Promyelocytic Leukaemia Protein nuclear bodies (PML-NBs) are dynamic nuclear protein aggregates. To gain insight in PML-NB function, reductionist and high throughput techniques have been employed to identify PML-NB proteins. Here we present a manually curated network of the PML-NB interactome based on extensive literature review including database information. By compiling 'the PML-ome', we highlighted the presence of interactors in the Small Ubiquitin Like Modifier (SUMO) conjugation pathway. Additionally, we show an enrichment of SUMOylatable proteins in the PML-NBs through an in-house prediction algorithm. Therefore, based on the PML network, we hypothesize that PML-NBs may function as a nuclear SUMOylation hotspot.

Ligand-induced clustering of type I cytokine receptor subunits leads to trans-phosphorylation and activation of associated cytosolic janus kinases (JAKs). In turn, JAKs phosphorylate tyrosine residues in the receptor tails, leading to recruitment and activation of signalling molecules. Among these, signal transducers and activators of transcription (STATs) are important in the direct transmission of signals to the nucleus. Here, we show that incorporation of an interaction trap in a signalling-deficient receptor allows the identification of protein-protein interactions, using a STAT-dependent complementation assay. Mammalian protein-protein interaction trap (MAPPIT) adds to existing yeast two-hybrid procedures, as originally explored by Fields and Song, and permits the detection of both modification-independent and of phosphorylation-dependent interactions in intact human cells. We also demonstrate that MAPPIT can be used to screen complex complementary DNA libraries, and using this approach, we identify cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signalling-2 (SOCS-2) as interaction partners of the phosphotyrosine 402 (Tyr 402)-binding motif in the erythropoietin receptor (EpoR). Importantly, this approach places protein-protein interactions in their normal physiological context, and is especially applicable to the in situ analysis of signal transduction pathways.

Background: Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C 4 (RP)-LC protein separation) followed by C 18 (RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.

The remarkable ability of tumour necrosis factor (TNF), especially in combination with interferon, selectively to kill or inhibit malignant cell lines is so far unmatched by any other combination of cytokines. But clinical trials in cancer patients have on the whole been disappointing, and it has been estimated that a TNF dose would be effective only at 5-25 times the maximum tolerated dose. High TNF concentrations give a much more pronounced antitumour activity in mice, in which murine TNF is about 50-fold more systemically toxic than human TNF. But there is little or no species specificity in cytotoxicity of murine TNF and human TNF on human as well as on murine cell lines. This dual action of TNF may be explained by the existence of two types of receptor for TNF: the smaller, TNF-R55, is present on most cells and particularly on those susceptible to the cytotoxic action of TNF; the larger, TNF-R75, is also present on many cell types, especially those of myeloid origin, and is strongly expressed on stimulated T and B lymphocytes. In mice, human TNF binds only to murine TNF-R55 (ref. 15), which can then mediate cytotoxic activity on malignant cells. As human TNF does not bind to murine TNF-R75, the latter must be responsible for the much enhanced systemic toxicity of murine TNF. Human TNF can, however, become toxic in mice when a second pathway is activated. There is no reciprocal situation in the human system: human and murine TNF bind almost equally well to the two human TNF receptors. Here we describe human TNF mutants that sill interact with the human TNF-R55 receptor but which have largely lost their ability to bind to human TNF-R75. Activation of TNF-R55 is sufficient to trigger cytotoxic activity towards transformed cells. One representative human TNF mutant retains its antitumour activity in nude mice carrying tumours derived from human cancers. Under the appropriate conditions, such human TNF mutants are expected to induce less systemic toxicity in man, while still exerting their direct antitumour effect.

BackgroundThe spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection.Methodology/Principal FindingsTo better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance.ConclusionsThis is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.

Summary When the tumour necrosis factor-alpha (TNF-a) gene was cloned the protein became available for use in clinical trials as an antineoplastic agent. However, side effects have severely limited its application in cancer treatment. Studies on the species specificity of TNF have indicated that the p75 TNF receptor (TNFR75) may play an important role in the generation of these side effects in humans. Using human TNF mutants with selective receptor-binding properties it has been demonstrated in neutrophils and endothelium that TNFR75 is involved in the mediation ofthe proinflammatory activity of TNF by facilitating the p55 TNF receptor {TNFR55). However, only TNFR55 appears to be involved in mediating TNF cytotoxicity. Therefore the potential exists for the successful reintroduction of TNF-a. in the form of TNFR55-selective mutants, into the clinical arena with the promise of reduced side effects.

Human tumour necrosis factor alpha (TNF‐alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF‐alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF‐alpha can be dissociated. The TNFR55‐selective mutants (R32W, E146K and R32W‐S86T) which bind poorly to TNFR75 displayed similar potency to wild‐type TNF in causing cytotoxicity of a human laryngeal carcinoma‐derived cell line (HEp‐2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55‐selective mutants exhibited lower proinflammatory activity than wild‐type TNF. Specifically, TNF‐alpha's priming of human neutrophils for superoxide production and antibody‐dependent cell‐mediated cytotoxicity, platelet‐activating factor synthesis and adhesion to endothelium were reduced by up to 170‐fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E‐selectin expression, neutrophil transmigration and IL‐8 secretion were also reduced by up to 280‐fold. On the other hand, D143F, a TNFR75‐selective mutant tested either alone or in combination with TNFR55‐selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75‐transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cervicovaginal fluid has an important function in the homeostasis and immunity of the lower female genital tract. Analysis of the cervicovaginal fluid proteome may therefore yield important information about the pathogenesis of numerous gynecological pathologies. Additionally, cervicovaginal fluid has great potential as a source of biomarkers for these conditions.This review provides a detailed discussion about the human cervicovaginal proteome and the proteomics studies performed to characterize this biological fluid. Furthermore, infection-correlated pathological conditions of the female genital tract are discussed for which cervicovaginal fluid has been used in order to identify potential biomarkers. Recent years, numerous studies have analyzed cervicovaginal fluid samples utilizing antibody-based technologies, such as ELISA or Western blotting, to identify biomarkers for preterm birth, premature preterm rupture of membranes, bacterial vaginosis and cervical cancer. The present article will discuss the importance of proteomic technologies as alternative techniques to gain additional meaningful information about these conditions. In addition, the review focuses on recent proteomic studies on cervicovaginal fluid samples for the identification of potential biomarkers. We conclude that the use of proteomic technology for analysis of human cervicovaginal fluid samples is promising and may lead to the discovery of new biomarkers which can improve disease prevention and therapy development.