Transglutaminase type 2 affects cell migration through post-translational modification of platelet-derived growth factor-BB.

Migration is a key cellular function with important implications in cell physiology. Impairment of such function is observed in angiogenesis, cancer, central nervous system development, and many other physiological and pathological events. Serum is considered among the most potent physiological chemotactic stimuli. Transglutaminase 2 (TG2) is involved in most of the mentioned processes, suggesting the hypothesis that TG2 may modulate cell movement and chemotaxis by acting on serum factors. Cell biology and biochemistry studies confirmed this hypothesis, showing that human serum contains potent chemotactic signals significantly impaired by activated TG2. Bioinformatics studies indicated that one potent serum factor potential substrate of TG2-dependent transamidation is platelet-derived growth factor-BB (PDGF-BB). Cell biology and immunometric experiments carried out with U87MG human glioma cell line showed that human recombinant PDGF-BB pre-incubated with calcium-activated TG2 lost about 70 % of its chemotactic activity and antigenicity. These data indicate that PDGF-BB is a substrate of TG2-transamidating activity, and such modification may play a key role in the modulation of PDGF's chemotactic features. Further, these findings suggest a novel point of view to study the extracellular functions of TG2 and to understand how protein signals, such as growth factors and cytokines, act in the extracellular space to reach their specific targets.

PDGFR-alpha inhibits melanoma growth via CXCL10/IP-10: a multi-omics approach.

Melanoma is the most aggressive skin-cancer, showing high mortality at advanced stages. Platelet Derived Growth Factor Receptor-alpha (PDGFR-alpha) potently inhibits melanoma- and endothelium-proliferation and its expression is significantly reduced in melanoma-biopsies, suggesting that melanoma progression eliminates cells expressing PDGFR-alpha. In the present study transient overexpression of PDGFR-alpha in endothelial (HUVEC) and melanoma (SKMel-28, A375, Preyer) human-cells shows strong anti-proliferative effects, with profound transcriptome and miRNome deregulation. PDGFR-alpha overexpression strongly affects expression of 82 genes in HUVEC (41 up-, 41 down-regulated), and 52 genes in SKMel-28 (43 up-, 9 down-regulated). CXCL10/IP-10 transcript showed up to 20 fold-increase, with similar changes detectable at the protein level. miRNA expression profiling in cells overexpressing PDGFR-alpha identified 14 miRNAs up- and 40 down-regulated, with miR-503 being the most down-regulated (6.4 fold-reduction). miR-503, miR-630 and miR-424 deregulation was confirmed by qRT-PCR. Interestingly, the most upregulated transcript (i.e., CXCL10/IP-10) was a validated miR-503 target and CXCL10/IP-10 neutralization significantly reverted the anti-proliferative action of PDGFR-alpha, and PDGFR-alpha inhibition by Dasatinb totally reverted the CXCL10/IP10 induction, further supporting a functional interplay of these factors. Finally, integration of transcriptomics and miRNomics data highlighted several pathways affected by PDGFR-alpha.This study demonstrates for the first time that PDGFR-alpha strongly inhibits endothelial and melanoma cells proliferation in a CXCL10/IP-10 dependent way, via miR-503 down-regulation.

Tissue transglutaminase activity protects from cutaneous melanoma metastatic dissemination: an in vivo study.

The role of tissue transglutaminase (TG-2, TGase-2) in cancer development is still a fascinating field of research. The available reports do not elucidate fully its mechanism of action, due to the limitations of in vitro approaches. Therefore, to understand TG-2 role in cancer, we carried out an in vivo study with a more direct approach. TG-2 was in vivo overexpressed in a murine model of melanoma (intravenous injection of B16 melanoma cells in C57BL/6N mice) by means of a plasmid carrying the TG-2 cDNA. The evaluation of the frequency and size of the metastases indicated that the number of melanoma lung foci was more markedly reduced by TG-2 overexpression than the metastatic size. Then, TG-2 overexpressing mice showed a prolonged survival with respect to control mice. Further analyses were carried by means of proteomic analysis of melanoma cell lysates and meta-analysis of published transcriptomic datasets. Proteomic analysis of cell lysates from a human melanoma cell line compared to human keratinocytes showed significant differences in the expression of TG-2 substrates known to be involved in proliferation/differentiation and cancer progression. Taken together, these findings indicate a protective role of TG-2 enzymatic activity in melanoma progression in vivo.

3D modelling of three pro-inflammatory molecules in selected fish species.

The inflammatory response is the reaction of all Metazoan organisms to pathogen invasion that initiates when pathogen-derived molecules are recognized by specific pattern recognition receptors expressed mainly on cells of the innate immune system. The successive expression of pro-inflammatory cytokines and chemokines limits pathogen spread, and attracts and activates immune cells to help in the elimination of the invaders. In this paper we focused on the analyses of the 3D structures of three pro-inflammatory molecules (interleukin-1ß, tumor necrosis factor-α, interleukin-8) from selected Teleost fish species (Oncorhynchus mykiss, Dicentrarchus labrax, Chionodraco hamatus) generated using as template models those of experimental homologous proteins. These structures were discussed with the aim to investigate the differences between them and mammalian counterparts and, moreover, to verify the presence of the structural requirements for their biological activities, known mainly in mammals.

Functional hypothesis on miraculin' sweetness by a molecular dynamics approach.

Miraculin differs from other sweet-tasting proteins because it is a taste-modifier having the unusual property of modifying sourness into sweetness. Its dimer is covalently linked by an inter-chain disulphide bond, and shows its taste-modifying activity at acidic pH, with maximum at pH 3.0, while it is flat at neutral pH. Previous studies suggested the importance of two histidine residues for the taste-modifying activity of miraculin. In this work, we have conducted molecular dynamics simulations on wild type miraculin and on three mutated dimers (H29A, H59A and H29A/H59A) both at neutral and acidic pH to investigate the structural and functional role of these two His residues. Our results suggested that at acidic pH the presence of two charged His at the interface induced a structural rearrangement of the two monomers, thus leading to their relative opening and the following adaptation of their conformation to the receptor surface. On the other hand the simulations on three mutants showed that the mutated dimers had a closed form, and highlighted the important role of H29 in stabilizing/destabilizing the dimer arrangement and also a cooperative effect of the two histidines.

Molecular and structural characterisation of a macrophage migration inhibitory factor from sea bass (Dicentrarchus labrax L.).

The macrophage migration inhibitory factor (MIF) is a cytokine produced in numerous cell types, mainly T lymphocytes and macrophages, in response to inflammatory stimuli. In this paper we report the identification of a cDNA encoding a MIF molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and its 3D structure obtained by template-based modelling. The sea bass MIF cDNA consists of 609bp that translates in one reading frame to give the entire molecule containing 115 amino acids. The sequence contains three cysteine residues in conserved positions compared to human MIF and most Teleost fishes, with the exception of zebrafish and carp. The Cys(57)-Ala(58)-Leu(59)-Cys(60) motif, present inside the stretch important for JAB1-interaction and mediator of the thiol-protein oxidoreductase activity of MIF, is conserved in sea bass, together with the Pro(2) residue that is crucial for the tautomerase catalytic activity. Real-time PCR analyses revealed that MIF is constitutively expressed in all selected tissues and organs, with the highest mRNA level observed in thymus. MIF expression was induced after 4h in vitro stimulation of head kidney leukocytes with LPS and decreased after 24h. The predicted 3D model of sea bass MIF has been used to verify the presence of structural requirements for its known biological activities.

Platelet-derived growth factor-receptor alpha strongly inhibits melanoma growth in vitro and in vivo.

Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Ralpha may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Ralpha respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Ralpha. Proliferation was rescued by PDGF-Ralpha inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Ralpha mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Ralpha was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Ralpha show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Balpha and a marked increase of p38gamma, mitogen-activated protein kinase kinase 3, and signal regulatory protein alpha1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Ralpha reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Ralpha strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.

Human prion protein helices: studying their stability by molecular dynamics simulations.

To study the intrinsic structural properties of three in human prion helices and analyse their stability, we conducted molecular dynamics simulations, applied helix propensity predictions and evaluated the energetic contribution of the helical regions to the PrP protein stability. Our results suggest that three helices present different stability and the helix 2 results the least stable.

Redesigning the reactive site loop of the wheat subtilisin/chymotrypsin inhibitor (WSCI) by site-directed mutagenesis. A protein-protein interaction study by affinity chromatography and molecular modeling.

A site-directed mutagenesis strategy was employed to obtain four mutants of wheat subtilisin/chymotrypsin inhibitor (WSCI), with the aim to produce inactive forms of this protein. The mutants were expressed in Escherichia coli as fusion proteins and, after the tag removal, were purified to homogeneity. Three mutants, containing a single mutation at the sequence positions 49 or 50, were named E49S, E49P and Y50G, respectively. These mutants exhibited anti-subtilisin activities comparable to that of the wild type protein; instead, anti-chymotrypsin activity was detectable only for the mutant E49S. A fourth mutant (M48P-E49G), containing a double amino acid substitution at the inhibitor reactive site (P1-P1'), was inactive against both subtilisin and chymotrypsin. In order to investigate the interactions between the putative susceptible enzymes and the mutated forms of WSCI, we performed time-course hydrolysis experiments by incubating samples of the mutants with subtilisin-agarose and chymotrypsin-agarose, respectively. These experiments yielded information on the E/I complex formation, as well as on the timing of the cleavage pattern of some of these mutants. Molecular modeling studies were carried out with the 3D models of the mutants and of their putative complexes with subtilisin and chymotrypsin. In terms of inter- and intra-chain H-bond networks, the observations made for each theoretical E/I complex were found to be fully coherent with experimental data (kinetic and time-course hydrolysis) and supplied specific modalities of interaction of each mutant with the enzyme counterpart.

Molecular characterisation and structural analysis of an interferon homologue in sea bass (Dicentrarchus labrax L.).

The interferons (IFNs) are a large family of soluble cytokines involved in the immune response against viral pathogens. Three families of IFNs have been identified in mammals (type I, type II and type III) and, recently, homologues of type I and type II genes have been found in various teleost fish species. In this paper we report the cloning of a cDNA encoding an type I IFN molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and gene structure and, finally, its 3D structure obtained by template-based modelling. The sea bass IFN cDNA consists of 1047bp that translates in one reading frame to give the entire molecule containing 185 amino acids. The analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential N-glycosylation sites. The sea bass IFN gene contains four introns as with other type I IFN teleost genes, except medaka that contains three introns. Real time PCR was performed after poly I:C stimulation of DLEC cell line to investigate the expression of sea bass IFN and Mx and an induction was observed for both genes. The predicted 3D structure of sea bass IFN is characterized by an "all-alpha" domain that shows an "up-down bundle" architecture made of six helices (ABB'CDE). The two cysteine residues present in the sequence (i.e. Cys(23) and Cys(126)) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. Our results will give the opportunity to investigate more in detail antiviral immune responses in sea bass and add to studies on the evolution of the IFN system in teleosts and vertebrates more generally.

Prediction of the protein structural class by specific peptide frequencies.

We evaluated the i-peptides occurrence frequency in the protein sequences belonging to the two datasets which include proteins with a sequence similarity lower than 25% and 40%, respectively. We worked out a new structural class prediction algorithm using the most frequent i-peptides (with i=2, 3, 4), which characterize the four structural classes. Using the tri-peptides, much more able to gain structural information from sequences compared to the di-peptides, the best results were obtained. Compared to the other methods, similarly founded on peptide occurrence frequencies, our method achieves the best prediction accuracy. We compared it also with methods founded on more sophisticated computational approaches.

The N-terminal 1-16 peptide derived in vivo from protein seminal vesicle protein IV modulates alpha-thrombin activity: potential clinical implications.

We have previously shown that seminal vesicle protein IV (SV-IV) and its 1-70 N-terminal fragment have anti-inflammatory activity and modulate anti-thrombin III (AT) activity. Moreover, mass spectrometry analysis of purified SV-IV has shown that the protein was found to be highly heterogeneous and 14% of the total SV-IV molecules are truncated forms, of particular interest the 1-16, 1-17, and 1-18 peptides. In this work we report experimental data which demonstrate that the 1-16 peptide (P1-16) possesses a marked effect on the AT activity by preventing the formation of the thrombin-AT complex. We found that the formation of thrombin-AT complex is markedly decreased in the presence of P1-16 used at equimolar concentration with thrombin as evaluated with SDS-PAGE. We also monitored the conformational changes of thrombin in the presence of different P1-16 concentrations, and calculated the K(d) of thrombin/P1-16 system by circular dichroism technique. The probable interaction sites of P1-16 with thrombin have been also evaluated by molecular graphics and computational analyses. These results have potential implications in the treatment of sterility and thrombotic diseases.

Molecular modelling of co-receptor CD8 alpha alpha and its complex with MHC class I and T-cell receptor in sea bream (Sparus aurata).

T-cells are the main actors of cell-mediated immune defence; they recognize and respond to peptide antigens associated with MHC class I and class II molecules. In this paper, we investigated by molecular modelling methods in the teleost sea bream (Sparus aurata) the interaction among the molecules of the tertiary complex CD8/MHC-I/TCR, which determines the T-cell-mediated immunological response to foreign molecules. First, we predicted the three-dimensional structure of CD8 alpha alpha dimer and MHC-I, and, successively, we simulated the CD8 alpha alpha/MHC-I complex. Finally, the 3D structure of the CD8/MHC-I/TCR complex was simulated in order to investigate the possible changes that can influence TCR signalling events.

CALCOM: a software for calculating the center of mass of proteins.

UNLABELLED: The center of mass of a protein is an artificial point useful for detecting important and simple features of proteins structure, shape and association.CALCOM is a software which calculates the center of mass of a protein, starting from PDB protein structure files. In the case of protein complexes and of protein-small ligand complexes, the position of protein residues or of ligand atoms respect to each protein subunit can be evaluated, as well as the distance among the center of mass of the protein subunits, in order to compare different conformations and evaluate the relative motion of subunits. AVAILABILITY: THE SERVICE IS AVAILABLE AT THE URL: http://bioinformatica.isa.cnr.it/CALCOM/.

Time-course analysis of genome-wide gene expression data from hormone-responsive human breast cancer cells.

BACKGROUND: Microarray experiments enable simultaneous measurement of the expression levels of virtually all transcripts present in cells, thereby providing a 'molecular picture' of the cell state. On the other hand, the genomic responses to a pharmacological or hormonal stimulus are dynamic molecular processes, where time influences gene activity and expression. The potential use of the statistical analysis of microarray data in time series has not been fully exploited so far, due to the fact that only few methods are available which take into proper account temporal relationships between samples. RESULTS: We compared here four different methods to analyze data derived from a time course mRNA expression profiling experiment which consisted in the study of the effects of estrogen on hormone-responsive human breast cancer cells. Gene expression was monitored with the innovative Illumina BeadArray platform, which includes an average of 30-40 replicates for each probe sequence randomly distributed on the chip surface. We present and discuss the results obtained by applying to these datasets different statistical methods for serial gene expression analysis. The influence of the normalization algorithm applied on data and of different parameter or threshold choices for the selection of differentially expressed transcripts has also been evaluated. In most cases, the selection was found fairly robust with respect to changes in parameters and type of normalization. We then identified which genes showed an expression profile significantly affected by the hormonal treatment over time. The final list of differentially expressed genes underwent cluster analysis of functional type, to identify groups of genes with similar regulation dynamics. CONCLUSIONS: Several methods for processing time series gene expression data are presented, including evaluation of benefits and drawbacks of the different methods applied. The resulting protocol for data analysis was applied to characterization of the gene expression changes induced by estrogen in human breast cancer ZR-75.1 cells over an entire cell cycle.

A CD4 homologue in sea bass (Dicentrarchus labrax): molecular characterization and structural analysis.

CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell co-receptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4h and a decrease after 24h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals.

Molecular modelling of miraculin: Structural analyses and functional hypotheses.

Miraculin is a plant protein that displays the peculiar property of modifying taste by swiching sour into a sweet taste. Its monomer is flavourless at all pH as well as at high concentration; the dimer form elicits its taste-modifying activity at acidic pH; a tetrameric form is also reported as active. Two histidine residues, located in exposed regions, are the main responsible of miraculin activity, as demonstrated by mutagenesis studies. Since structural data of miraculin are not available, we have predicted its three-dimensional structure and simulated both its dimer and tetramer forms by comparative modelling and molecular docking techniques. Finally, molecular dynamics simulations at different pH conditions have indicated that at acidic pH the dimer assumes a widely open conformation, in agreement with the hypotheses coming from other studies.

ESBRI: a web server for evaluating salt bridges in proteins.

UNLABELLED: Salt bridges can play important roles in protein structure and function and have stabilizing and destabilizing effects in protein folding. ESBRI is a software available as web tool which analyses the salt bridges in a protein structure, starting from the atomic coordinates. In the case of protein complexes, the salt bridges between protein chains can be evaluated, as well as those among specific charged amino acids and the different protein subunits, in order to obtain useful information regard the protein-protein interaction. AVAILABILITY: The service is available at the URL: http://bioinformatica.isa.cnr.it/ESBRI/

PreSSAPro: a software for the prediction of secondary structure by amino acid properties.

PreSSAPro is a software, available to the scientific community as a free web service designed to provide predictions of secondary structures starting from the amino acid sequence of a given protein. Predictions are based on our recently published work on the amino acid propensities for secondary structures in either large but not homogeneous protein data sets, as well as in smaller but homogeneous data sets corresponding to protein structural classes, i.e. all-alpha, all-beta, or alpha-beta proteins. Predictions result improved by the use of propensities evaluated for the right protein class. PreSSAPro predicts the secondary structure according to the right protein class, if known, or gives a multiple prediction with reference to the different structural classes. The comparison of these predictions represents a novel tool to evaluate what sequence regions can assume different secondary structures depending on the structural class assignment, in the perspective of identifying proteins able to fold in different conformations. The service is available at the URL http://bioinformatica.isa.cnr.it/PRESSAPRO/.