Miloslav Šanda

Hemoglobin Digestion in Blood-Feeding Ticks: Mapping a Multipeptidase Pathway by Functional Proteomics

Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.

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.

Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil

ABSTRACT Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.

Crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata

The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the hosts defence during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 A (1 A=0.1 nm) and was used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the pro-inflammatory cytokines tumour necrosis factor alpha and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the hosts adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.

Insulin analogues with modifications at position B26. Divergence of binding affinity and biological activity.

In this study, we prepared several shortened and full-length insulin analogues with substitutions at position B26. We compared the binding affinities of the analogues for rat adipose membranes with their ability to lower the plasma glucose level in nondiabetic Wistar rats in vivo after subcutaneous administration, and also with their ability to stimulate lipogenesis in vitro. We found that [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2 were very potent insulin analogues with respect to their binding affinities (214 and 465%, respectively, compared to that of human insulin), but they were significantly less potent than human insulin in vivo. Their full-length counterparts, [NMeHisB26]-insulin and [NMeAlaB26]-insulin, were less effective than human insulin with respect to binding affinity (10 and 21%, respectively) and in vivo activity, while [HisB26]-insulin exhibited properties similar to those of human insulin in all of the tests we carried out. The ability of selected analogues to stimulate lipogenesis in adipocytes was correlated with their biological potency in vivo. Taken together, our data suggest that the B26 residue and residues B26-B30 have ambiguous roles in binding affinity and in vivo activity. We hypothesize that our shortened analogues, [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2, have different modes of interaction with the insulin receptor compared with natural insulin and that these different modes of interaction result in a less effective metabolic response of the insulin receptor, despite the high binding potency of these analogues.

Structure–activity relationship of CART (cocaine- and amphetamine-regulated transcript) peptide fragments

CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of (125)I-CART(61-102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61-102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74-86) and CART(62-86) containing disulfide bridges kept partial binding potency of the original molecule with K(i) in 10(-5) and 10(-4)M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.

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.

Non-Equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface.

Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (Kd = 8.8 μm). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in Kd values of 142 and 587 μm, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R6 form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R6-specific intra/intermolecular interactions for hexamer stability.

Profiling and characterization of volatile secretions from the European stink bug Graphosoma lineatum (Heteroptera: Pentatomidae) by two-dimensional gas chromatography/time-of-flight mass spectrometry.

An efficient method combining the headspace solid-phase microextraction (HS-SPME) sampling procedure and comprehensive two-dimensional gas-chromatography/time-of-flight mass spectrometry (GC×GC/TOF-MS) was established to study the volatile secretion components of stink bugs (Heteroptera: Pentatomidae). The combined power of this approach is illustrated by the identification of fifty-seven compounds in the secretion of a European stink-bug representative, Graphosoma lineatum. (E)-4-oxohex-2-enal and (E)-dec-2-enal were found to be the major components in the adult bug secretions followed by lower amounts of n-alkenal (C₅-C₁₂), n-alkenyl acetate (C₅-C₁₁), n-alkane (C₁₁-C₁₇) homologs, dienals and other compounds. More than thirty known compounds have been identified that had not been described before in G. lineatum adults. Of these compounds, (E)-4-oxohex-2-enal is of particular interest, since its isolation and identification, while calling some previous reports into question, clearly demonstrates a potential ability of our approach to yield artifact-free secretion profiles.

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.