Melissa Sondej

The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal Secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Solution structure of the phosphoryl transfer complex between the signal transducing proteins HPr and IIA(glucose) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The solution structure of the second protein–protein complex of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system, that between histidine‐containing phosphocarrier protein (HPr) and glucose‐specific enzyme IIAGlucose (IIAGlc), has been determined by NMR spectroscopy, including the use of dipolar couplings to provide long‐range orientational information and newly developed rigid body minimization and constrained/restrained simulated annealing methods. A protruding convex surface on HPr interacts with a complementary concave depression on IIAGlc. Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIAGlc. Formation of the unphosphorylated complex, as well as the phosphorylated transition state, involves little or no change in the protein backbones, but there are conformational rearrangements of the interfacial side chains. Both HPr and IIAGlc recognize a variety of structurally diverse proteins. Comparisons with the structures of the enzyme I–HPr and IIAGlc–glycerol kinase complexes reveal how similar binding surfaces can be formed with underlying backbone scaffolds that are structurally dissimilar and highlight the role of redundancy and side chain conformational plasticity.

High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr.

The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts ofEscherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant ∼108 m −1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the K m for glycogen and an increase in the V max for phosphate, indicating a mixed type activation. The stimulatory effect of E. coliHPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.

Glycoprofiling of the Human Salivary Proteome

IntroductionGlycosylation is an important component for a number of biological processes and is perhaps the most abundant and complicated of the known post-translational modifications found on proteins.MethodsThis work combines two-dimensional (2-D) polyacrylamide gel electrophoresis and lectin blotting to map the salivary glycome and mass spectrometry to identity the proteins that are associated with the glycome map. A panel of 15 lectins that recognize six sugar-specific categories was used to visualize the type and extent of glycosylation in saliva from two healthy male individuals. Lectin blots were compared to 2-D gels stained either with Sypro Ruby (protein stain) or Pro-Q Emerald 488 (glycoprotein stain).ResultsEach lectin shows a distinct pattern, even those belonging to the same sugar-specific category. In addition, the glycosylation profiles generated from the lectin blots show that most salivary proteins are glycosylated and that the profiles are more widespread than is demonstrated by the glycoprotein-stained gel. Finally, the coreactivity between lectins was measured to determine what types of glycan structures are associated with one another and also the population variation of the lectin reactivity for 66 individuals were reported.ConclusionsThis starting 2-D gel glycosylation reference map shows that the scientifically accepted, individual oligosaccharide variability is not limited to a few large glycoproteins such as MUC5B, but are found on most members of the salivary proteome. Finally, in order to see the full range of oligosaccharide distribution, multiple reagents or lectins are needed.

Effects of green tea extract on lung cancer A549 cells: proteomic identification of proteins associated with cell migration.

Green tea polyphenols exhibit multiple antitumor activities, and the mechanisms of action are not completely understood. Previously, we reported that green tea extract (GTE)‐induced actin remolding is associated with increased cell adhesion and decreased motility in A549 lung cancer cells. To identify the cellular targets responsible for green tea‐induced actin remodeling, we performed 2‐DE LC‐MS/MS of A549 cells before and after GTE exposure. We have identified 14 protein spots that changed in expression (≥2‐fold) after GTE treatment. These proteins are involved in calcium‐binding, cytoskeleton and motility, metabolism, detoxification, or gene regulation. In particular we found upregulation of several genes that modulate actin remodeling and cell migration, including lamin A/C. Our data indicated that GTE‐induced lamin A/C upregulation appears to be at the transcriptional level and the increased expression results in the decrease in cell motility, as confirmed by siRNA. The result of the study demonstrates that GTE alters the levels of many proteins involved in growth, motility and apoptosis of A549 cells and their identification may explain the multiple antitumor activities of GTE.