Metabolic characterisation of transglutaminase 2 inhibitor effects in breast cancer cell lines.

Transglutaminase 2 (TG2), which mediates post-translational modifications of multiple intracellular enzymes, is involved in the pathogenesis and progression of cancer. We used 1 H-NMR metabolomics to study the effects of AA9, a novel TG2 inhibitor, on two breast cancer cell lines with distinct phenotypes, MCF-7 and MDA-MB-231. AA9 can promote apoptosis in both cell lines, but it is particularly effective in MD-MB-231, inhibiting transamidation reactions and decreasing cell migration and invasiveness. This metabolomics study provides evidence of a major effect of AA9 on MDA-MB-231 cells, impacting glutamate and aspartate metabolism, rather than on MCF-7 cells, characterised by choline and O-phosphocholine decrease. Interestingly, AA9 treatment induces myo-inositol alteration in both cell lines, indicating action on phosphatidylinositol metabolism, likely modulated by the G protein activity of TG2 on phospholipase C. Considering the metabolic deregulations that characterise various breast cancer subtypes, the existence of a metabolic pathway affected by AA9 further points to TG2 as a promising hot spot. The metabolomics approach provides a powerful tool to monitor the effectiveness of inhibitors and better understand the role of TG2 in cancer.

Salivary Cytokines as Biomarkers for Oral Squamous Cell Carcinoma: A Systematic Review.

The prognosis of patients with oral squamous carcinoma (OSCC) largely depends on the stage at diagnosis, the 5-year survival rate being approximately 30% for advanced tumors. Early diagnosis, including the detection of lesions at risk for malignant transformation, is crucial for limiting the need for extensive surgery and for improving disease-free survival. Saliva has gained popularity as a readily available source of biomarkers (including cytokines) useful for diagnosing specific oral and systemic conditions. Particularly, the close interaction between oral dysplastic/neoplastic cells and saliva makes such fluid an ideal candidate for the development of non-invasive and highly accurate diagnostic tests. The present review has been designed to answer the question: "Is there evidence to support the role of specific salivary cytokines in the diagnosis of OSCC?" We retrieved 27 observational studies satisfying the inclusion and exclusion criteria. Among the most frequent cytokines investigated as candidates for OSCC biomarkers, IL-6, IL-8, TNF-α are present at higher concentration in the saliva of OSCC patients than in healthy controls and may therefore serve as basis for the development of rapid tests for early diagnosis of oral cancer.

Thermal stability, ligand binding and allergenicity data of Mus m 1.0102 allergen and its cysteine mutants.

The presented data were obtained with the lipocalin allergen Mus m 1.0102 and its cysteine mutants MM-C138A, MM-C157A and MM-C138,157A, whose structural features and unfold reversibility investigations are presented in the research article entitled "The allergen Mus m 1.0102: cysteine residues and molecular allergology" [1]. The data were obtained by means of a Dynamic Light Scattering-based thermal stability assay, a Fluorescence-based ligand-binding assay and a basophil degranulation test, and describe proteins' fold stability, ligand binding ability and allergenic potential, respectively. Analysis of the collected data produced the temperatures corresponding to the onset of the protein unfolding, the dissociation constants for N-Phenyl-1-naphthylamine ligand and the profiles of ß-hexosaminidase release from RBL SX-38 cells, sensitized with the serum of selected allergic patients and incubated with increasing antigens concentrations. These data allow for comparison of the lipocalin allergen Mus m 1.0102 with its conserved cysteines mutants and, with regard to their potential application in allergy diagnostics and immunotherapy, they contribute to the process of recombinant allergen characterization and standardization.

The allergen Mus m 1.0102: Cysteine residues and molecular allergology.

Mus m 1.0102 is a member of the mouse Major Urinary Protein family, belonging to the Lipocalins superfamily. Major Urinary Proteins (MUPs) are characterized by highly conserved structural motifs. These include a disulphide bond, involved in protein oxidative folding and protein structure stabilization, and a free cysteine residue, substituted by serine only in the pheromonal protein Darcin (MUP20). The free cysteine is recognized as responsible for the onset of inter- or intramolecular thiol/disulphide exchange, an event that favours protein aggregation. Here we show that the substitution of selected cysteine residues modulates Mus m 1.0102 protein folding, fold stability and unfolding reversibility, while maintaining its allergenic potency. Recombinant allergens used for immunotherapy or employed in allergy diagnostic kits require, as essential features, conformational stability, sample homogeneity and proper immunogenicity. In this perspective, recombinant Mus m 1.0102 might appear reasonably adequate as lead molecule because of its allergenic potential and thermal stability. However, its modest resistance to aggregation renders the protein unsuitable for pharmacological preparations. Point mutation is considered a winning strategy. We report that, among the tested mutants, C138A mutant acquires a structure more resistant to thermal stress and less prone to aggregation, two events that act positively on the protein shelf life. Those features make that MUP variant an attractive lead molecule for the development of a diagnostic kit and/or a vaccine.

Dissection of the Structural Features of a Fungicidal Antibody-Derived Peptide.

The synthetic peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM antibodies, proved to exert a significant activity in vitro against yeast strains, including multidrug resistant isolates. Alanine substitution of positively charged residues led to a decrease in candidacidal activity. A more dramatic reduction in activity resulted from cysteine replacement. Here, we investigated the conformational properties of T11F and its alanine-substituted derivatives by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Peptide interaction with Candida albicans cells was studied by confocal and scanning electron microscopy. T11F and most of its derivatives exhibited CD spectra with a negative band around 200 nm and a weaker positive band around 218 nm suggesting, together with NMR coupling constants, the presence of a polyproline II (PPII) helix, a conformational motif involved in a number of biological functions. Analysis of CD spectra revealed a critical role for phenylalanine in preserving the PPII helix. In fact, only the F11A derivative presented a random coil conformation. Interestingly, the loss of secondary structure influenced the rate of killing, which turned out to be significantly reduced. Overall, the obtained results suggest that the PPII conformation contributes in characterising the cell penetrating and fungicidal properties of the investigated peptides.

Red blood cells metabolome changes upon treatment with different X-ray irradiation doses.

The upholding of red blood cells (RBC) quality and the removal of leukocytes are two essential issues in transfusion therapy. Leukodepletion provides optimum results, nonetheless there are cases where irradiation is recommended for some groups of hematological patients such as the ones with chronic graft-vs-host disease, congenital cellular immunodeficiency, and hematopoietic stem cell transplant recipients. The European guidelines suggest irradiation doses from 25 to 50 Gray (Gγ). We evaluated the effect of different prescribed doses (15 to 50 Gγ) of X-ray irradiation on fresh leukodepleted RBCs bags using a novel protocol that provides a controlled irradiation. Biochemical assays integrated with RBCs metabolome profile, assessed by nuclear magnetic resonance spectroscopy, were performed on RBC units supernatant, during 14 days storage. Metabolome analysis evidenced a direct correlation between concentration increase of three metabolites, glycine, glutamine and creatine, and irradiation dose. Higher doses (35 and 50 Gγ) effect on RBC mean corpuscular volume, hemolysis, and ammonia concentration are considerable after 7 and 14 days of storage. Our data show that irradiation with 50 Gγ should be avoided and we suggest that 35 Gγ should be the upper limit. Moreover, we suggest for leukodepleted RBCs units the irradiation with the prescribed dose of 15 Gγ, value at center of bag, and ranging between 13.35-15 Gγ, measured over the entire bag volume, may guarantee the same benefits of a 25 Gγ dose assuring, in addition, a better quality of RBCs.

The allergen Mus m 1.0102: Dissecting the relationship between molecular conformation and allergenic potency.

BACKGROUND: The species Mus musculus experiences an obligate proteinuria: predominant are the Major Urinary Proteins (MUPs), that, collectively known as the major mouse allergen Mus m 1, are among the most important aeroallergens for mouse allergic patients. The production of a soluble and stable hypoallergenic form of Mus m 1 is essential for the development of immunotherapeutic protocols to treat allergic symptoms. METHODS: We introduced the substitution C138S in recombinant Mus m 1.0102, an allergenic isoform of Mus m 1. Solubility, conformation, stability and ability to refold after chemical denaturation were investigated with dynamic light scattering, circular dichroism, fluorescence and NMR spectroscopy. An in vitro degranulation assay was used to evaluate the protein allergenic potential, and compare it with Mus m 1.0102 and with an hypoallergenic variant bearing the substitution Y120A. RESULTS: Mus m 1.0102-C138S retains a native-like fold revealing, however, local conformational alterations that influence some of its physical and allergenic properties: it is monodispersed, thermostable up to 56°C, able to reversibly unfold and it exhibits an enhanced allergenicity. CONCLUSIONS: The unique free thiol group affects the solution structural stability of the native protein. Because the mutant C138S does not aggregate over time it is a good lead protein to develop diagnostic and therapeutic applications. GENERAL SIGNIFICANCE: We elucidated the relationship between unfolding reversibility and sulphydryl reactivity. We ascribed the enhanced allergenicity of the mutant C138S to an increased accessibility of its allergenic determinants, an enticing feature to further investigate the structural elements of the allergen-IgE interface.

Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins.

Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

¹H, ¹5N, and ¹³C resonance assignment of cerato-populin, a fungal PAMP from Ceratocystis populicola.

Plant pathogenic fungi secrete several non-catalytic proteins involved in various aspects of the pathogenesis process. Amongst these, cerato-populin (Pop1) produced by Ceratocystis populicola; a protein orthologous of cerato-platanin (CP), the core member of the CP family. These two proteins interact with host and non-host plants. In plane leaves they induce synthesis of phytoalexins, disruption of intercellular and intracellular leaf tissue, cell plasmolysis, programmed cell death, over-expression of defence-related genes, H2O2 and NO production, activation of MAPK cascade and plant resistance. All these features point to CP and Pop1 as defence inducers, though Pop1 shows a reduced efficiency. Pop1/CP similarity is 73%. CD spectroscopy highlights some secondary structure differences between Pop1 and CP. Indeed, the region between the first two cysteines (C20-C57), that in CP includes the ß2-strand and it is involved in GlcNAc (N-acetyl-D-glucosamine) interaction, in Pop1 is predicted to be fully disordered.

Peptides of the constant region of antibodies display fungicidal activity.

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Killer peptide: a novel paradigm of antimicrobial, antiviral and immunomodulatory auto-delivering drugs.

The incidence of life-threatening viral and microbial infections has dramatically increased over recent decades. Despite significant developments in anti-infective chemotherapy, many issues have increasingly narrowed the therapeutic options, making it imperative to discover new effective molecules. Among them, small peptides are arousing great interest. This review will focus in particular on a killer peptide, engineered from an anti-idiotypic recombinant antibody that mimics the activity of a wide-spectrum antimicrobial yeast killer toxin targeting ß-glucan cell-wall receptors. The in vitro and in vivo antimicrobial, antiviral and immunomodulatory activities of killer peptide and its ability to spontaneously and reversibly self-assemble and slowly release its active dimeric form over time will be discussed as a novel paradigm of targeted auto-delivering drugs.

Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine.

Recent discoveries suggest cysteine-stabilized toxins and antimicrobial peptides have structure-activity parallels derived by common ancestry. Here, human antimicrobial peptide hBD-2 and rattlesnake venom-toxin crotamine were compared in phylogeny, 3D structure, target cell specificity, and mechanisms of action. Results indicate a striking degree of structural and phylogenetic congruence. Importantly, these polypeptides also exhibited functional reciprocity: (i) they exerted highly similar antimicrobial pH optima and spectra; (ii) both altered membrane potential consistent with ion channel-perturbing activities; and (iii) both peptides induced phosphatidylserine accessibility in eukaryotic cells. However, the Na(v) channel-inhibitor tetrodotoxin antagonized hBD-2 mechanisms, but not those of crotamine. As crotamine targets eukaryotic ion channels, computational docking was used to compare hBD-2 versus crotamine interactions with prototypic bacterial, fungal, or mammalian Kv channels. Models support direct interactions of each peptide with Kv channels. However, while crotamine localized to occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 interacted with prokaryotic and eukaryotic Kv channels but did not occlude either. Together, these results support the hypothesis that antimicrobial and cytotoxic polypeptides have ancestral structure-function homology, but evolved to preferentially target respective microbial versus mammalian ion channels via residue-specific interactions. These insights may accelerate development of anti-infective or therapeutic peptides that selectively target microbial or abnormal host cells.

Solution structure of the phytotoxic protein PcF: the first characterized member of the Phytophthora PcF toxin family.

The PcF protein from Phytophthora cactorum is the first member of the "PcF toxin family" from the plant pathogens Phytophthora spp. It is able to induce withering in tomato and strawberry leaves. The lack of sequence similarity with other proteins hampers the identification of the molecular mechanisms responsible for its toxicity. Here, we show that the six cysteines form a disulphide pattern that is exclusive for PcF and essential for the protein withering activity. The NMR solution structure identifies a novel fold among protein effectors: a helix-loop-helix motif. The presence of a negatively charged surface suggests that it might act as a site of electrostatic interaction. Interestingly, a good fold match with Ole e 6, a plant protein with allergenic activity, highlighted the spatial superimposition of a stretch of identical residues. This finding suggests a possible biological activity based on molecular mimicry.

The solution structure of the outer membrane lipoprotein OmlA from Xanthomonas axonopodis pv. citri reveals a protein fold implicated in protein-protein interaction.

The outer membrane lipoprotein A (OmlA) belongs to a family of bacterial small lipoproteins widely distributed across the beta and gamma proteobacteria. Although the role of numerous bacterial lipoproteins is known, the biological function of OmlA remains elusive. We found that in the citrus canker pathogen, Xanthomonas axonopodis pv. citri (X. citri), OmlA is coregulated with the ferric uptake regulator (Fur) and their expression is enhanced when X. citri is grown on citrus leaves, suggesting that these proteins are involved in plant-pathogen interaction. To gain insights into the function of OmlA, its conformational and dynamic features were determined by nuclear magnetic resonance. The protein has highly flexible N- and C- termini and a structurally well defined core composed of three beta-strands and two small alpha-helices, which pack against each other forming a two-layer alpha/beta scaffold. This protein fold resembles the domains of the beta-lactamase inhibitory protein BLIP, involved in protein-protein binding. In conclusion, the structure of OmlA does suggest that this protein may be implicated in protein-protein interactions required during X. citri infection.

Development of a bacterial cloning vector for expression of scorpion toxins for biotechnological studies.

Scorpion venoms contain toxic peptides that recognize K(+) channels of excitable and non-excitable cells. These toxins comprise three structurally distinct groups designated alpha-KTx, beta-KTx, and gamma-KTx. It is highly desirable to develop systems for the expression of these toxins for further physiological and structural studies. In this work, an expression vector (pTEV3) was constructed by inserting protein D (major capsid of phage lambda) and TEV protease recognition site into plasmid pET21d DNA sequences. Three alpha-KTx toxins (OsK2, PbTx1, and BmKK3) were cloned into vector pTEV3 and expressed as soluble fusion proteins. The fractions containing the purified fusion proteins (protein D-toxin) were treated with TEV protease to remove protein D. The resulting toxins were analyzed by MALDI-TOF Mass Spectrometry. The results showed that the vector is appropriate for the expression of the target toxins in soluble form and that ion exchange purification of these toxins by flow-through recovery is possible. Analysis by MALDI-TOF Mass Spectrometry of Osk2 demonstrated that this toxin was expressed in its native form, as suggested by the values expected for the presence of two disulfide bridges.

Interaction of local anesthetics with a peptide encompassing the IV/S4-S5 linker of the Na+ channel.

The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.

Cerato-platanin, the first member of a new fungal protein family: cloning, expression, and characterization.

The ascomycete Ceratocystis fimbriata, the causal agent of "canker stain disease," secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP similar to proteins of the hydrophobin family.

Lytic activity and structural differences of amphipathic peptides derived from trialysin.

Trialysin is a pore-forming protein found in the saliva of Triatoma infestans (Hemiptera, Reduviidae), the insect vector of Chagas' disease. The protein is active against a broad range of cell types from bacteria to eukaryotic cells. Recognizing that the N-terminus of trialysin harbors the lytic motif [Amino, R., Martins, R. M., Procopio, J., Hirata, I. Y., Juliano, M. A., and Schenkman, S. (2002) J. Biol. Chem. 277, 6207-6213], we designed a set of peptides scanning this region to investigate the structural basis of its biological function. Peptides encompassing residues 1-32 (P6), 1-27 (P7), and 6-32 (P5) efficiently induced lysis of the protozoan parasite Trypanosoma cruzi and Escherichia coli in the 0.4-9.0 microM range, while much higher concentrations were required to cause hemolysis. Other more internal peptides, including peptide P2 (residues 21-47) and others up to residue 52, were less effective. P6 turned out to be the most active of all. P7 has a significantly higher activity than P5 against E. coli, while P5 has a hemolytic activity comparable to that of P6. CD spectroscopy showed that all tested peptides acquire a comparable helical content in solvent mixtures or in detergent micelles. The solution structure of P2 and P5-P7 was determined in a 30% trifluoroethanol/water mixture by nuclear magnetic resonance. All peptides exhibit a structure characterized by a central helical fold, and except for P2, which does not show a continuous hydrophobic surface, they are amphipathic. The structural models show that P5 and P7 extend their structural similarities with the most active peptide, P6, in either the C-terminus or the N-terminus. Amino acid substitutions in the N-terminus of P6 improved hemolysis but did not change the activity against T. cruzi. These results suggest that while amphipathicity is essential for the lytic activity, the selectivity of the active peptides for specific organisms appears to be associated with the structural features of their N- and C-termini.

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II.

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.

Modeling the Trypanosoma cruzi Tc85-11 protein and mapping the laminin-binding site.

Trypanosoma cruzi expresses a set of glycoproteins encoded by the gp85/trans-sialidase gene superfamily. In this report a structure model is proposed for a cloned member of the superfamily, the Tc85-11 protein. The structure consists of an N-terminus beta-propeller and a C-terminus beta-sandwich interconnected by an alpha-helix. The recombinant protein, corresponding to the N-domain (Tc85-N), binds to laminin in a selective manner. Six synthetic 20-mer peptides from the N-domain adhere onto the surface of LLC-MK(2) cells and two of these peptides specifically inhibit the Tc85-N/laminin interaction, indicating that they are the laminin-binding sites of the molecule. Thus, Tc85-11 and other related members of the family appear to be good candidates to play an important role in T. cruzi infection via a laminin mediated host-parasite interaction.