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The antibody Trastuzumab (Tz) targeting ERBB2 has improved the prognosis of patients with breast cancer (BCa) that overexpress this receptor. Resistance to Tz negatively impacts prognosis. Several mechanisms have been reported to cause Tz resistance. The objective of this study was to identify common mechanisms in in vitro models of acquired BCa Tz resistance. In particular, we used three widely available ERBB2+ BCa cell lines adapted to grow in Tz. We performed several analyses to address possible changes in phenotype, proliferation, and ERBB2 membrane expres-sion common to the three Tz-R cell lines compared to wt, but none was found. High-resolution mass spectrometry analysis identified, instead, a common set of differentially expressed proteins (DEPs) in Tz-R vs. wt cells. Furthermore, bioinformatic tools revealed that all three Tz-R cell models shared a modulation of proteins involved in the metabolism of lipids, organophosphate biosynthetic process, and macromolecule methylation. Ultrastructural analysis confirmed altera-tion of lipid droplets in resistant cells. Our data strongly support the hypothesis that complex metabolic adaptation, including lipid metabolism, protein phosphorylation, and possibly chro-matin remodeling, may fuel Tz resistance. The identification of a common set of 10 DEPs in all three TZ-resistant cell lines may provide novel targets for therapeutic intervention.

To date, the 5-year overall survival rate of 60% for early-stage non-small cell lung cancer (NSCLC) is still unsatisfactory. Therefore, reliable prognostic factors are needed. Growing evidence shows that cancer progression may depend on an interconnection between cancer cells and the surrounding tumor microenvironment; hence, circulating molecules may represent promising markers of cancer recurrence. In order to identify a prognostic score, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs (Exo-miR) and peptides, in 67 radically resected NSCLCs. The miRnome profile selected the Exo-miR-130a-3p as the most overexpressed in relapsed patients. Peptidome analysis identified four progressively more degraded forms of fibrinopeptide A (FpA), which were depleted in progressing patients. Notably, stepwise Cox regression analysis selected Exo-miR-130a-3p and the greatest FpA (2-16) to build a score predictive of recurrence, where high-risk patients had 18 months of median disease-free survival. Moreover, in vitro transfections showed that higher levels of miR-130a-3p lead to a deregulation of pathways involved in metastasis and angiogenesis, including the coagulation process and metalloprotease increase which might be linked to FpA reduction. In conclusion, by integrating circulating markers, the identified risk score may help clinicians predict early-stage NSCLC patients who are more likely to relapse after primary surgery.

Macrocyclic compounds meso-(p-acetamidophenyl)-calix[4]pyrrole and meso-(m-acetamidophenyl)-calix[4]pyrrole have previously been reported to exhibit cytotoxic properties towards lung cancer cells. Here, we report pre-clinical in vitro and in vivo studies showing that these calixpyrrole derivatives can inhibit cell growth in both PC3 and DU145 prostatic cancer cell lines. We explored the impact of these compounds on programmed cell death, as well as their ability to inhibit cellular invasion. In this study we have demonstrated the safety of these macrocyclic compounds by cytotoxicity tests on ex-vivo human peripheral blood mononuclear cells (PBMCs), and by in vivo subcutaneous administration. Preliminary in vivo tests demonstrated no hepato-, no nephro- and no genotoxicity in Balb/c mice compared to controls treated with cisplatin. These findings suggest these calixpyrroles might be novel therapeutic tools for the treatment of prostate cancer and of particular interest for the treatment of androgen-independent castration-resistant prostate cancer.

Imidazolidine-2-thione substructure represents a pharmaceutically attractive scaffold, being included in different antimicrobial, anticancer and pesticide agents. To further evaluate the pharmaceutical potential of this chemical moiety, imidazolidine-2-thione was reacted with atypical Vilsmeier adducts, obtained by the condensation between dimethylacetamide and various acyl chlorides endowed with different electronic and steric properties. The formation of mono-acylated or di-acylated thiourea derivatives emerged to be affected by the nature of the considered acyl chloride reagent. Computational semi-empirical simulations were carried out to rationalize the relevant factor influencing the outcome of the reaction. As acylthioureas are pharmacologically relevant compounds, the chemical versatility of mono-acylated derivatives were evaluated by reacting benzoyl imidazolidin-2-thione with acyl chlorides. A small library of asymmetric di-acylthioureas was prepared and the obtained derivatives did not show any cytotoxicity on SKOV-3 and MCF-7 cancer cell lines. Additionally, in silico studies predicted good pharmacokinetics properties and promising drug-like characteristics for mono- and di-acylated thioureas. These considerations further support the value of the prepared compounds as interesting non-cytotoxic chemical scaffold useful in the medicinal chemistry field. Graphical abstract

A series of highly functionalized pyrazole derivatives has been prepared by a one-pot, versatile and regioselective procedure. Pyrazoles 1-29 were tested in cell-based assay to assess their antiproliferative activity against a panel of tumour cells. Additionally, the cytotoxicity of prepared compounds was evaluated against normal human fibroblasts. The antiproliferative activity of the synthesized molecules emerged to be affected by the nature of the substituents of the pyrazole scaffold and derivatives 21-23 proved to inhibit the growth of melanoma and cervical cancer cells. Compound 23 was identified as the most active derivative and docking simulations predicted its ability to interact with estrogen receptors.

Breast cancer is still considered a high-incidence disease, and numerous are the research efforts for the development of new useful and effective therapies. Among anticancer drugs, carbazole compounds are largely studied for their anticancer properties and their ability to interfere with specific targets, such as microtubule components. The latter are involved in vital cellular functions, and the perturbation of their dynamics leads to cell cycle arrest and subsequent apoptosis. In this context, we report the anticancer activity of a series of carbazole analogues 1–8. Among them, 2-nitrocarbazole 1 exhibited the best cytotoxic profile, showing good anticancer activity against two breast cancer cell lines, namely MCF-7 and MDA-MB-231, with IC50 values of 7 ± 1.0 and 11.6 ± 0.8 μM, respectively. Furthermore, compound 1 did not interfere with the growth of the normal cell line MCF-10A, contrarily to Ellipticine, a well-known carbazole derivative used as a reference molecule. Finally, in vitro immunofluorescence analysis and in silico studies allowed us to demonstrate the ability of compound 1 to interfere with tubulin organization, similarly to vinblastine: a feature that results in triggering MCF-7 cell death by apoptosis, as demonstrated using a TUNEL assay.

Colorectal cancer is a malignancy with a high incidence. Currently, the drugs used in chemotherapy are often accompanied by strong side effects. Natural secondary metabolites can interfere with chemotherapeutic drugs and intensify their cytotoxic effects. This study aimed to profile the secondary metabolites from the methanol extract of Scabiosa atropurpurea and investigate their in vitro activities, alone or in combination with the chemotherapeutic agent doxorubicin. MTT assay was used to determine the cytotoxic activities. Annexin-V/PI double-staining analysis was employed to evaluate the apoptotic concentration. Multicaspase assay, quantitative reverse transcription real-time PCR (RT-qPCR), and ABC transporter activities were also performed. LC-MS analysis revealed 31 compounds including phenolic acids, flavonoids, and saponins. S. atropurpurea extract intensified doxorubicin anti-proliferative effects against resistant tumor cells and enhanced the cytotoxic effects towards Caco-2 cells after 48 h. The mRNA expression levels of Bax, caspase-3, and p21 were increased significantly whereas Bcl-2 expression level was decreased. Furthermore, the methanol extract reversed P-glycoprotein or multidrug resistance-associated protein in Caco-2 cells. In conclusion, S. atropurpurea improved chemosensitivity and modulated multidrug resistance in Caco-2 cells which makes it a good candidate for further research in order to develop a new potential cancer treatment.

Herein, we report the synthesis and the multiple anti-tumor properties of new gold and silver carbene complexes. The chemical modifications, grounded on our previous studies, led us to identify a good lead complex, gold-based, whose biological features are very exciting and promising in the anti-cancer research and could be further developed. Indeed, the bis-[4,5-dichloro-(N-methyl-N&rsquo, (2-hydroxy-2-phenyl)ethyl-imidazole-2-ylidene)gold(I)]+[dichloro-gold]&minus, (AuL7) complex possesses the ability to interfere with at least three important and different intracellular targets, namely the human topoisomerases I and II and tubulin, which are able to modulate metabolic processes not directly correlated each other. We proved that the modifications of the ligands structure in AuL7, with respect to another already published complex, i.e., bis-[4,5-dichloro-(N-methyl-N&rsquo, (cyclopentane-2ol)-imidazole-2-ylidine)gold(I)]+[dichloro-gold]&minus, (AuL4), produce a different behavior toward tubulin-polymerization process, since AuL7 is a tubulin-polymerization inhibitor and AuL4 a stabilizer, with the final same result of hampering the tumor growth. Taken together, our outcomes designate AuL7 as a promising compound for the development of multi-targeted anti-cancer therapies.

Chemotherapy is used for the treatment of all stages of breast cancer, including the metastatic stage of the disease. Treatment regimens are generally tailored for each patient's particular situation. However, chemotherapeutic agents are the leading cause of serious drug-related adverse effects; moreover, drug resistance often occurs. In this study, we designed and synthesized a new series of N-alkylcarbazoles derived from ellipticine, an alkaloid with a carbazole skeleton initially used in the treatment of metastatic breast cancer and later dismissed because of poor aqueous solubility and severe side effects. After evaluating the binding modes of our class of newly synthesized compounds with human topoisomerase II (hTopo II), we performed hTopo II decatenation assays, identifying compound 4 f (2-(4-((3-chloro-9H-carbazol-9-yl)pentyl)piperazin-1-yl)-N,N,N-trimethylethanammonium iodide) as a good inhibitor. Moreover, 4 f and 4 g (2-(4-((3-chloro-9H-carbazol-9-yl)hexyl)piperazin-1-yl)-N,N,N-trimethylethanammonium iodide) showed a good anti-proliferative activity toward breast cancer cells, causing apoptosis by activation of the caspase pathway. Interestingly, the activity of these two compounds on triple-negative MDA-MB-231 cells, which tend to be highly metastatic and aggressive, is strictly connected to the observed inhibition of hTopo II.

Drugs delivered by proper carriers enter into the cells much more rapidly and carry out their action much more promptly than in the free forms. A high drug concentration can be sustained for longer periods of time at the target site in the cell. In in vivo conditions, this would translate into a reduction of systemic toxicity, dosage and frequency of dosing. Dendritic polymers significantly affect drug delivery in terms of reaching the target site, modifying the bio-distribution of the drug, and enhancing the efficacy of different drugs including anticancer compounds. 2-({[2-({[(2-tolyl)amino]carbonothioyl}oxy)ethyl]amino}carbo-nyl)benzoic acid 1 is a thiocarbamate derivative belonging to an already reported class of non-nucleoside HIV-1 reverse transcriptase inhibitors. In in vitro assay it showed no cytotoxic effects but was endowed with very low solubility and poor activity against wild-type HIV-1 (EC50 = 27 μM). With the aim at improving its water solubility, 1 has been successfully incorporated inside non-toxic amino acids-modified core-shell hetero dendrimers. IR, NMR, zeta potential, mean size of particles, buffer capacity and in vitro release profile of prepared materials were reported. All dendriplexes were evaluated in cell-based assays to assess their cytotoxic profile. The obtained complexes, which harmonize a peripheral polycationic character and a buffer capacity which presuppose efficient cells penetration and increased residence time with a not PAMAM structured biodegradable scaffold, were well water-soluble and could rationally appear as a promising set of prodrugs for safe in vivo administrations.

In the few last years, nanosystems have emerged as a potential therapeutic approach to improve the efficacy and selectivity of many drugs. Cyclodextrins (CyDs) and their nanoparticles have been widely investigated as drug delivery systems. The covalent functionalization of CyD polymer nanoparticles with targeting molecules can improve the therapeutic potential of this family of nanosystems. In this study, we investigated cross-linked γ- and β-cyclodextrin polymers as carriers for doxorubicin (ox) and oxaliplatin (Oxa). We also functionalized γ-CyD polymer bearing COOH functionalities with arginine-glycine-aspartic or arginine moieties for targeting the integrin receptors of cancer cells. We tested the Dox and Oxa anti-proliferative activity in the presence of the precursor polymer with COOH functionalities and its derivatives in A549 (lung, carcinoma) and HepG2 (liver, carcinoma) cell lines. We found that CyD polymers can significantly improve the antiproliferative activity of Dox in HepG2 cell lines only, whereas the cytotoxic activity of Oxa resulted as enhanced in both cell lines. The peptide or amino acid functionalized CyD polymers, loaded with Dox, did not show any additional effect compared to the precursor polymer. Finally, studies of Dox uptake showed that the higher antiproliferative activity of complexes correlates with the higher accumulation of Dox inside the cells. The results show that CyD polymers could be used as carriers for repositioning classical anticancer drugs such as Dox or Oxa to increase their antitumor activity.

Pyrimido-pyrimidine derivatives have been developed as rigid merbarone analogues. In a previous study, these compounds showed potent antiproliferative activity and efficiently inhibited topoisomerase II&alpha, To further extend the structure&ndash, activity relationships on pyrimido-pyrimidines, a novel series of analogues was synthesized by a two-step procedure. Analogues 3&ndash, 6 bear small alky groups at positions 1 and 3 of the pyrimido-pyrimidine scaffold whereas at position 6a (4-chloro)phenyl substituent was inserted. The basic side chains introduced at position 7 were selected on the basis of the previously developed structure&ndash, activity relationships. The antiproliferative activity of the novel compounds proved to be affected by both the nature of the basic side chain and the substituents on the pyrimido-pyrimidine moiety. Derivatives 5d and 5e were identified as the most promising molecules still showing reduced antiproliferative activity in comparison with the previously prepared pyrimido-pyrimidine analogues. In topoisomerase II&alpha, 5d docking complex, the ligand would poorly interact with the enzyme and assume a different orientation in comparison with 1d bioactive conformation.

The indole scaffold has been recognized, over the years, as a model for the synthesis of compounds with anticancer activity by dint of its substantiated ability to act via multiple mechanisms, which also involves the inhibition of enzymes engaged in DNA replication. In this regard, a new series of indole and pyranoindole derivatives have been prepared, some of which showed good antitumor activity and proved their inhibitory effects on the tubulin target. The anticancer activity of the newly synthesized compounds has been evaluated on breast cancer cell lines, as MCF-7 and MDA-MB231, cervical cancer cells line HeLa and Ishikawa endometrial cancer cell line. Among the compounds under study, 7 exhibited a good antitumor activity on HeLa cell line (IC50 = 3.6 ± 0.5), leading to cell death by apoptosis due to the inhibition of tubulin polymerization, which demonstrated that the compound can explicate its function in a similar way to Vinblastine, a well-known inhibitor of tubulin polymerization. The data were also confirmed by in silico assays. No cytotoxicity against normal cells has been detected. Furthermore, in order to investigate the antioxidant properties, DPPH and ABTS tests were performed, together with fluorescence assays on 3T3-L1 cells. All our findings taken together led us to consider compound 7 a favourable candidate for the battle against cancer.

meso-(p-acetamidophenyl)-calix[4]pyrrole 3 was found to exhibit remarkable cytotoxicity towards A549 cancer cells. A comparative study including the isomer of 3meso-(m-acetamidophenyl)-calix[4]pyrrole 5, as well as molecules containing ‘fragments’ of these structures, demonstrated that both the calix[4]pyrrole and the acetamidophenyl units are essential for high cytotoxicity. Although calix[4]pyrroles and other anion-complexing ionophores have recently been reported to induce apoptosis by perturbing cellular chloride concentrations, in our study an alternative mechanism has emerged, as proven by the isolation of covalent DNA adducts revealed by the 32P postlabelling technique. Preliminary pharmacokinetic studies indicate that 3 is able to cross the Blood-Brain-Barrier, therefore being a potential drug that could kill primary and brain metastatic cancer cells simultaneously.

F1Fo-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H+ flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H+ within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F1 and FO domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.

Estrogens influence multiple physiological processes and are implicated in many diseases as well. Cellular responses to estrogens are mainly mediated by the estrogen receptors (ER)α and ERβ, which act as ligand-activated transcription factors. Recently, a member of the G protein-coupled receptor (GPCR) superfamily, namely GPER/GPR30, has been identified as a further mediator of estrogen signalling in different pathophysiological conditions, including cancer. Today, computational methods are commonly used in all areas of health science research. Among these methods, virtual ligand screening has become an established technique for hit discovery and optimization. The absence of an established three-dimensional structure of GPER promoted studies of structure-based drug design in order to build reliable molecular models of this receptor. Here, we discuss the results obtained through the structure-based virtual ligand screening for GPER, which allowed the identification and synthesis of different selective agonist and antagonist moieties. These compounds led significant advances in our understanding of the GPER function at the cellular, tissue, and organismal levels. In particular, selective GPER ligands were critical toward the evaluation of the role elicited by this receptor in several pathophysiological conditions, including cancer. Considering that structure-based approaches are fundamental in drug discovery, future research breakthroughs with the aid of computer-aided molecular design and chemo-bioinformatics could generate a new class of drugs that, acting through GPER, would be useful in a variety of diseases as well as in innovative anticancer strategies.

Estrogens regulate numerous pathophysiological processes, mainly by binding to and activating estrogen receptor (ER)α and ERβ. Increasing amounts of evidence have recently demonstrated that G-protein coupled receptor 30 (GPR30; also known as GPER) is also involved in diverse biological responses to estrogens both in normal and cancer cells. The classical ER and GPER share several features, including the ability to bind to identical compounds; nevertheless, some ligands exhibit opposed activity through these receptors. It is worth noting that, owing to the availability of selective agonists and antagonists of GPER for research, certain differential roles elicited by GPER compared with ER have been identified. Here, we provide evidence on the molecular mechanisms through which a calixpyrrole derivative acts as a GPER antagonist in different model systems, such as breast tumor cells and cancer-associated fibroblasts (CAFs) obtained from breast cancer patients. Our data might open new perspectives toward the development of a further class of selective GPER ligands in order to better dissect the role exerted by this receptor in different pathophysiological conditions. Moreover, calixpyrrole derivatives could be considered in future anticancer strategies targeting GPER in cancer cells.

Transcriptional and posttranscriptional processes regulate expression of genetic networks in response to environmental cues. The extracellular signal-activated p38 MAP kinase (p38) pathway plays a fundamental role in conversion of myoblasts to differentiated myocytes. p38 phosphorylates specific transcription factors and chromatin-associated proteins promoting assembly of the myogenic transcriptome. Here, we demonstrate that p38 alpha and beta isoforms also control muscle-gene expression posttranscriptionally, by stabilizing critical myogenic transcripts. KSRP, an important factor for AU-rich element (ARE)-directed mRNA decay, undergoes p38-dependent phosphorylation during muscle differentiation. KSRP phosphorylated by p38 displays compromised binding to ARE-containing transcripts and fails to promote their rapid decay, although it retains the ability to interact with the mRNA degradation machinery. Overexpression of KSRP selectively impairs induction of ARE-containing early myogenic transcripts, without affecting p38-mediated transcriptional responses. Our results uncover an unanticipated role for KSRP in establishing a biochemical link between differentiation-activated p38 signaling and turnover of myogenic mRNAs.

International audience; Despite tremendous successes of GPCR crystallography, the receptors with available structures represent only a small fraction of human GPCRs. An important role of the modeling community is to maximize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock assessment was established to stimulate and monitor the progress in molecular modeling and ligand docking for GPCRs. The four targets in the present third assessment round presented new and diverse challenges for modelers, including prediction of allosteric ligand interaction and activation states in 5-hydroxytryptamine receptors 1B and 2B, and modeling by extremely distant homology for smoothened receptor. Forty-four modeling groups participated in the assessment. State-of-the-art modeling approaches achieved close-to-experimental accuracy for small rigid orthosteric ligands and models built by close homology, and they correctly predicted protein fold for distant homology targets. Predictions of long loops and GPCR activation states remain unsolved problems.

The formation of a fibrin network following fibrinogen enzymatic activation is the central event in blood coagulation and has important biomedical and biotechnological implications. A non-covalent polymerization reaction between macromolecular monomers, it consists basically of two complementary processes: elongation/branching generates an interconnected 3D scaffold of relatively thin fibrils, and cooperative lateral aggregation thickens them more than 10-fold. We have studied the early stages up to the gel point by fast fibrinogen:enzyme mixing experiments using simultaneous small-angle X-ray scattering and wide-angle, multi-angle light scattering detection. The coupled evolutions of the average molecular weight, size, and cross section of the solutes during the fibrils growth phase were thus recovered. They reveal that extended structures, thinner than those predicted by the classic half-staggered, double-stranded mechanism, must quickly form. Following extensive modeling, an initial phase is proposed in which single-bonded "Y-ladder" polymers rapidly elongate before undergoing a delayed transition to the double-stranded fibrils. Consistent with the data, this alternative mechanism can intrinsically generate frequent, random branching points in each growing fibril. The model predicts that, as a consequence, some branches in these expanding "lumps" eventually interconnect, forming the pervasive 3D network. While still growing, other branches will then undergo a Ca(2+)/length-dependent cooperative collapse on the resulting network scaffolding filaments, explaining their sudden thickening, low final density, and basic mechanical properties.

Sir2-like proteins (Sirtuins) are a class of enzymes conserved throughout the kingdoms of life. In fact, from Archaea to Mammals, these (class III) NAD + -dependent deacetylases catalyse the removal of the acetyl moiety from a substrate protein. Sirtuins show a conserved central catalytic domain with two more variable amino- and carboxy-terminal flanking regions. Amino acid comparison of these central conserved catalytic core sequences allows us to divide Sirtuins into five different classes (I, II, III, IV and U). These proteins differ in their subcellular localization (i.e. in Eukaryotes they can be found in the nucleus, cytoplasm or mitochondria). In humans there are seven Sirtuins (SIRT1-7) that are implicated in various physiological processes including aging and age-related disorders such as neoplasms, cardiovascular, metabolic and neurodegenerative diseases, and inflammation. Nowadays, the estimated life expectancy is definitely longer than in the past thus, we may consider all aging-related problems as having a strong social impact. Consequently, Sirtuins are emerging, particularly from a pharmacological point of view, as new and valuable drug targets.

This report describes the genomic organization of the 5'-region of the human tenascin-C (TN) gene and the functional characterization of its promoter. Approximately 2300 base pairs of the TN gene 5'-flanking region have been cloned and sequenced. This genomic region contains several potential binding sites for transcription factors. By primer extension and S1 nuclease analysis we have localized the transcription start site. The first exon of the TN gene (179 base pairs long) is present in the two major TN transcripts, showing that the expression of these two mRNAs is regulated by a single promoter. The 220 bases upstream to the transcription start site are equally active in directing the expression of chloramphenicol acetyltransferase (CAT) reporter gene in TN producer and nonproducer cells. Using deletion fragments of the human 5'-flanking region we have shown the presence of putative "silencer" elements in the -220 to -2300 region active in both TN producer and nonproducer cell lines. Furthermore, we have demonstrated that the selective transcription in TN producing cells requires the presence of a 1.3-kilobase portion of the TN gene intron 1 in the CAT expression vectors. These findings indicate that complex mechanisms control the transcriptional regulation of TN gene.

G-Protein Coupled Receptor (GPCR) superfamily, which comprises approximately 900 members, is the largest family of protein targets with proven therapeutic value. Although at least 500 GPCRs have been identified as therapeutically relevant, only thirteen GPCRs have been structurally characterized in apo-form or in complex with ligands. GPCRs share relatively low sequence similarity making hard the process of homology modelling, nevertheless some successful hits have been determined. Recently, the G-protein-coupled estrogen receptor 1 (GPER, formerly known as GPR30) has attracted increasing interest due to its ability in mediating estrogen signaling in different normal and cancer tissues. In this regard, the identification of selective GPER ligands has provided valuable tools in order to differentiate the specific functions elicited by this novel estrogen receptor respect to those exerted by the classical estrogen receptors (ERs). In this review, we focus on GPER examining "in silico" docking simulations and evaluating the different binding modes of diverse natural and synthetic ligands.

Functionally different tenascin (TN) isoforms may be generated by alternative splicing of the TN primary transcript. In fact, it has been demonstrated that only the larger TN isoform containing the alternatively spliced region induces loss of focal adhesion in cultured cells and seems able to facilitate cell migration. Recent studies have shown that the higher molecular mass TN isoform is a marker of stromal cell proliferation in hyperplastic and neoplastic breast tissues. This finding prompted us to study the pattern of TN alternative splicing in proliferating and non-proliferating cultured fibroblasts. Here, we show that the mitogenic stimulation of fibroblasts with serum or cytokines leads to an early and striking modification in the steady-state levels of the two major TN mRNAs. We also show that de novo protein synthesis is not necessary for this modification, indicating that it is a "primary response" event. Similarly, mitogenic stimulation induces changes both in synthesis and accumulation of the different TN isoforms.

Although the action of estrogens has been traditionally explained by the binding to and transactivation of the nuclear estrogen receptor (ER)α and ERβ, recently the G protein-coupled receptor GPR30/GPER has been involved in the rapid estrogen signaling. We investigated the ability of two original molecules, which were named GPER-L1 and GPERL2, to bind to and activate the GPER transduction pathway in cancer cells. Competition assays, docking simulations, transfection experiments, real-time PCR, immunoblotting, gene silencing technology and growth assays were performed to ascertain the selective action of GPER-L1 and GPER-L2 in activating the GPER-mediated signaling. Both compounds, which did not show any ability to bind to and activate the classical ERs, were able to bind to GPER and to trigger the rapid activation of the GPER/EGFR/ERK transduction pathway which led to the up-regulation of GPER-target genes. Notably, GPER-L1 and GPER-L2 induced the proliferation of SkBr3 breast and Ishikawa endometrial cancer cells at nM concentrations through GPER, hence providing further evidence on their capability to elicit relevant biological responses mediated by GPER. The identification and characterization of these novel compounds as selective GPER agonists represent a valuable tool to further dissect the pharmacology of this novel estrogen receptor and to better differentiate the specific functions elicited by each estrogen receptor subtype in cancer cells.

Microtubules (MTs), which are highly dynamic assemblies of the protein tubulin, play important and diverse roles in eukaryotic cells. MT dynamics are regulated during the cell cycle by interacting with a large number of endogenous cellular regulators. In addition, many anti-tumour drugs and natural ligands that interact directly with tubulin are able to either stabilise or destabilise MTs and to disrupt the normal dynamics. Herein, we compare the structures of tubulin when complexed with different ligands in order to analyse: (i) various binding-sites of the protein and different positions of ligands within the microtubule (ii) the diverse effect on the microtubule dynamics. The structures and data given are essential for understanding tubulin-ligand interactions and their influence on the regulation of the microtubule system.

Non receptor protein tyrosine kinases are targets in the treatment of a number of diseases. This review focuses on the role of Fes tyrosine kinase and on the design of inhibitors of this protein. Fes and its homologously related protein Fer are the only two members of a distinct class of non receptor tyrosine kinases and they seem to play a role in cytoskeletal rearrangements and inside-out signaling associated with receptor-ligand, cell-matrix and cell-cell interactions. The knowledge of the three dimensional structure of this protein, in fact, has informed drug design, while at the same time it has helped to shed some light on the molecular mechanism at the basis of kinase activation and functions.

Synthesis of the active [Ni–Fe]-hydrogenase in prokaryotes requires a series of ancillary maturation factors. Among them, the HypF maturation factor is a multidomain 82 kDa protein, whose N-terminal domain displays sequence and structural similarities to acylphosphatases. Acylphosphatases are small enzymes that are able to catalyze carboxyl-phosphate bond hydrolysis in acylphosphates, as well as in nucleoside di- and tri-phosphates and in arylphosphates. Here, we present a crystallographic comparison between microgravity and earth-grown crystals of the HypF N-terminal domain. Both crystals were of excellent quality, thereby allowing us to collect very high resolution datasets. A detailed analysis of data collection and refinement statistics, together with an analysis of the diffraction pattern showed that microgravity would appear to further improve the internal order of crystals.

Paired-like homeodomain 2 (Pitx2), first identified as the gene responsible for the Axenfeld-Rieger syndrome, encodes a protein factor that, controlling cell proliferation in a tissue-specific manner, has a crucial role in morphogenesis. During embryonic development, Pitx2 exerts a role in the expansion of muscle progenitors and is expressed at all stages of myogenic progression. In this study, we show that Pitx2 is phosphorylated by the protein kinase Akt2 and is necessary to ensure proper C2C12 myoblast proliferation and differentiation. Pitx2 associates with a ribonucleoprotein complex that includes the mRNA stabilizing factor HuR and sustains Ccnd1 (also known as Cyclin D1) expression, thereby prolonging its mRNA half-life. When the differentiation program is initiated, phosphorylation by Akt2 impairs the ability of Pitx2 to associate with the Ccnd1 mRNA-stabilizing complex that includes HuR and, as a consequence, Ccnd1 mRNA half-life is shortened. We propose that unphosphorylated Pitx2 is required to favor HuR-mediated Ccnd1 mRNA stabilization, thus sustaining myoblast proliferation. Upon Akt2-phosphorylation, the complex Pitx2/HuR/Ccnd1 mRNA dissociates and Ccnd1 mRNA is destabilized. These events contribute to the switch of C2C12 cells from a proliferating to a differentiating phenotype.

Hemoglobins at high concentration have been isolated long ago from some insect larvae living in hypoxic environments. Conversely, a monomeric hemoglobin has been discovered recently in the fruit fly Drosophila melanogaster as intracellular protein expressed both in larvae and in the adult fly. Such a finding indicates that the oxygen supply in insects may be more complex than previously thought, relying not only on O2 diffusion through the tubular tracheal system, but also on carrier-mediated transport and storage. We present here the crystal structure of recombinant D. melanogaster hemoglobin at 1.20 A resolution. Spectroscopic data show that the protein displays a hexacoordinated heme, whose axial ligands are the proximal and distal His residues. Such bis-His ligation of the heme has sizable effects on the protein local structure. Three protein matrix cavities, comparable in size but not in topological locations with those of sperm whale myoglobin, are spread through the protein matrix; one of these can host a xenon atom. Additionally, D. melanogaster hemoglobin binds one molecule of 3-(cyclohexylamino)propanesulfonic acid (CAPS) buffer at a surface pocket, next to the EF hinge. Despite the high resolution achieved, no sequence/structure features specifically supporting the heme hexa- to pentacoordination transition required for diatomic ligand binding could be recognized.

NKp46, a natural killer (NK) cell-specific receptor, has been recently identified as one of the triggering receptors involved in NK cell activation mediated by non-HLA class I ligands. The structure of the NKp46 extracellular receptor region, here reported, consists of two Ig-like domains assembled similarly to leukocyte immunoglobulin-like receptors (LIRs) and killer inhibitory receptors (KIRs). The extensive NKp46 residue substitutions at sites structurally related to those mediating interaction with HLA antigens in LIRs and KIRs indicate that NKp46 recognition processes in vivo should involve non-HLA ligands. NKP46 is shown to stem from an ancestral KIR/LIR family. However, the absence of close paralogues, such as those found for LIR and KIR, indicates that NKp46 is the unique member of a distinct Ig-like subfamily and suggests a specific role, which appears to be maintained across primates and rodents.

Natural killer (NK) cells direct cytotoxicity against tumor or virally infected cells. NK cell activation depends on a fine balance between inhibitory and activating receptors. NKp44 is a cytotoxicity activating receptor composed of one Ig-like extracellular domain, a transmembrane segment, and a cytoplasmic domain. The 2.2 Å crystal structure shows that the NKp44 Ig domain forms a saddle-shaped dimer, where a charged surface groove protrudes from the core structure in each subunit. NKp44 Ig domain disulfide bridge topology defines a new Ig structural subfamily. The data presented are a first step toward understanding the molecular basis for ligand recognition by natural cytotoxicity receptors, whose key role in the immune system is established, but whose cellular ligands are still elusive.

The large extracellular loop of human CD81, a tetraspanin mediating hepatitis C virus envelope protein E2 binding to human cells, has been crystallized in a hexagonal form. The three-dimensional structure, solved and refined at 2.6 A resolution (R-factor = 22.8%), shows that the protein adopts a dimeric assembly, based on an association interface built up by tetraspanin-conserved residues. Structural comparisons with the tertiary structure of human CD81 large extracellular loop, previously determined in a different crystal form, show marked conformational fluctuations in the molecular regions thought to be involved in binding to the viral protein, suggesting rules for recognition and assembly within the tetraspan web.