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Purpose Ovarian cancer is the fifth leading cause of cancer-related deaths in women. Standard treatment consists of tumor debulking surgery followed by platinum and paclitaxel chemotherapy; yet, despite the initial response, about 70–75% of patients develop resistance to chemotherapy. Gold compounds represent a family of very promising anticancer drugs. Among them, we previously investigated the cytotoxic and pro-apoptotic properties of Au(NHC) and Au(NHC)2PF6, i.e., a monocarbene gold(I) complex and the corresponding bis(carbene) complex. Gold compounds are known to alter the redox state of cells interacting with free cysteine and selenocysteine residues of several proteins. Herein, a redox proteomic study has been carried out to elucidate the mechanisms of cytotoxicity in A2780 human ovarian cancer cells. Methods A biotinylated iodoacetamide labeling method coupled with mass spectrometry was used to identify oxidation-sensitive protein cysteines. Results Gold carbene complexes cause extensive oxidation of several cellular proteins; many affected proteins belong to two major functional classes: carbohydrate metabolism, and cytoskeleton organization/cell adhesion. Among the affected proteins, Glyceraldehyde-3-phosphate dehydrogenase inhibition was proved by enzymatic assays and by ESI–MS studies. We also found that Au(NHC)2PF6 inhibits mitochondrial respiration impairing complex I function. Concerning the oxidized cytoskeletal proteins, gold binding to the free cysteines of actin was demonstrated by ESI–MS analysis. Notably, both gold compounds affected cell migration and invasion. Conclusions In this study, we deepened the mode of action of Au(NHC) and Au(NHC)2PF6, identifying common cellular targets but confirming their different influence on the mitochondrial function.

The aim of this study was to characterize the salivary proteome and metabolome of highly trained female and male young basketball players, highlighting common and different traits. A total of 20 male and female basketball players (10 female and 10 male) and 20 sedentary control subjects (10 female and 10 male) were included in the study. The athletes exercised at least five times per week for 2 h per day. Saliva samples were collected mid-season, between 9:00 and 11:00 a.m. and away from sport competition. The proteome and metabolome were analyzed by using 2DE and GC–MS techniques, respectively. A computerized 2DE gel image analysis revealed 43 spots that varied in intensity among groups. Between these spots, 10 (23.2%) were differentially expressed among male athletes and controls, 22 (51.2%) between female basketball players and controls, 11 spots (25.6%) between male and female athletes, and 13 spots (30.2%) between male and female controls. Among the proteins identified were Immunoglobulin, Alpha-Amylase, and Dermcidin, which are inflammation-related proteins. In addition, several amino acids, such as glutamic acid, lysine, ornithine, glycine, tyrosine, threonine, and valine, were increased in trained athletes. In this study, we highlight that saliva is a useful biofluid to assess athlete performance and confirm that the adaptation of men and women to exercise has some common features, but also some different sex-specific behaviors, including differential amino acid utilization and expression of inflammation-related proteins, which need to be further investigated. Moreover, in the future, it will be interesting to examine the influence of sport-type on these differences.

Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. In the present study, we describe, the pattern of differentially abundant spots that is associated to the worsening of dystrophy phenotype induced by chronic exercise. Our proteomic analysis pointed out 34 protein spots with different abundance between sedentary and exercised mdx mice. These proteins belong mostly to glucose metabolism, energy production and sarcomere structure categories. Interestingly exercise induced an increase of typical fast twitch fiber proteins (Troponin T fast skeletal muscle, Troponin I fast skeletal muscle and Myozenin-1) combined with an increase of several glycolytic enzymes. Concerning energy transfer, Adenylate kinase, showed a marked decrease when compared with non-exercised mdx. The decline of this enzyme correlates with increased Creatin kinase enzyme, suggesting that a compensatory energy metabolism mechanism could be activated in mdx mouse skeletal muscle following exercise. In addition, we analysed muscles from exercised mdx mice treated with two natural anti-oxidant compounds, apocynin and taurine, that in our previous study, were proved to be beneficial on some pathology related parameters, and we showed that these compounds can counteract exercise-induced changes in the abundance of several proteins. Significance Mdx mouse model of Duchenne muscular dystrophy shows a phenotype of the disorder milder than in human sufferers. This phenotype can be worsened by a different protocols of chronic exercise. These protocols can mimic the muscle progressive damage observed in humans, can allow studying the effects of inadequate training on dystrophic muscles and have been largely used to assess the ability of a drug to reduce the damage induced by exercise. In this study, we describe for the first time, the pattern of protein variation associated with the worsening of dystrophy phenotype induced by chronic exercise. Our proteomic analysis pointed out 34 protein spots with different amount between sedentary and exercised mdx mice. These proteins belong mostly to glucose metabolism, energy production and sarcomere structure categories and their variation indicates that mdx exercised muscle are not able to carry out the metabolic changes associated to fast-to-slow transition typically observed in aerobically trained muscle.

The imbalance between the reactive oxygen (ROS) and nitrogen (RNS) species production and their handling by the antioxidant machinery (low molecular weight antioxidant molecules and antioxidant enzymes), also known as oxidative stress, is a condition caused by physiological and pathological processes. Moreover, oxidative stress may be due to an overproduction of free radicals during physical exercise. Excess of radical species leads to the modification of molecules, such as proteins - the most susceptible to oxidative modification - lipids and DNA. With regard to the oxidation of proteins, carbonylation is an oxidative modification that has been widely described. Several studies have detected changes in the total amount of protein carbonyls following different types of physical exercise, but only few of these identified the specific amino acidic residues targets of such oxidation. In this respect, proteomic approaches allow to identify the proteins susceptible to carbonylation and in many cases, it is also possible to identify the specific protein carbonylation sites. This review focuses on the role of protein oxidation, and specifically carbonyl formation, for plasma and skeletal muscle proteins, following different types of physical exercise performed at different intensities. Furthermore, we focused on the proteomic strategies used to identify the specific protein targets of carbonylation. Overall, our analysis suggests that regular physical activity promotes a protection against protein carbonylation, due to the activation of the antioxidant defence or of the turnover of protein carbonyls. However, we can conclude that from the comprehensive bibliography analysed, there is no clearly defined specific physiological role about this post-translational modification of proteins.

Amino polystyrene nanospheres are shown to be efficient and controllable delivery devices, capable of transporting several bioactive cargoes. Recently, the design of a new device for prodrug activation, using these nanospheres with palladium encapsulated onto them, has been developed successfully. To study the influence of the cellular uptake of these nanodevices, we investigated the cellular response of human embryonic kidney cells (HEK-293T) and murine fibroblasts (L929) treated with empty or palladium-conjugated amino polystyrene nanospheres. To identify differentially expressed proteins, we performed an exhaustive proteomic analysis. In accordance with genomic data previously obtained, the uptake of the empty nanospheres did not induce significant variation in protein expression levels. Following the treatment with palladium-conjugated nanospheres, some changes in protein profiles in both cell lines were observed; these alterations affect proteins involved in cell metabolism and intracellular transport. No key regulator of the cell cycle result was differentially expressed after the treatment, confirming that these innovative drug delivery systems are harmless and well tolerated by the cells.

// Francesca Magherini 1 , Tania Fiaschi 1 , Elisa Valocchia 1 , Matteo Becatti 1 , Alessandro Pratesi 2 , Tiziano Marzo 2, 3 , Lara Massai 2 , Chiara Gabbiani 3 , Ida Landini 4 , Stefania Nobili 5 , Enrico Mini 5 , Luigi Messori 2 , Alessandra Modesti 1 and Tania Gamberi 1 1 Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy 2 Department of Chemistry “Ugo Schiff”, University of Florence, Florence, Italy 3 Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy 4 Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy 5 Department of Health Sciences, University of Florence, Florence, Italy Correspondence to: Tania Gamberi, email: tania.gamberi@unifi.it Luigi Messori, email: luigi.messori@unifi.it Keywords: gold(I)-N-heterocyclic carbene complexes; A2780 human ovarian cancer cells; proteomics; mitochondrial metabolism; thioredoxin reductase Received: August 13, 2017 Accepted: May 19, 2018 Published: June 15, 2018 ABSTRACT Au(NHC) and Au(NHC) 2 , i.e. a monocarbene gold(I) complex and the corresponding bis(carbene) complex, are two structurally related compounds, endowed with cytotoxic properties against several cancer cell lines. Herein, we explore the molecular and cellular mechanisms at the basis of their cytotoxicity in A2780 human ovarian cancer cells. Through a comparative proteomic analysis, we demonstrated that the number of modulated proteins is far larger in Au(NHC) 2 -treated than in Au(NHC)-treated A2780 cells. Both gold compounds mainly affected proteins belonging to the following functional classes: protein synthesis, metabolism, cytoskeleton and stress response and chaperones. Particularly, Au(NHC) 2 gave rise to an evident upregulation of several glycolytic enzymes. Moreover, only Au(NHC) 2 triggered a net impairment of respiration and a metabolic shift towards glycolysis, suggesting that mitochondria are relevant cellular targets. We also found that both carbenes, similarly to the gold(I) compound auranofin, caused a strong inhibition of the seleno-enzyme thioredoxin reductase (TrxR). In conclusion, we highlighted that coordination of two carbene ligands to the same gold(I) center greatly enhances the antiproliferative effects of the resulting compound in comparison to the monocarbene derivative. Moreover, TrxR inhibition and metabolic impairment seem to play a major role in the Au(NHC) 2 cytotoxicity. Overall, these antiproliferative effects were also confirmed on other two human ovarian cancer cell lines ( i.e. SKOV3 and IGROV1).

Aubipyc is an organogold(III) compound endowed with encouraging anti-proliferative properties in vitro that is being evaluated pre-clinically as a prospective anticancer agent. A classical proteomic approach is exploited here to elucidate the mechanisms of its biological actions in A2780 human ovarian cancer cells. Based on 2-D gel electrophoresis separation and subsequent mass spectrometry identification, a considerable number of differentially expressed proteins were highlighted in A2780 cancer cells treated with Aubipyc. Bioinformatic analysis of the groups of up-regulated and down-regulated proteins pointed out that Aubipyc primarily perturbs mitochondrial processes and the glycolytic pathway. Notably, some major alterations in the glycolytic pathway were validated through Western blot and metabolic investigations. Biological significance This is the first proteomic analysis regarding Aubipyc cytotoxicity in A2780/S ovarian cancer cell line. Aubipyc is a promising gold(III) compound which manifests an appreciable cytotoxicity toward the cell line A2780, being able to overcome resistance to platinum. The proteomic study revealed for Aubipyc different cellular alterations with respect to cisplatin as well as to other gold compound such as auranofin. Remarkably, the bioinformatic analysis of proteomic data pointed out that Aubipyc treatment affected, directly or indirectly, several glycolytic enzymes. These data suggest a new mechanism of action for this gold drug and might have an impact on the use of gold-based drug in cancer treatment.

The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on S. cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild-type BY4741 strain grown on fermentable and on nonfermentable carbon sources. The analysis showed that on nonfermentable medium, the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol, this mutant displayed an increase of some glycolytic and fermentative enzymes such as glyceraldehyde-3-phosphate dehydrogenase 1, enolase 2, pyruvate decarboxylase 1, and alcohol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on nonfermentable carbon source, the lack of Sco1p causes a metabolic shift from respiration to fermentation.

Understanding the relationship between physical exercise, reactive oxygen species and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Unbalanced ROS levels can lead to oxidation of cellular macromolecules and a major class of protein oxidative modification is carbonylation. The aim of this investigation was to study muscle protein expression and carbonylation patterns in trained and untrained animal models. We analyzed two muscles characterized by different metabolisms: tibialis anterior and soleus. Whilst tibialis anterior is mostly composed of fast-twitch fibers, the soleus muscle is mostly composed of slow-twitch fibers. By a proteomic approach we identified 15 protein spots whose expression is influenced by training. Among them in tibialis anterior we observed a down-regulation of several glycolitic enzymes. Concerning carbonylation, we observed the existence of a high basal level of protein carbonylation. Although this level shows some variation among individual animals, several proteins (mostly involved in energy metabolism, muscle contraction, and stress response) appear carbonylated in all animals and in both types of skeletal muscle. Moreover we identified 13 spots whose carbonylation increases after training.

The antimicrobial peptide esculentin 1-21 (Esc 1-21) is a shorter synthetic version of the 46-residue peptide occurring in the Rana esculenta skin secretion. Here we propose an integrated proteomic and transcriptomic approach to interpret the biological effects of this peptide on Saccharomyces cerevisiae. We further investigated the response to this peptide by correlating the results of the transcriptome and proteome analysis with phenotypic effects. The results show that S. cerevisiae adapts to Esc 1-21 using the High Osmolarity Glycerol (HOG) pathway involved in osmotic tolerance and cell wall maintenance. Comparative proteomics reveals that Esc 1-21 causes downregulation of enzymes of the lower glycolytic pathway and in genes involved in spindle body formation and remodelling of cell-wall synthesis. Moreover the peptide induces downexpression of protein actin within 45 min and cells pre-treated with peptide show less sensitivity to osmotic stress and increased sensitivity to heat shock stress. The results obtained with the two different methodologies are in agreement at the cellular process levels. A combined approach may help elucidate the main aspects related to the effects of this peptide on the eukaryotic cell. The employment of different technologies may reveal the potential and limitations of each adapted approach in a prospective application for drug screening.

The cellular alterations produced in cisplatin-resistant A2780 ovarian cancer cells (A2780/R) upon treatment with the cytotoxic organogold(III) complex Aubipyc were investigated in depth through a classical proteomic approach. We observed that A2780/R cell exposure to a cytotoxic concentration of Aubipyc for 24 hours results in a conspicuous number of alterations at the protein level that were carefully examined. Notably, we observed that several affected proteins belong to the glucose metabolism system further supporting the idea that the cytotoxic effects of Aubipyc in A2780/R cells are mostly mediated by an impairment of glucose metabolism in excellent agreement with previous observations on the parent cisplatin-sensitive cell line.

Purpose Extracellular proteins are easily accessible, which presents a subproteome of molecular targets that have high diagnostic and therapeutic potential. Efforts have been made to catalog the cardiac extracellular matridome and analyze the topology of identified proteins for the design of therapeutic targets. Although many bioinformatics tools have been developed to predict protein topology, topology has been experimentally validated for only a very small portion of membrane proteins. The aim of this study was to use a glycoproteomics and MS approach to identify glycoproteins in the extracellular matridome of the infarcted left ventricle (LV) and provide experimental evidence for topological determination. Experimental design Glycoproteomics analysis was performed on eight biological replicates of LV samples from wild-type mice at 7 days following myocardial infarction using SPE of glycopeptides, followed by mass spectrometric identification of N-linked glycosylation sites for topology assessment. Results We identified hundreds of glycoproteins, and the identified N-glycosylation sites provide novel information on the correct topology for membrane proteins present in the infarct setting. Conclusions and clinical relevance Our data provide the foundation for future studies of the LV infarct extracellular matridome, which may facilitate the discovery of drug targets and biomarkers.

Starting from Hippocrates, at the Age of Pericles, the One Health initiative takes inspiration from the Greek father of medicine and is based on his approach which recognizes that human health, animal health and environmental health are part of a whole body. Chiron, the wisest of all centaurs, is the classical mythological representation of an integrated view between man and the environment. Thus, he is the tangible example of the Hippocratic dyad where healthcare is achieved by the integration of man with nature. As a mythological Chiron in modern systems medicine, the integrated body of evidence in proteomics investigations is providing key molecular and analytical knowledge to achieve an evidence based approach. Hereafter we introduce some examples published in this themed proteomics issue.

Are protein functions continuous or discretized? Proteomics investigations are starting to address this non-trivial awesome question focusing upon determining the nature of biological molecular relationships. In the following editorial we present a number of experimental studies published in this themed Proteomics Issue demonstrating the development of a new analogical vision for the interpretation of genotype-phenotype relationships. New metrics and languages are evolving, which may complement the insufficiency based on a binary digital interpretation of biological phenomena, providing new tools for the interpretation of large scale-experimental studies.

Endothelial cell caveolar-rafts are considered functional platforms that recruit several pro-angiogenic molecules to realize an efficient angiogenic program. Here we studied the differential caveolar-raft protein composition of endothelial colony-forming cells following stimulation with VEGF, which localizes in caveolae on interaction with its type-2 receptor. Endothelial colony-forming cells are a cell population identified in human umbilical blood that show all the properties of an endothelial progenitor cell and a high proliferative rate. Two-dimensional gel electrophoresis analysis was coupled with mass spectrometry to identify candidate proteins. The twenty-eight differentially expressed protein spots were grouped according to their function using Gene Ontology classification. In particular, functional categories relative to cell death inhibition and hydrogen peroxide metabolic processes resulted enriched. In these categories, Peroxiredoxin-2 and 6, that control hydrogen peroxide metabolic processes, are the main enriched molecules together with the anti-apoptotic 78 kDa glucose regulated protein. Some of the proteins we identified had never before identified as caveolar-raft components. Other identified proteins include calpain small subunit-1, known to mediates angiogenic response to VEGF, gelsolin, which regulates stress fiber assembly, and annexin A3, an angiogenic mediator that induces VEGF production. We validated the functional activity of the above proteins, showing that the siRNA silencing of these resulted in the inhibition of capillary morphogenesis. Overall, our data show that VEGF stimulation triggers the caveolar-raft recruitment of proteins that warrant a physiological amount of reactive oxygen species to maintain a proper angiogenic function of endothelial colony-forming cells and preserve the integrity of the actin cytoskeleton.

Previous studies by us and other groups characterized protein expression variation following long-term moderate training, whereas the effects of single bursts of exercise are less known. Making use of a proteomic approach, we investigated the effects of acute swimming exercise (ASE) on protein expression and carbonylation patterns in two hind limb muscles: the Extensor Digitorum Longus (EDL) and the Soleus, mostly composed of fast-twitch and slow-twitch fibres, respectively. Carbonylation is one of the most common oxidative modifications of proteins and a marker of oxidative stress. In fact, several studies suggest that physical activity and the consequent increase in oxygen consumption can lead to increase in reactive oxygen and nitrogen species (RONS) production, hence the interest in examining the impact of RONS on skeletal muscle proteins following ASE. Results indicate that protein expression is unaffected by ASE in both muscle types. Unexpectedly, the protein carbonylation level was reduced following ASE. In particular, the analysis found 31 and 5 spots, in Soleus and EDL muscles respectively, whose carbonylation is reduced after ASE. Lipid peroxidation levels in Soleus were markedly reduced as well. Most of the decarbonylated proteins are involved either in the regulation of muscle contractions or in the regulation of energy metabolism. A number of hypotheses may be advanced to account for such results, which will be addressed in future studies.

DIGE (difference in gel electrophoresis) proteomics is exploited here to gain insight into the molecular mechanisms of two established ruthenium-based antimetastatic agents, namely trans-[tetrachloro (DMSO) (imidazole)ruthenate(III)] (NAMI-A) and [Ru(eta(6)-toluene)Cl-2(PTA)] (RAPTA-T), where PTA is 1,3,5-triaza-7-phosphaadamantane. Following 24 h exposure of A2780/S human ovarian carcinoma cells to pharmacologically relevant concentrations of either ruthenium compound, 2D-DIGE proteomic analysis evidenced only few differentially expressed proteins with respect to controls. Successive mass spectrometry measurements, MALDI-TOF (matrix assisted laser desorption ionization-time of flight) or LC-ESI/MS-MS (liquid chromatography-electrospray ionization/multi-stage mass spectrometry), allowed identification of most altered protein spots, some of which were associated to perturbations in specific cellular functions. Direct insight into the cellular effects of the investigated metallodrugs is thus achieved. Notably, the patterns of protein alterations induced by NAMI-A and RAPTA-T are quite similar to each other while being deeply different from those of cisplatin. To the best of our knowledge this is the first proteomic study on human cancer cells investigating responses to antimetastatic ruthenium drugs. The key role of new "omic" approaches for deciphering the elusive and complex biochemical mechanisms through which anticancer metallodrugs produce their pharmacological effects is further documented. (C) 2012 Elsevier Inc. All rights reserved.

Cupriavidus metallidurans CH34 is a completely sequenced soil-borne beta-proteobacterium with known genome and proteome. Comparative 2-D electrophoresis and protein mass spectrometry were used to compare the proteome of C. metallidurans CH34 from liquid culture and after incubation for 1, 3, and 12 days in microcosms containing quartz sand, kaolinite, montmorillonite, or an artificial soil. Results showed that proteome from liquid culture was similar to CH34 proteins extracted from sand and kaolinite, whereas the proteins extracted from artificial soil differed significantly and no proteins were detected from C. metallidurans CH34 incubated in the montmorillonite microcosms. Protein recovery decreased on prolonging incubation time in all microcosms. Mass spectrometry identification showed that the trend of lower recovery upon incubation time was independent on the putative function of protein. These results suggest that the soil solid phase influences the protein recovery and soil proteomic analysis and that distinction between protein recovery and protein expression in soil will be a challenging for soil proteomic researchers.

Cytotoxic gold compounds hold today great promise as new pharmacological agents for treatment of human ovarian carcinoma; yet, their mode of action is still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2D-DIGE analysis to identify differential protein expression in a cisplatin-sensitive human ovarian cancer cell line (A2780/S) following treatment with two representative gold(iii) complexes that are known to be potent antiproliferative agents, namely AuL12 and Au(2)Phen. Software analysis using DeCyder was performed and few differentially expressed protein spots were visualized between the three examined settings after 24 h exposure to the cytotoxic compounds, implying that cellular damage at least during the early phases of exposure is quite limited and selective, reflecting the attempts of the cell to repair damage and to survive the insult. The potential of novel proteomic methods to disclose mechanistic details of cytotoxic metallodrugs is herein further highlighted. Different patterns of proteomic changes were highlighted for the two metallodrugs with only a few perturbed protein spots in common. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified. Two of these were validated by western blotting: Ubiquilin-1, responsible for inhibiting degradation of proteins such as p53 and NAP1L1, a candidate marker identified in primary tumors. Ubiquilin-1 resulted over-expressed following both treatments and NAP1L1 was down-expressed in AuL12-treated cells in comparison with control and with Au(2)Phen-treated cells. In conclusion, we performed a comprehensive analysis of proteins regulated by AuL12 and Au(2)Phen, providing a useful insight into their mechanisms of action.

Our aim was to identify the key proteins involved in the pathogenesis of AAAs. To explore the possible pathogenetic mechanisms involved in AAA, we analyzed by proteomics modifications in plasma proteome of patients with AAA. Therefore, the present study analyzed the soluble plasma proteins using two dimensional electrophoresis (2-DE) and mass spectrometry (MS). We identified 33 protein spots, 31 of which show an up-regulation in AAA patients whilst the expression level of 2 protein spots is reduced. We confirm a number of biomarkers associated with AAA that have been previously identified by various authors. We identified a significant increase of a class of proteins such as fibrinogen, α1-antitrypsin and haptoglobin in plasma from AAA patients. The presence of these proteins in human AAA plasma may be related to the inflammatory processes active in these subjects. We have seen a negative correlation between the vitamin D-binding protein (DBP) and hemoglobin subunit β and AAA presence. DBP levels have been found to increase in AAA wall tissues by others and this discrepancy with our results could be due to the different analysis source. We wanted to analyze the factors measurable in plasma-associated rather than in tissue-associated markers because the application of circulating biomarkers in diagnostic laboratories would be relatively simple. DBP is very important for vascular remodelling and it may have an important role in the protection of vascular walls. In plasma tissue this protein reduces platelet aggregation and extends coagulation time. No one protein identified in this study has the biologic plausibility to be used singularly as a biomarker of aneurysmal disease due to inadequate specificity. The effect of using multiple biomarkers combined with clinical factors requires investigation in carefully designed population-based studies and these studies need to select the criteria of choice to define healthy controls very carefully. Clearer identification of various markers is needed, possibly using other proteomic techniques to screen for new candidates such as gel-free proteomic technology that enables us to handle larger groups of subject compared to gel-based proteomic technology.

Regular physical activity is associated with a reduced risk of coronary heart disease, as it probably modifies the balance between free-radical generation and antioxidant activity. On the other hand, however, acute physical activity increases oxygen uptake and leads to a temporary imbalance between the production of reactive oxygen and nitrogen species (RONS) and their disposal: this phenomenon is called oxidative stress. Proteins are one of the most important oxidation targets during physical exercise and carbonylation is one of the most common oxidative protein modifications. In cells there is a physiological level of oxidized proteins that doesn't interfere with cell function; however, an increase in oxidized protein levels may cause a series of cellular malfunctions that could lead to a disease state. For this reason the quantification of protein oxidation is important to distinguish a healthy state from a disease state. Several studies have demonstrated an increase of carbonylated plasma proteins in athletes after exercise, but none have identified targets of this oxidation. Recently a process of protein decarbonylation has been discovered, this may indicate that carbonylation could be involved in signal transduction. The aim of our research was to characterize plasma protein carbonylation in response to physical exercise in trained male endurance athletes. We analyzed by proteomic approach their plasma proteins at resting condition and after two different kinds of physical exercise (PE). We used 2D-GE followed by western blot with specific antibodies against carbonylated proteins. The 2D analysis identified Haptoglobin as potential protein target of carbonylation after PE. We also identified Serotransferrin and Fibrinogen whose carbonylation is reduced after exercise. These methods have allowed us to obtain an overview of plasma protein oxidation after physical exercise.

volume of the Italian Proteomics Association 5th Annual national conference

Since the discovery of cisplatin and its introduction in the clinics, metal compounds have been intensely investigated in view of their possible application in cancer therapy. In this frame, a deeper understanding of their mode of action, still rather obscure, might turn crucial for the design and the obtainment of new and better anticancer agents. Due to the extreme complexity of the biological systems, it is now widely accepted that innovative and information-rich methods are absolutely needed to afford such a goal. Recently, both proteomic and metallomic strategies were successfully implemented for the elucidation of specific mechanistic features of anticancer metallodrugs within an innovative “Systems Biology” perspective. Particular attention was paid to the following issues: i) proteomic studies of the molecular basis of platinum resistance; ii) proteomic analysis of cellular responses to cytotoxic metallodrugs; iii) metallomic studies of the transformation and fate of metallodrugs in cellular systems. Notably, those pioneering studies, that are reviewed here, allowed a significant progress in the understanding of the molecular mechanisms of metal based drugs at the cellular level. A further extension of those studies and a closer integration of proteomic and metallomic strategies and technologies might realistically lead to rapid and significant advancements in the mechanistic knowledge of anticancer metallodrugs.

We have recently shown that a group of structurally diverse gold compounds are highly cytotoxic toward a panel of 36 human tumor cell lines through a variety of biochemical mechanisms. A classic proteomic approach is exploited here to gain deeper insight into those mechanisms. This investigation is focused on Auoxo6, a novel binuclear gold(III) complex, and auranofin, a clinically established gold(I) antiarthritic drug. First, the 72-h cytotoxicity profiles of Auoxo6 and auranofin were determined against A2780 human ovarian carcinoma cells. Subsequently, protein extraction from gold-treated A2780 cells sensitive to cisplatin and 2D gel electrophoresis separation were carried out according to established procedures. Notably, both metallodrugs caused relatively modest changes in protein expression in comparison with controls as only 11 out of approximately 1,300 monitored spots showed appreciable quantitative changes. Very remarkably, six altered proteins were in common between the two treatments. Eight altered proteins were identified by mass spectrometry; among them was ezrin, a protein associated with the cytoskeleton and involved in apoptosis. Interestingly, two altered proteins, i.e., peroxiredoxins 1 and 6, are known to play crucial roles in the cell redox metabolism. Increased cleavage of heterogeneous ribonucleoprotein H was also evidenced, consistent with caspase 3 activation. Overall, the results of the present proteomic study point out that the mode of action of Auoxo6 is strictly related to that of auranofin, that the induced changes in protein expression are limited and selective, that both gold compounds trigger caspase 3 activation and apoptosis, and that a few affected proteins are primarily involved in cell redox homeostasis.

Background Postoperative organ dysfunction in conventional surgery for abdominal aortic aneurysm (AAA) is associated with a complex inflammatory reaction, with activation of coagulation and fibrinolysis. A prospective,observational study was performed to define the complex plasma proteomic changes after AAA repair and to identify factor(s) that may affect myocardial function in uncomplicated procedures. Methods Ten patients undergoing infrarenal AAA repair were investigated. Eight subjects subjected to major abdominal surgery served as controls. Hemodynamic changes were continuously monitored by using the pressure recording analytical method technique. The time course of plasma proteins was investigated after induction of anesthesia and at different times after surgery (6 h, 12 h, 24 h, 36 h) by using two-dimensional difference gel electrophoresis, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and Western blot. The effects of plasma on the functional properties of isolated rat ventricular myocytes were also investigated. Results In AAA patients alone, 18 spots were found to change more than two-fold in expression level, spot identification revealing an increased thrombin generation 6 h after surgery. At the same time cardiac cycle efficiency significantly reduced versus baseline (-0.5 +/- 0.9 vs. 0.18 +/- 0.3 in AAA patients, P < 0.01; 0.4 +/- 0.1 vs. 0.2 +/- 0.3 in control surgery, not significant; P < 0.01 group x time interaction at ANOVA). Plasma obtained 6 h after AAA surgery dose-dependently inhibited contractile function of control rat myocytes (percent shortening fell by 51% with 10% of AAA plasma and was abolished with 20% of AAA plasma, P < 0.001 for both). The inhibitory response was abolished by thrombin antagonism. Conclusions These findings show for the first time the possible role of thrombin generation within the complex activation of inflammatory response in causing hemodynamic instability in the early postoperative period after AAA surgery.

Several human diseases are associated with the deposition of stable ordered protein aggregates known as amyloid fibrils. In addition, a large wealth of data shows that proteins not involved in amyloidoses, are able to form, in vitro, amyloid-like prefibrillar and fibrillar assemblies indistinguishable from those grown from proteins associated with disease. Previous studies showed that early prefibrillar aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) are cytotoxic, inducing early mitochondria membrane depolarization, activation of caspase 9 and eventually cell death. To gain knowledge on the molecular basis of HypF-N aggregate cytotoxicity, we performed a differential proteomic analysis of NIH-3T3 cells exposed to HypF-N prefibrillar aggregates in comparison with control cells. Two-dimensional gel electrophoresis followed by protein identification by MALDI-TOF MS, allowed us to identify 21 proteins differentially expressed. The changes of the expression level of proteins involved in stress response (Hsp60 and 78 kDa glucose-regulated protein) and in signal transduction (Focal adhesion kinase1) appear particularly interesting as possible determinants of the cell fate. The levels of some of the differently expressed proteins were modified also in similar studies carried out on cells exposed to Abeta or alpha-synuclein aggregates, supporting the existence of shared features of amyloid cytotoxicity.

H-Ras oncogene requires deregulation of additional oncogenes or inactivation of tumor suppressor proteins to increase cell proliferation rate and transform cells. In fact, the expression of the constitutively activated H-RasV12 induces cell growth arrest and premature senescence, which act like barriers in pre-neoplastic lesions. In our experimental model, human fibroblasts transfected with H-RasV12 show a dramatic modification of morphology. H-RasV12 expressing cells also show premature senescence followed by cell death, induced by autophagy and apoptosis. In this context, we provide evidence that in H-RasV12 expressing cells, the premature senescence is associated with cellular redox imbalance as well as with altered post-translation protein modification. In particular, redox imbalance is due to a strong reduction of total antioxidant capacity, and significant decrease of glutathione level. As the reversible addition of glutathione to cysteinyl residues of proteins is an important post-translational regulative modification, we investigated S-glutathionylation in cells expressing active H-Ras. In this contest we observed different S-glutathionylation patterns in control and H-RasV12 expressing cells. Particularly, the GAPDH enzyme showed S-glutathionylation increase and significant enzyme activity depletion in H-Ras V12 cells. In conclusion, we proposed that antioxidant defense reduction, glutathione depletion and subsequent modification of S-glutathionylation of target proteins contribute to arrest cell growth, leading to death of fibroblasts expressing constitutively active H-Ras oncogene, thus acting as oncogenic barriers that obstacle the progression of cell transformation.