, resulting in decreased complement activation in solution and attenuated deposition of C3 fragments and C5b-9 membrane attack complexes on cell surfaces. Accordingly, extracellular NS1 may function to minimize immune system targeting of West Nile virus by decreasing complement recognition of infected cells.flavivirus ͉ immune evasion ͉ innate immunity ͉ pathogenesis

BackgroundIdeally, disease modifying therapies for Alzheimer disease (AD) will be applied during the ‘preclinical’ stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome.Methods and FindingsCSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85–0.94 95% confidence interval [CI]) and 0.88 (0.81–0.94 CI), respectively.ConclusionsFour novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions.

The Haemophilus influenzae HMW1 adhesin mediates adherence to respiratory epithelial cells, a critical early step in the pathogenesis of H. influenzae disease. In recent work, we demonstrated that HMW1 undergoes glycosylation. In addition, we observed that glycosylation of HMW1 is essential for HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence to host epithelium. In this study, we examined HMW1 proteolytic fragments by mass spectrometry, achieved 89% amino acid sequence coverage, and identified 31 novel modification sites. All of the modified sites were asparagine residues, in all but one case in the conventional consensus sequence of N-linked glycans, viz. NX(S/T). Liquid chromatography-tandem mass spectrometry analysis using a hybrid linear quadrupole ion trap Fourier transform ion cyclotron mass spectrometer, accurate mass measurements, and deuterium exchange studies established that the modifying glycan structures were mono-or dihexoses rather than the N-acetylated chitobiosyl core that is characteristic of N-glycosylation. This unusual carbohydrate modification suggests that HMW1 glycosylation requires a glycosyltransferase with a novel activity.Until recently, glycosylation of proteins was believed to be limited to eukaryotes. However, glycoproteins are now being recognized increasingly in prokaryotes as well, including a variety of pathogenic bacteria. Examples of bacterial glycoproteins include the flagellins of Pseudomonas aeruginosa, Campylobacter jejuni, and Campylobacter coli; the type IV pili of P. aeruginosa, Neisseria gonorrhoeae, and Neisseria meningitidis; and non-pilus adhesins in Chlamydia trachomatis, enterotoxigenic Escherichia coli, and diffusely adhering E. coli (1-9).Nonencapsulated (nontypeable) strains of Haemophilus influenzae are a common cause of human respiratory tract disease and initiate infection by colonizing the upper respiratory tract. Approximately 75-80% of isolates express two related high molecular weight proteins called HMW1 and HMW2 that promote efficient adherence to respiratory epithelial cells and facilitate the process of colonization (10, 11). The HMW1 and HMW2 adhesins are encoded by highly homologous chromosomal loci that appear to represent a gene duplication event and contain three genes, designated hmw1A, hmw1B, and hmw1C and hmw2A, hmw2B, and hmw2C, respectively (12, 13). HMW1 and HMW2 are synthesized as preproproteins and are secreted by the two-partner secretion system (14 -16). Amino acids 1-68 direct the preproproteins to the Sec apparatus, where they are cleaved by signal peptidase I (15). Subsequently, amino acids 69 -441 target the proproteins to the outer membrane and interact with the HMW1B or HMW2B outer membrane translocator protein, undergoing removal by an unknown process (15, 16 -18). Following translocation across the outer membrane, the mature HMW1 and HMW2 proteins remain noncovalently associated with the bacterial surface, with small amounts released into the culture supernatant (15,16).In recent work, we est...

Standardized, comprehensive platforms for the discovery of protease substrates have been extremely difficult to create. Screens for protease specificity are now frequently based on the cleavage patterns of peptide substrates, which contain small recognition motifs that are required for the cleavage of the scissile bond within an active site. However, these studies do not identify in vivo substrates, nor can they lead to the definition of the macromolecular features that account for the biological specificity of proteases. To use properly folded proteins in a proteomic screen for protease substrates, we used 2D difference gel electrophoresis and tandem MS to identify substrates of an apoptosis-inducing protease, granzyme B. We confirmed the cleavage of procaspase-3, one of the key substrates of this enzyme, and identified several substrates that were previously unknown, as well as the cleavage site for one of these substrates. We were also able to observe the kinetics of substrate cleavage and cleavage product accumulation by using the 2D difference gel electrophoresis methodology. ''Protease proteomics'' may therefore represent an important tool for the discovery of the native substrates of a variety of proteases.

BackgroundBiomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimer's disease. Cerebrospinal fluid (CSF) is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance) among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF) and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) for CSF biomarker discovery by (1) identifying reparable sources of technical variability, (2) assessing subject variance and residual technical variability for numerous CSF proteins, and (3) testing its ability to segregate samples on the basis of desired biomarker characteristics.Methods/ResultsFourteen aliquots of pooled CSF and two aliquots from six cognitively normal individuals were randomized, enriched for low-abundance proteins by MAF, digested endoproteolytically, randomized again, and analyzed by nano-LC-MS. Nano-LC-MS data were time and m/z aligned across samples for relative peptide quantification. Among 11,433 aligned charge groups, 1360 relatively abundant ones were annotated by MS2, yielding 823 unique peptides. Analyses, including Pearson correlations of annotated LC-MS ion chromatograms, performed for all pairwise sample comparisons, identified several sources of technical variability: i) incomplete MAF and keratins; ii) globally- or segmentally-decreased ion current in isolated LC-MS analyses; and iii) oxidized methionine-containing peptides. Exclusion of these sources yielded 609 peptides representing 81 proteins. Most of these proteins showed very low coefficients of variation (CV<5%) whether they were quantified from the mean of all or only the 2 most-abundant peptides. Unsupervised clustering, using only 24 proteins selected for high subject variance, yielded perfect segregation of pooled and individual samples.ConclusionsQuantitative label-free LC-MS/MS can measure scores of CSF proteins with low technical variability and can segregate samples according to desired criteria. Thus, this technique shows potential for biomarker discovery for neurological diseases.