Molecular basis for the reversible ADP-ribosylation of guanosine bases.

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins.

Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation.

The mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ.

Moonlighting Proteins and Cardiopathy in the Spatial Response of MCF-7 Breast Cancer Cells to Tamoxifen.

Background: The purpose of this study is to apply quantitative high-throughput proteomics methods to investigate dynamic aspects of protein changes in nucleocytoplasmic distribution of proteins and of total protein abundance for MCF-7 cells exposed to tamoxifen (Tam) in order to reveal the agonistic and antagonistic roles of the drug.

Experimental Design: The MS-based global quantitative proteomics with the analysis of fractions enriched in target subcellular locations is applied to measure the changes in total abundance and in the compartmental abundance/distribution between the nucleus and cytoplasm for several thousand proteins differentially expressed in MCF-7 cells in response to Tam stimulation.

Results: The response of MCF-7 cells to the Tam treatment shows significant changes in subcellular abundance rather than in their total abundance.

Rapid aggregation and assembly in aqueous solution of A beta (25-35) peptide.

The highly toxic A beta (25-35) is a peculiar peptide that differs from all the other commonly studied beta-amyloid peptides because of its extremely rapid aggregation properties and enhanced neurotoxicity. We investigated A beta (25-35) aggregation in H2O at pH 3.0 and at pH 7.

Robust MS quantification method for phospho-peptides using 18O/16O labeling.

Background: Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-beta treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.

Increased expression of the oligopeptidase THOP1 is a neuroprotective response to Abeta toxicity.

In a comprehensive proteomics study aiming at the identification of proteins associated with amyloid-beta (Abeta)-mediated toxicity in cultured cortical neurons, we have identified Thimet oligopeptidase (THOP1). Functional modulation of THOP1 levels in primary cortical neurons demonstrated that its overexpression was neuroprotective against Abeta toxicity, while RNAi knockdown made neurons more vulnerable to amyloid peptide. In the TgCRND8 transgenic mouse model of amyloid plaque deposition, an age-dependent increase of THOP1 expression was found in brain tissue, where it co-localized with Abeta plaques.

Sequential detergent fractionation of primary neurons for proteomics studies.

Proteomics studies employing primary neurons are difficult due to the neurons' characteristics. We have developed a detergent-based fractionation method which reduces complexity of the protein extracts, is sufficiently fast to allow differential proteomics analysis after treatments of neurons for short time periods, can be applied to small numbers of cells directly in culture plates, and allows differential extraction of proteins in a compartment-specific manner. The sequential use of detergent-containing buffers on neurons in culture plates yields four extracts enriched in cytosolic, membrane-bound or enclosed, nuclear, and cytoskeletal proteins.

Target deconvolution strategies in drug discovery.

Recognition of some of the limitations of target-based drug discovery has recently led to the renaissance of a more holistic approach in which complex biological systems are investigated for phenotypic changes upon exposure to small molecules. The subsequent identification of the molecular targets that underlie an observed phenotypic response--termed target deconvolution--is an important aspect of current drug discovery, as knowledge of the molecular targets will greatly aid drug development. Here, the broad panel of experimental strategies that can be applied to target deconvolution is critically reviewed.

Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology.

Although protein phosphorylation is probably the most studied post-translational modification occurring in cells, the number of proteins, which are the target of this modification, is still largely unknown. Increasing the coverage of the phosphoproteome as well as the detection of variation at the phosphorylation level would be very helpful for understanding the mechanisms of cell life and the modifications of the cell state leading to pathological conditions such as neurodegeneration. In order to further investigate variations occurring at the phosphorylation level, we have initiated the creation of a reference map of phosphorylated proteins in rat cortical neurons, employing a combination of phosphatase treatment and 2-DE/differential in gel electrophoresis technology.

Phosphoproteome analysis.

Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made.

Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study.

We used two-dimensional (2-D) electrophoresis to analyze the protein composition of fluid recovered by bronchoalveolar lavage (BALF) from patients with sarcoidosis and idiopathic pulmonary fibrosis, two forms of interstitial lung disease with different cellular composition and cytokine profile in BALF. They are also characterized by different pathogenesis and clinical evolution, idiopathic pulmonary fibrosis being less favorable than sarcoidosis due to rapidly progressive pulmonary fibrosis. Thirty-eight proteins or protein fragments, never previously assigned in BALF samples, were identified by various methods including mass fingerprinting of tryptic digests.

Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering.

Signalling by immunoreceptors is orchestrated at specific plasma membrane microdomains, referred to as lipid rafts. Here we present a proteomics approach to the temporal analysis of protein association with lipid rafts following T-cell antigen receptor (TCR) triggering. We show that TCR engagement promotes the temporally regulated recruitment of proteins participating in the TCR signalling cascade to lipid rafts.

Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland.

The study of the development of the mammary gland at the molecular level in the animal is difficult because of the complex tissue organization of the gland. We have previously developed an in vitro system for genetic analysis of mammary cell differentiation, based on the cell line LA7 clonally derived from a rat mammary adenocarcinoma. This cell line, after induction with DMSO, differentiates forming structures called domes.

Identification of Chlamydia trachomatis antigens recognized by human CD4+ T lymphocytes by screening an expression library.

Identification of the immunogenic proteins that induce Chlamydia trachomatis (CT)-specific T cell responses is crucial to the development of protective vaccines and understanding the mechanisms of chlamydia-induced pathology. To characterize the targets of the human T cell response we have used chlamydia-reactive human T cell clones as cellular probes to screen a CT genomic library expressed in Escherichia coli using peripheral blood mononuclear cells to present antigens. The library was screened with three chlamydia-reactive T cell clones of unknown specificity and three novel stimulatory chlamydia antigens were identified.

Proteomic dissection of dome formation in a mammary cell line: role of tropomyosin-5b and maspin.

In this work we extended the study of genes controlling the formation of specific differentiation structures called "domes" formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the beta-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells.

Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two-dimensional electrophoresis map with patient sera.

Western blots of two-dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera.

Charge heterogeneity of macrophage migration inhibitory factor (MIF) in human liver and breast tissue.

Macrophage migration inhibitory factor (MIF) is an ubiquitous protein playing various immunological and hormonal roles. Theoretical electrophoretic coordinates calculated from protein sequence in the SWISS-PROT database (AC P14174) are 12 kDa and pI 8.24.

A two-dimensional protein map of human amniotic fluid at 17 weeks' gestation.

Using updated technical procedures (immobilized pH gradients for isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: IPG/SDS-PAGE) we provide a two-dimensional (2-D) map of amniotic fluid (AF) proteins. This map comprises over 800 silver-stained spots. Over 150 spots have been identified by matching on the net with human plasma and cerebrospinal fluid maps available from SWISS 2DPAGE database; several additional spots were assigned by immunoblotting and/or microanalytical techniques.

Acute-phase proteins in perinatal human plasma.

Plasma from eight newborns (4 pre-term and 4 full-term) with early-onset (< 72 h) sepsis and six apparently healthy controls was analyzed. The presence of spots identified as haptoglobin and serum amyloid A protein was the electrophoretic result most consistently associated with disease. Time course monitoring showed rises, peaks and declines of spot intensity as expected for acute-phase proteins induced by transient stimuli.

Two-dimensional electrophoretic patterns of acute-phase human serum proteins in the course of bacterial and viral diseases.

Acute-phase serum proteins were analyzed by two-dimensional electrophoresis with isoelectric focusing in 3-10 immobilized pH gradients. Most spots were identified by reference to the plasma map in the SWISS-2DPAGE database. Serum amyloid A protein spots were identified by immunoblotting with specific antiserum and by matching determined with predicted values of electrophoretic parameters.

The human leucine zipper-containing guanine-nucleotide exchange protein elongation factor-1 delta.

Copy-DNA clones containing the complete coding region of the human elongation factor-1 delta (EF-1 delta) mRNA have been isolated and characterized. We present the deduced amino acid sequence and observe in it a leucine zipper motif seen recently in EF-1 delta from Artemia and Xenopus laevis. The human EF-1 delta sequence shows a strong conservation in its C-terminal domain.