Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry

The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.     

A structural analysis of the catalytic mechanism of methionine sulfoxide reductase A from Neisseria meningitidis

The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product—methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.     

Determination of urinary iodine by inductively coupled plasma mass spectrometry.

BACKGROUND: Mild iodine deficiency is endemic in many countries of Europe including Belgium. Fast, accurate and specific methods for quantification of urinary iodine are needed. We describe in this report a specific ICP-MS method for the quantification of urinary iodine. METHOD: Samples and iodate calibrators were diluted 20 times into aqueous solution containing triton X-100, 1.5% HCl and (103)Rh as an internal standard. Prior digestion or oxidation was not necessary. Results were compared with those obtained by Sandell-Kolthoff (S-K) spectrophotometric method. RESULTS: Comparison of both methods showed good agreement. The Passing-Bablok regression between both methods was ICP-MS=0.986 (S-K)-7.51. The Bland-Altman difference plot showed a small but significant mean difference of -13.3 microg/L for ICP-MS. The between-day coefficient of variation (CV) was 13% at 89 microg/L. Limit of detection was 4 microg/L and limit of quantification was 20 microg/L. No carryover effect has been observed on series containing up to 50 samples. CONCLUSION: The ICP-MS method described here is fast, accurate and specific for the quantification of urinary iodine. Compared to the S-K method the urinary iodine concentrations measured by the ICP-MS method were slightly, but significantly lower. Consequently, the results of studies using S-K method should be compared with caution with those using the ICP-MS method.     

Beta-naphthoflavone inhibits the induction of hepatic oestrogen-dependent proteins by 17alpha-ethynylestradiol in mosquitofish (Gambusia holbrooki)

The interactive effects of an aryl hydrocarbon receptor (AhR) agonist and of a xenoestrogen on biomarker responses were studied in the liver of male mosquitofish (Gambusia holbrooki). Hepatic 7-ethoxyresorufin O-deethylase (EROD) enzymatic activity was measured as a biomarker of exposure to the model AhR agonist beta-naphthoflavone (bNF). Hepatic proteins indicating the exposure of males to the synthetic oestrogen 17alpha-ethynylestradiol (EE2) were monitored by Western blot analysis using immunoserum prepared for this study. After a semi-static exposure only to waterborne EE2, Western blot analysis of liver homogenate revealed the induction of two protein bands (a double band at 205 kDa and a single band at 125 kDa). The interaction between bNF and EE2 was investigated by analysing, on the one hand, EROD activity and, on the other hand, immunoreactivity corresponding to the two oestrogen-dependent protein bands in the liver of fish exposed to different concentrations of bNF for 2 days, then to the same concentrations of bNF plus 0.1 µg l^-1 EE2 for 5 days. EE2 changed neither the basal activity of EROD nor its rate of induction with 1.0 and 4.0 µg l^-1 bNF. On the other hand, the induction of oestrogen-dependent proteins with 0.1 µg l^-1 EE2 was inhibited by exposure to 4.0 µg l^-1 bNF. These results together with literature data suggest that field monitoring of xenoestrogen contamination through the analysis of oestrogen-dependent protein in male fish as a biomarker should take into account the possible negative interference of AhR agonists.     

Nonuniformity of calcium efflux from pancreatic acinar cells and its analysis by mathematical model of calcium diffusion and buffering in extracellular solution

We studied spatial and temporal patterns of Ca²⁺ extrusion from pancreatic acinar cells evoked by acetylcholine(ACh)-induced activation of plasma membrane calcium pumps. Using a modification of an earlier developed model, we estimated the time course of extracellular calcium concentration changes near the basal pole of a cell in the case, when calcium ions are released from the same site on the cell surface, and in the case when they are extruded from the apical pole and diffuse to the basal one. It is concluded that at the first stage of ACh-induced Ca²⁺ extrusion the appearance of Ca²⁺ elevation near the basal pole of the cells cannot be explained as a result of diffusion, but is mainly determined by Ca²⁺ efflux from this pole. The results also show that there are plasma membrane calcium pumps in both apical and basal parts of pancreatic acinar cells, but the activity of the pumps is substantially higher in the apical region.     

Corticofugal influences on the neurons of different regions of the hypothalamus

Responses of the neurons of the lateral and ventromedial hypothalamic regions (HL andHvm, respectively), as well as of the area of the dorsal hypothalamus (aHd) and the projection region of the medial forelimb bundle (MFB), evoked by stimulation of the proreal cortex (field 8), cingular cortex (field 24), pyriform lobula (periamigdalar cortex), and hippocampus (CA3) were studied in acute experiments on cats under ketamine anesthesia. Distributions of the latent periods of the responses recorded from hypothalamic neurons at stimulation of the above cortical structures were analyzed. The responses were classified into primary excitatory and primary inhibitory. Stimulation of the proreal gyrus evoked four times more excitatory responses than inhibitory responses. With stimulation of the cingular gyrus, the ratio of excitatory/inhibitory responses was 1.5∶1. Stimulation of the pyriform cortex evoked activatory and inhibitory responses with a similar probability. With hippocampal stimulation, inhibitory responses appeared two times more frequently than excitatory reactions. The hypothalamus was found to be a zone of wide convergence: one-half of all responding neurons in theHL andHvm responded to stimulations of two or more tested cortical zones. In 26% of the cells, only excitatory convergence was observed, while in 10% only inhibitory convergence was found; 21% of the cells revealed mixed convergence.     

Purification of bovine cathepsin B: proteomic characterization of the different forms and production of specific antibodies.

Cathepsin B (EC 3.4.22.1) has been highly purified (14,225 fold) from bovine kidney by a rapid procedure that included the preparation of an enriched lysosomal extract, a selective fractionation with ammonium sulphate, size-exclusion chromatography, two cation-exchange chromatographies, and anion-exchange chromatography on diethylaminoethyl-Sephacel. After the last purification step, two hydrolytic peaks against Z-Phe-Arg-AMC were separated from each other, a minor peak corresponding to the cathepsin B single-chain form and a major one representing the double-chain form of cathepsin B. The single-chain form showed a molecular mass of 31 kDa on sodium dodecyl sulphate - polyacrylamide gel electrphoresis (PAGE) under reducing conditions, whereas the heavy chain of the double-chain form appeared as a doublet with molecular masses of 23.4 and 25 kDa, respectively. The identity of the different cathepsin B isoforms and the quality of the final enzyme preparation were confirmed by using two types of antibodies, one against a synthetic peptide sequence and one against purified cathepsin B. The proteomic study confirmed the identity of the different SDS-PAGE protein bands as cathepsin B isoforms and allowed the comparison and study of some structural differences between them at the level of their primary structures.