A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization

We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1‐PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N‐hydroxysuccinimidobiotin (NHS‐biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS‐biotin when compared with the [PB1‐PA] heterodimer. Mutational analysis of residues in two such regions—at K265 and K481 of PB1, which were about three‐ and twofold, respectively, less accessible to biotinylation in the PB1‐PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q‐POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein–protein interaction interfaces. The Q‐POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs.     

A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa

In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2′-aminohexylcarbamoyl-c-di-GMP (2′-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2′-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.     

Immobilization of carbohydrate epitopes for surface plasmon resonance using the Staudinger ligation

The detection of carbohydrate-protein interactions is often performed using techniques that require surface immobilization of the lectin or the glycan. A commonly used assay for lectin binding is surface plasmon resonance (SPR). We describe an implementation of the Staudinger ligation as a method to immobilize carbohydrate epitopes to a biosensor surface. This was accomplished by first introducing an azide functionality to a carboxymethyldextran surface, followed by reaction with a phosphane-modified carbohydrate ligand. The chemistry employed is extremely mild and was easily adapted to a commercial biosensor system. Using this approach, we investigated the binding of jacalin and wheat germ agglutinin (WGA) to galactose, lactose, and N-acetyl-lactosamine. We observed that WGA binding shows evidence of multivalent interaction with the surface. Additionally, we found that jacalin binding was influenced by the presence of a flexible and hydrophobic galactosyl aglycone.     

Investigations into human serum sensitivity expressed by stocks of Trypanosoma brucei evansi

Trypanosoma bruceievansi, a widely distributed species of trypanosome infecting different livestock species in many countries in Africa, Asia and South America, has recently been reported as a pathogen causing a case of human trypanosomiasis in India. To date, there is little information regarding the natural resistance of animal-infective stocks of T. b. evansi to normal human serum (NHS). In this study, we investigated the degree of sensitivity to NHS of 15 stocks of T. b. evansi from different geographical origins and found that 10 of the stocks were completely susceptible to the action of NHS; parasites disappeared from the blood of infected mice within a few hours and the mice remained free from infection for more than 1 month. The remaining five stocks were partially resistant to NHS; although parasites initially disappeared from the circulation more than 50% of the mice showed relapse infection 10-18 days later. Studies on one stock, T. b. evansi STIB 810, showed that the changes in parasitaemia in the infected mice were correlated with the amount of NHS inoculated (correlation factor −0.584 and P = 0.001). When this stock was passaged 25 times in mice in the presence of NHS it was found that the trypanosomes’ serum resistance increased compared with the parent stock from which they were derived; 40% of the passaged parasites survived after in vitro incubation with 50% NHS for 7 h, while only 1% of individual trypanosomes of the parent stock survived under the same conditions. These findings show, to our knowledge for the first time, that human serum sensitivity varies amongst stocks of T. b. evansi, that some of them naturally display resistance to NHS and that, furthermore, T. b. evansi serum resistance can be increased by sub-passage in the presence of NHS.     

Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate)

The synthesis and properties of a new cleavable protein cross-linking reagent, [³⁵S]dithiobis(succinimidyl propionate), are detailed. Free primary and secondary aliphatic amino groups are quantitatively acylated by the reagent in either organic or aqueous media within two minutes at 23 °C. By contrast, the half-time for hydrolysis of the active ester termini in buffer at pH 7 is four to five hours, so that protein cross-linkage can be optimized by application of low concentrations of reagent. Accessible amino groups of hemoglobin are acylated with extreme rapidity of 0 °C in pH 7 buffer when [³⁵S]dithiobis(succinimidyl propionate) is applied in 0.4 to 9-fold molar excess. Submicrogram quantities of the cross-linked hemoglobin subunits which result are detectable by monitoring the ³⁵S distribution in sodium dodecyl sulfate-polyacrylamide gels. In addition to amine acylation, two of the six thiol groups in hemoglobin, tentatively located at cysteine 93 of the β chains, are reversibly modified at 0 °C by mercaptan-disul-fide interchange with the reagent or its bis amide analogs. This equilibrium-controlled, pH-dependent reaction occurs at a slower rate than acylation, and is blocked by short preincubation of the protein with N-ethylmaleimide or by addition of 3,3′- dithiodipropionamide (or other disulfides) to the reaction mixture. Disulfides introduced into hemoglobin by acylation and interchange are quantitatively cleaved by reduction for 30 minutes at 37 °C with 10 mm-dithioerythritol buffered at pH 8.5.The properties of high reactivity under mild conditions, long solution half-life, and the radioactive label make [³⁵S]dithiobis(succinimidyl propionate) a particularly useful and versatile probe of extended structures in a variety of biological systems.     

Follicular cells versus oocytes: cell population dynamics in the developing ovary

The aim of the current paper is to evaluate the correlation of germ and follicular cells kinetics during ovarian morphogenesis. Thus, immunohistochemical detection of PCNA and Ki-67 proteins has been examined using PC10 (Dako) and NCL-Ki-67 (Novocastra) antibodies in the developing ovaries of Wistar rat embryos and neonates [14.5, 18.5, 20.5days post-coitum (dpc), birth (day 0), 1, 3, 5, 7day post-partum (dpp)]. Estimation of reactive/total cell ratio, per cell type (germ and follicular cells) and visual field was achieved using the Image Pro Plus Software. The statistical interpretation of the results has shown that, before birth, using the PCNA antibody, the percentage of labeled/total germ cells (labeling index, LI) increases from 71.19% at 14.5dpc to 75.66% at 18.5dpc. It then decreases to 73.26% at 20.5dpc. At birth, the labeling index drops significantly (28.57%). Immediately after birth, the percentage of labeled/total germ cells increases, reaching 43.58% at 1dpp. Subsequently, a further decrease in the percentage of reactive cells is observed resulting to a maximum drop of the LI at 7dpp (18.41%). Using the Ki-67 antibody, the percentage of labeled/total germ cells is generally lower although the fluctuation is similar with that observed using the first marker of cell proliferation. Using the PCNA antibody, the LI of follicular cells in the developing ovary, increases from 0.70% (at 14.5dpc) to 28.94% (at 18.5dpc) and then drops to 18.03% (at 20.5dpc). At birth, the percentage of reactive follicular cells, reaches 27.66% and remains high thereafter. Similar results are obtained using the Ki-67 antibody. In conclusion, follicular cell reaction ratio, using both antibodies (PCNA and Ki-67), increases continuously throughout the examined period with a maximum value at 7dpp, suggesting a kinetics profile similar to that observed for Sertoli cells in the testis. In all age groups, PCNA labeling is more intense than Ki-67, a result that may be attributed to selective staining at different periods of the cell cycle.     

Epithelial-to-mesenchymal transition in fibrosis: Collagen type I expression is highly upregulated after EMT,but does not contribute to collagen deposition

The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concerning the relative contribution of epithelial cells to the pool of myofibroblasts. We hypothesized that EMT-derived cells might directly contribute to collagen deposition. To study this, EMT was induced in human epithelial lung and renal cell lines in vitro by means of TGF-β1 stimulation, and we compared the collagen type I (COL1A1) expression levels of transdifferentiated cells with that of myofibroblasts obtained by TGF-β1 stimulation of human dermal and lung fibroblasts. COL1A1 expression levels of transdifferentiated epithelial cells appeared to be at least one to two orders of magnitude lower than that of myofibroblasts. This was confirmed at immunohistochemical level: in contrast to myofibroblasts, collagen type I deposition by EMT-derived cells was not or hardly detectable. We postulate that, even when type II EMT occurs in vivo, the direct contribution of EMT-derived cells to collagen accumulation is rather limited.     

Proteomic analysis of UVC irradiation-induced damage of plasma proteins: Serum amyloid P component as a major target of photolysis

Ultraviolet-C (UVC) irradiation is a pathogen inactivation method used for disinfection of pharmaceutical products derived from human blood. Previous studies have shown that UVC can potentially damage proteins through photolysis or can generate reactive species resulting in protein thiol oxidation. In this study, two fluorescence-based quantitative proteomic approaches were used to assess the effects of a novel UVC-disinfection strategy on human plasma fractions. We show minimal changes in protein content, but gross alterations in protein thiol reactivity, indicative of oxidative damage. We identify a number of the damaged proteins by mass spectrometry, including serum amyloid P component, and further demonstrate UVC-induced photolysis of its disulphide bond.