Irena Selicharová

Changes in the proteomes of the hemocytes and fat bodies of the flesh fly Sarcophaga bullata larvae after infection by Escherichia coli

BackgroundInsects have an efficient self-defense system that is based on innate immunity. Recent findings have disclosed many parallels between human and insect innate immunity, and simultaneously fine differences in the processes between various species have been revealed. Studies on the immune systems of various insect species may uncover the differences in their host defense strategies.ResultsWe analyzed the proteomes of the hemocytes and fat bodies of Sarcophaga bullata larvae after infection by Escherichia coli. The 2-DE gels of the hemocytes and fat bodies of infected larvae were compared with those of aseptically injured larvae. Our analysis included the construction of protein maps of the hemocyte cells and cells from fat bodies, the identification of the changed proteins, in response to infection, using LC-MS/MS, and the estimation of the trends in expression of these proteins at three time points (30 min, 6 hours and 22 hours) after infection. In total, seven changed spots were found in the hemocytes, and four changed spots were found in the fat bodies. Three types of trends in protein expression were observed. Cofilin and transgelin were undetectable at 30 min after infection but were continuously up-regulated in the induced larvae after 22 hours. A prophenoloxidase isoform and lectin subunit α were slightly up-regulated at 30 min after infection, and their protein levels reached the highest points after 6 hours but decreased after 22 hours. T-Complex subunit α, GST, ferritin-like protein and an anterior fat body protein (regucalcin homologue) were down-regulated at 22 hours after infection.ConclusionsMany proteins identified in our study corresponded to the proteins identified in other insects. Compared to the former studies performed in insects, we presented 2-D protein maps of the hemocytes and fat bodies and showed the trends in expression of the immune-elicited proteins.

Two-dimensional electrophoretic comparison of metastatic and non-metastatic human breast tumors using in vitro cultured epithelial cells derived from the cancer tissues

BackgroundBreast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. Identification of proteins resembling the tumor biology can improve the diagnosis, prediction, treatment selection, and targeting of therapy. Since the beginning of the post-genomic era, the focus of molecular biology gradually moved from genomes to proteins and proteomes and to their functionality. Proteomics can potentially capture dynamic changes in protein expression integrating both genetic and epigenetic influences.MethodsWe prepared primary cultures of epithelial cells from 23 breast cancer tissue samples and performed comparative proteomic analysis. Seven patients developed distant metastases within three-year follow-up. These samples were included into a metastase-positive group, the others formed a metastase-negative group. Two-dimensional electrophoretical (2-DE) gels in pH range 4–7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses (R/maanova package) and data-mining analysis (GUHA). For identification of proteins in selected spots, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed.ResultsThree protein spots were significantly altered between the metastatic and non-metastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased.ConclusionWe have performed an extensive proteomic study of mammary epithelial cells from breast cancer patients. We have found differentially expressed proteins between the samples from metastase-positive and metastase-negative patient groups.

Rational steering of insulin binding specificity by intra-chain chemical crosslinking.

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22–B30 segment, using the CuI-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26–B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone’s B-chain C-terminus for its IR-B specificity.

Quantification of homocysteine‐related metabolites and the role of betaine–homocysteine S‐methyltransferase in HepG2 cells

We optimized and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of six metabolites of homocysteine metabolism: homocysteine, methionine, cysteine, S-adenosylmethionine, S-adenosylhomocysteine and betaine. The detection limits for these metabolites were in the nanomolar range, and the intra- and inter-day precisions were lower than 20% of the relative standard deviations. The method was specifically designed for the determination of the intracellular concentrations of the metabolites in cultured cells. To study the role of betaine-homocysteine S-methyltransferase (BHMT), HepG2 cells and HepG2 cells that were stably transfected with BHMT ((BHMT) HepG2) were treated with homocysteine or with a specific inhibitor of BHMT, and metabolite levels were subsequently measured. Severely compromised methyl group metabolism in the HepG2 cells, which is typical of cancer-derived cells, prevented clear evaluation of the changes caused by the external manipulations of homocysteine metabolism. However, the ease of handling these cells and the almost unlimited source of experimental material supplied by cells in permanent culture allowed us to develop a reliable methodology. The precautions concerning intracellular metabolite determinations using LC-MS/MS in cultured cells that are expressed in this work will have global validity for future metabolomics studies.

Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins

BackgroundTransformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest.MethodsWe compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots.ResultsCorrelation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics.ConclusionsOur results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.

Effects of hyperhomocysteinemia and betaine-homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome.

Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1mM and 2mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine-homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.

Targeted Metabolomics for Homocysteine-Related Metabolites in Primary Hepatocytes

Liquid chromatography-tandem mass spectrometry has become the most convenient method to identify and quantify low molecular weight metabolites from various sources. Metabolomics studies of hepatocytes hold promise for the identification of the mechanisms of toxicant-related disease processes. In this chapter, we present a rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantification of intracellular concentrations of nine homocysteine-based metabolites, namely homocysteine, methionine, cysteine, dimethylglycine, cystathionine, S-adenosylmethionine, S-adenosylhomocysteine, choline, and betaine. The method is specifically designed for the analysis of cultured primary hepatocytes.