Daniela Cecconi

Protein nitration during defense response in Arabidopsis thaliana

Nitric oxide and reactive oxygen species play a key role in the plant hypersensitive disease resistance response, and protein tyrosine nitration is emerging as an important mechanism of their co‐operative interaction. Up to now, the proteins targeted by this post‐translational modification in plants are still totally unknown. In this study, we analyzed for the first time proteins undergoing nitration during the hypersensitive response by analyzing via 1D‐ and 2D‐western blot the protein extracts from Arabidopsis thaliana plants challenged with an avirulent bacterial pathogen (Pseudomonas syringae pv. Tomato). We show that the plant disease resistance response is correlated with a modulation of nitration of proteins involved in important cellular process, such as photosynthesis, glycolysis and nitrate assimilation. These findings shed new light on the signaling functions of nitric oxide and reactive oxygen species, paving the way on studies on the role of this post‐translational modification in plants.

Proteomic analysis of Arabidopsis halleri shoots in response to the heavy metals cadmium and zinc and rhizosphere microorganisms.

Arabidopsis halleri has the rare ability to colonize heavy metal‐polluted sites and is an emerging model for research on adaptation and metal hyperaccumulation. The aim of this study was to analyze the effect of plant–microbe interaction on the accumulation of cadmium (Cd) and zinc (Zn) in shoots of an ecotype of A. halleri grown in heavy metal‐contaminated soil and to compare the shoot proteome of plants grown solely in the presence of Cd and Zn or in the presence of these two metals and the autochthonous soil rhizosphere‐derived microorganisms. The results of this analysis emphasized the role of plant–microbe interaction in shoot metal accumulation. Differences in protein expression pattern, identified by a proteomic approach involving 2‐DE and MS, indicated a general upregulation of photosynthesis‐related proteins in plants exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy‐demanding process. The analysis also showed that proteins involved in plant defense mechanisms were downregulated indicating that heavy metals accumulation in leaves supplies a protection system and highlights a cross‐talk between heavy metal signaling and defense signaling.

Phospho-proteomic analysis of mantle cell lymphoma cells suggests a pro-survival role of B-cell receptor signaling.

BackgroundMantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues.MethodsPhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines.ResultsPhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels.ConclusionOur data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes.

Characterization of the signaling pathway downstream p75 neurotrophin receptor involved in β-amyloid peptide-dependent cell death

The accumulation of β-amyloid (Aβ) peptide is a key pathogenic event in Alzheimer’s disease. Previous studies have shown that Aβ peptide can damage neurons by activating the p75 neurotrophin receptor (p75NTR). However, the signaling pathway leading to neuronal cell death is not completely understood. By using a neuroblastoma cell line devoid of neurotrophin receptors and engineered to express either a full-length or a death domain (DD)-truncated form of p75NTR, we demonstrated that Aβ peptide activates the mitogen-activated protein kinases (MAPKs) p38 and c-Jun N-terminal kinase (JNK). We also found that Aβ peptide induces the translocation of nuclear factor-κB (NF-κB). These events depend on the DD of p75NTR. β-Amyloid (Aβ) peptide was found not to be toxic when the above interactors were inhibited, indicating that they are required for Aβ-induced neuronal cell death. p75 neurotrophin receptor (p75NTR)-expressing cells became resistant to Aβ toxicity when transfected with dominant-negative mutants of MAPK kinases 3, 4, or 6 (MKK3, MKK4, or MKK6), the inhibitor of κBα, or when treated with chemical inhibitors of p38 and JNK. Furthermore, p75NTR-expressing cells became resistant to Aβ peptide upon transfection with a dominant-negative mutant of p53. These results were obtained in the presence of normal p38 and JNK activation, indicating that p53 acts downstream of p38 and JNK. Finally, we demonstrated that NF-κB activation is dependent on p38 and JNK activation. Therefore, our data suggest a signaling pathway in which Aβ peptide binds to p75NTR and activates p38 and JNK in a DD-dependent manner, followed by NF-κB translocation and p53 activation.

Proteomic changes involved in tenderization of bovine Longissimus dorsi muscle during prolonged ageing

To study proteomic changes involved in tenderization of bovine Longissimus dorsi four Charolaise heifers and four Charolaise bulls muscles were sampled at slaughter after early and long ageing (2-4°C for 12 and 26days respectively). Descriptive sensory evaluation of samples were performed and their tenderness evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged was analysed by cartesian and polar 2-D electrophoresis. Students t-test and Ranking-PCA analyses were performed to detect proteomic modulation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. This research has demonstrated that there are no differences between proteomic patterns of male and females Longissimus dorsi muscle, and that the extension of ageing beyond 12days, did not brings any concrete advantage in terms of sensory quality. Furthermore, the data presented here demonstrated that meat maturation caused changes of the abundance of proteins involved in metabolic, structural, and stress related processes.

Proteomics in pancreatic cancer research

In this review, we give an overview of the actual role of proteomic technologies in the study of pancreatic cancers (PCs). We describe PC proteomics on the basis of sample origins, i.e. tissues, body fluids, and PC cell lines. As regards PC tissues, we report the identification of a number of candidate biomarkers of precursor lesions that may allow early diagnosis of this neoplasia. Moreover, we describe cytoskeletal and hypoxia‐regulated proteins that confirm the involvement of cytoskeleton modifications and metabolism adaptations in carcinogenesis. We also discuss the most important biomarkers identified by proteomic analysis involved in local invasion and distant metastasis, and in the cross‐talk between pancreatic tumor and the surrounding stroma. Furthermore, we report novel candidate biomarkers identified in serum, plasma, and pancreatic juice of cancer patients compared with cancer‐free controls. Proteomic alterations in PC cell line models as compared to normal controls and studies on cell lines treated with drugs or new agents to understand their mechanism of pharmacological action or the onset of drug resistance are also presented. Finally, we discuss the recent improvements obtained in classical 2‐DE and high‐throughput proteomic strategies able to allow the overcoming of relevant proteomic drawbacks.

Proteomic analysis of the compatible interaction between Vitis vinifera and Plasmopara viticola

We analyzed the proteome of grapevine (Vitis vinifera) leaves 24, 48 and 96 h post infection (hpi) with the downy mildew pathogen Plasmopara viticola. Total proteins were separated on 2-DE gels. By MS analysis, we identified 82 unique grapevine proteins differentially expressed after infection. Upregulated proteins were often included in the functional categories of general metabolism and stress response, while proteins related to photosynthesis and energy production were mostly downregulated. As expected, the activation of a defense reaction was observed more often at the late time point, consistent with the establishment of a compatible interaction. Most proteins involved in resistance were isoforms of different PR-10 pathogenesis-related proteins. Although >50 differentially expressed protein isoforms were observed at 24 and 96 hpi, only 18 were detected at 48 hpi and no defense-related proteins were among this group. This profile suggests a transient breakdown in defense responses accompanying the onset of disease, further supported by gene expression analyses and by a western blot analysis of a PR-10 protein. Our data reveal the complex modulation of plant metabolism and defense responses during compatible interactions, and provide insight into the underlying molecular processes which may eventually yield novel strategies for pathogen control in the field.

Changes in amniotic fluid and umbilical cord serum proteomic profiles of foetuses with intrauterine growth retardation

Foetal growth is a result of a complex net of processes, requiring coordination within the maternal, placental, and foetal compartments, the imbalance or lack of which may lead to intrauterine growth restriction (IUGR). IUGR is the major cause of perinatal morbidity and mortality, and is also related to enhanced morbidity and metabolic abnormalities later in life. In the present study, the protein profiles of umbilical cord serum (UCS) and amniotic fluid (AF) of ten IUGR and ten appropriate for gestational age newborns have been analysed by 2‐DE, and nanoHPLC‐Chip/MS technology. A total of 18 and 13 spots were found to be differentially expressed (p<0.01) in UCS and AF respectively. The unique differentially expressed proteins identified by MS/MS analysis were 14 in UCS, and 11 in AF samples. Protein gene ontology classification indicate that 21% of proteins are involved in inflammatory response, 20% in immune response, while a smaller proportion are related to transport, blood pressure, and coagulation. These results support the conclusion that the IUGR condition alters the expression of proteins involved in the coagulation process, immune mechanisms, blood pressure and iron and copper homeostasis control, offering a new insight into IUGR pathogenesis.

Proteomic analysis of Oenococcus oeni freeze‐dried culture to assess the importance of cell acclimation to conduct malolactic fermentation in wine

Cultures of Oenococcus oeni, the most important malolactic bacterium, are used to induce malolactic fermentation in wine. Survival assays in two different wines confirmed that cells acclimated for 24 h in half‐strength wine‐like medium (acclimation medium) enhanced the malolactic performances. To investigate the effect of the pre‐incubation phase on cell physiology, a proteomic study was carried out. Total protein extracts of acclimated and non‐acclimated cell cultures (control) were analyzed by 2‐D‐PAGE. A total of 20 out of approximately 400 spots varied significantly. All the spots were identified by MS analysis and most of them were proteins involved in metabolism, transcription/translation processes and stress response. The results revealed the different physiological status between non‐acclimated and acclimated cells explaining, in part, their different behavior in wine. Regulation of stress proteins such as heat and cold shock proteins was involved. Moreover, the availability of sugars and amino acids (even if at low concentration) in acclimation medium determined a modulation of energy metabolism enhancing the resistance to stressful conditions (as those that cells find in wine when inoculated). Finally, this proteomic study increased knowledge concerning the physiological changes in freeze‐dried culture occurring with pre‐inoculation procedures.

Selenite biotransformation and detoxification by Stenotrophomonas maltophilia SeITE02: Novel clues on the route to bacterial biogenesis of selenium nanoparticles

A putative biosynthetic mechanism for selenium nanoparticles (SeNPs) and efficient reduction of selenite (SeO32-) in the bacterial strain Stenotrophomonas maltophilia SeITE02 are addressed here on the basis of information gained by a combined approach relying on a set of physiological, chemical/biochemical, microscopy, and proteomic analyses. S. maltophilia SeITE02 is demonstrated to efficiently transform selenite into elemental selenium (Se°) by reducing 100% of 0.5mM of this toxic oxyanion to Se° nanoparticles within 48h growth, in liquid medium. Since the selenite reducing activity was detected in the cytoplasmic protein fraction, while biogenic SeNPs showed mainly extracellular localization, a releasing mechanism of SeNPs from the intracellular environment is hypothesized. SeNPs appeared spherical in shape and with size ranging from 160nm to 250nm, depending on the age of the cultures. Proteomic analysis carried out on the cytoplasmic fraction identified an alcohol dehydrogenase homolog, conceivably correlated with the biogenesis of SeNPs. Finally, by Fourier Transformed Infrared Spectrometry, protein and lipid residues were detected on the surface of biogenic SeNPs. Eventually, this strain might be efficaciously exploited for the remediation of selenite-contaminated environmental matrices due to its high SeO32- reducing efficiency. Biogenic SeNPs may also be considered for technological applications in different fields.