HIV-1 Activates T Cell Signaling Independently of Antigen to Drive Viral Spread.

HIV-1 spreads between CD4 T cells most efficiently through virus-induced cell-cell contacts. To test whether this process potentiates viral spread by activating signaling pathways, we developed an approach to analyze the phosphoproteome in infected and uninfected mixed-population T cells using differential metabolic labeling and mass spectrometry. We discovered HIV-1-induced activation of signaling networks during viral spread encompassing over 200 cellular proteins. Strikingly, pathways downstream of the T cell receptor were the most significantly activated, despite the absence of canonical antigen-dependent stimulation. The importance of this pathway was demonstrated by the depletion of proteins, and we show that HIV-1 Env-mediated cell-cell contact, the T cell receptor, and the Src kinase Lck were essential for signaling-dependent enhancement of viral dissemination. This study demonstrates that manipulation of signaling at immune cell contacts by HIV-1 is essential for promoting virus replication and defines a paradigm for antigen-independent T cell signaling.

Proteome changes induced by laser in diabetic retinopathy.

BACKGROUND: Diabetic macular oedema (DMO) is the commonest cause of vision loss in people with diabetes. Laser photocoagulation can be effective in the treatment of DMO; however, its mechanism of action is still poorly understood. A better understanding of these mechanisms may allow the development of therapeutic approaches that could avoid the deleterious adverse events associated with photocoagulation. METHODS: We have used proteomic techniques to identify the protein changes induced by threshold intensity retinal laser treatment in a rodent model of diabetic retinopathy. Retinae were obtained from diabetic Dark Agouti rats 8 weeks following laser treatment. Extracted proteins from lasered and non-lasered diabetic retinae were separated and compared using two-dimensional gel electrophoresis. RESULTS: Image analysis revealed 24 protein spots with decreased expression after laser treatment and 9 spots with increased expression. On lasered retinal gels, four spots were uniquely expressed, with eight unique spots on non-lasered gels. Twenty-two protein spots of interest were identified using matrix-assisted desorption ionization-mass spectrometry with database matching. Following laser, Wnt-5 beta, LEK-1, GADPH, claudin-12 and calretinin were significantly down-regulated in expression. CONCLUSIONS: The present study provides a proteomic insight into the underlying biological basis for the therapeutic effects of laser for DMO. We provide further evidence of the involvement of Wnt pathway signalling in the neural retina in DMO, and for up to 2 months following laser treatment. Changes in LEK-1 and claudin-12 may have effector roles, and changes in glyceraldehyde-3-phosphate dehydrogenase and calretinin may reflect the altered retinal microenvironment resulting from laser treatment.

Pilot application of iTRAQ to the retinal disease Macular Telangiectasia.

We used the comparative proteomic technique iTRAQ coupled with offline 2DLC-MS/MS to analyze a rare specimen of the poorly understood, potentially blinding ophthalmic condition Macular Telangiectasia type 2 (MacTel type 2). We refined the technique using an internal standard consisting of pooled samples for each iTRAQ experiment to allow for multiple comparisons between different regions of the retina and different tissue donors. A total of 594 nonredundant proteins were identified in the retina and 168 in the vitreous, of which approximately half were found in significantly different abundance in the various comparisons made. The most prominent differences were found within the glycolytic pathway, where 8 proteins were reduced in the diseased macula compared with peripheral retina of the same eye, and 10 were also reduced in comparison with the macula of a control eye. Furthermore, Müller cell-associated proteins, including GFAP, VIME, and GLNA, were also reduced in the diseased macula, consistent with a link between the glycolytic pathway and Müller cells. These changes were validated by Western blotting and immunohistochemical studies. Proteomic analysis of the vitreous revealed an increase of proteins that were reduced in the retina. This supports proteomic analysis of the more easily available vitreous, which may reveal retina-specific protein changes associated with disease. Furthermore, our study has highlighted changes in the glycolytic pathway as a possible component of MacTel type 2 pathobiology.

The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate-washed membrane standard.

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress.

Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies.

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.

Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: new insight in the pathogenesis of diabetic retinopathy.

We have employed proteomics to establish a proteome map of the normal rat retina. This baseline map was then used for comparison with the early diabetic rat retinal proteome. Diabetic rat retinae were obtained from Dark Agouti rats after 10 wk of streptozotocin-induced hyperglycaemia. Extracted proteins from normal and diabetic rat retinae were separated and compared using 2-DE. A total of 145 protein spots were identified in the normal rat retina using MALDI-MS and database matching. LC-coupled ESI-MS increased the repertoire of identified proteins by 23 from 145 to 168. Comparison with early diabetic rat retinae revealed 24 proteins unique to the diabetic gels, and 37 proteins absent from diabetic gels. Uniquely expressed proteins identified included the HSPs 70.1A and 8, and platelet activating factor. There were eight spots with increased expression and 27 with decreased expression on diabetic gels. Beta catenin, phosducin and aldehyde reductase were increased in expression in diabetes whilst succinyl coA ligase and dihydropyrimidase-related protein were decreased. Identification of such changes in protein expression has given new insights and a more comprehensive understanding of the pathogenesis of diabetic retinopathy, widening the scope of potential avenues for new therapies for this common cause of blindness.

Stress-responsive proteins are upregulated in Streptococcus mutans during acid tolerance.

Streptococcus mutans is an important pathogen in the initiation of dental caries as the bacterium remains metabolically active when the environment becomes acidic. The mechanisms underlying this ability to survive and proliferate at low pH remain an area of intense investigation. Differential two-dimensional electrophoretic proteome analysis of S. mutans grown at steady state in continuous culture at pH 7.0 or pH 5.0 enabled the resolution of 199 cellular and extracellular protein spots with altered levels of expression. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 167 of these protein spots. Sixty-one were associated with stress-responsive pathways involved in DNA replication, transcription, translation, protein folding and proteolysis. The 61 protein spots represented isoforms or cleavage products of 30 different proteins, of which 25 were either upregulated or uniquely expressed during acid-tolerant growth at pH 5.0. Among the unique and upregulated proteins were five that have not been previously identified as being associated with acid tolerance in S. mutans and/or which have not been studied in any detail in oral streptococci. These were the single-stranded DNA-binding protein, Ssb, the transcription elongation factor, GreA, the RNA exonuclease, polyribonucleotide nucleotidyltransferase (PnpA), and two proteinases, the ATP-binding subunit, ClpL, of the Clp family of proteinases and a proteinase encoded by the pep gene family with properties similar to the dipeptidase, PepD, of Lactobacillus helveticus. The identification of these and other differentially expressed proteins associated with an acid-tolerant-growth phenotype provides new information on targets for mutagenic studies that will allow the future assessment of their physiological significance in the survival and proliferation of S. mutans in low pH environments.

Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance.

Two-dimensional gel electrophoretic analysis of the proteome of Streptococcus mutans grown at a steady state in a glucose-limited anaerobic continuous culture revealed a number of proteins that were differentially expressed when the growth pH was lowered from pH 7.0 to pH 5.0. Changes in the expression of metabolic proteins were generally limited to three biochemical pathways: glycolysis, alternative acid production and branched-chain amino acid biosynthesis. The relative level of expression of protein spots representing all of the enzymes associated with the Embden-Meyerhof-Parnas pathway, and all but one of the enzymes involved in the major alternative acid fermentation pathways of S. mutans, was identified and measured. Proteome data, in conjunction with end-product and cell-yield analyses, were consistent with a phenotypic change that allowed S. mutans to proliferate at low pH by expending energy to extrude excess H(+) from the cell, while minimizing the detrimental effects that result from the uncoupling of carbon flux from catabolism and the consequent imbalance in NADH and pyruvate production. The changes in enzyme levels were consistent with a reduction in the formation of the strongest acid, formic acid, which was a consequence of the diversion of pyruvate to both lactate and branched-chain amino acid production when S. mutans was cultivated in an acidic environment.

Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat.

The oral pathogen, Streptococcus mutans, was grown under glucose limitation in a chemostat at pH 7.0 and a dilution rate of 0.1 h(-1) to mimic the conditions prevailing in a healthy human oral cavity in between meal times. Solubilized cellular and extracellular proteins were separated by two-dimensional gel electrophoresis (2-DE) and, following tryptic digestion, 421 protein spots analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry or electrospray ionization-tandem mass spectrometry. Analyses of the mass spectral data showed that the proteins matched the translation products of 200 different open reading frames (ORFs) deduced from contigs of the S. mutans UA159 genome and thus represented proteins derived from approximately 11% of the total ORFs of the bacterium. Of the identified proteins, 172 (including one surface protein) were characterized in the cellular fraction, and the remaining 28 (including two surface proteins) were uniquely identified from the culture fluid. The expression and therefore the existence of 30 proteins previously designated as 'hypothetical' or with no known function was confirmed. 2-DE of whole cell lysates revealed only a single intrinsic membrane protein. This is consistent with proteomic analyses of other Gram-positive bacteria where hydrophilic proteins represent the vast majority of those characterized.