Alberto Milli

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.

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.

Proteomic analysis of dopamine and α-synuclein interplay in a cellular model of Parkinson’s disease pathogenesis

Altered dopamine homeostasis is an accepted mechanism in the pathogenesis of Parkinson’s disease. α‐Synuclein overexpression and impaired disposal contribute to this mechanism. However, biochemical alterations associated with the interplay of cytosolic dopamine and increased α‐synuclein are still unclear. Catecholaminergic SH‐SY5Y human neuroblastoma cells are a suitable model for investigating dopamine toxicity. In the present study, we report the proteomic pattern of SH‐SY5Y cells overexpressing α‐synuclein (1.6‐fold induction) after dopamine exposure. Dopamine itself is able to upregulate α‐synuclein expression. However, the effect is not observed in cells that already overexpress α‐synuclein as a consequence of transfection. The proteomic analysis highlights significant changes in 23 proteins linked to specific cellular processes, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis, and neuronal plasticity. A bioinformatic network enrichment procedure generates a significant model encompassing all proteins and allows us to enrich functional categories associated with the combination of factors analyzed in the present study (i.e. dopamine together with α‐synuclein). In particular, the model suggests a potential involvement of the nuclear factor kappa B pathway that is experimentally confirmed. Indeed, α‐synuclein significantly reduces nuclear factor kappa B activation, which is completely quenched by dopamine treatment.

Signal transduction pathways of mantle cell lymphoma: A phosphoproteome-based study

Mantle cell lymphoma (MCL) is an incurable hematologic malignancy whose pathogenesis is only partly understood. The aim of the present study was to define a “core phosphoproteome” in MCL cell lines that is representative of primary MCL in order to improve knowledge of the signal transduction pathways involved in its tumorigenesis. We have analyzed phosphorylated proteins in several MCL cell lines by immobilized metal affinity chromatography and separation by 2‐D PAGE, followed by RP‐HPLC coupled with MS/MS identification. These data were correlated with information on copy number gains obtained by SNP‐chip analysis. Several of the proteins identified could be linked to a specific signal transduction pathway, and have been recently recognized as important players in MCL pathogenesis, such as nuclear factor‐kappaB (NF‐κB) and phosphoinositide‐3 kinase‐mammalian target of rapamycin (PI3K‐mTOR). However, our data also implicate a number of novel proteins and pathways in the pathobiology of MCL, one of which is mitochondrial signaling. A second‐level analysis identified MAPK1, CK2, CK1, PKCzeta, and PKCepsilon as candidate upstream molecules. Our study provides new insights in MCL pathogenesis and helps to form the basis for testing new target‐specific therapeutics.

Application of partial least squares discriminant analysis and variable selection procedures: a 2D-PAGE proteomic study

Abstract2D gel electrophoresis is a tool for measuring protein regulation, involving image analysis by dedicated software (PDQuest, Melanie, etc.). Here, partial least squares discriminant analysis was applied to improve the results obtained by classic image analysis and to identify the significant spots responsible for the differences between two datasets. A human colon cancer HCT116 cell line was analyzed, treated and not treated with a new histone deacetylase inhibitor, RC307. The proteins regulated by RC307 were detected by analyzing the total lysates and nuclear proteome profiles. Some of the regulated spots were identified by tandem mass spectrometry. The preliminary data are encouraging and the protein modulation reported is consistent with the antitumoral effect of RC307 on the HCT116 cell line. Partial least squares discriminant analysis coupled with backward elimination variable selection allowed the identification of a larger number of spots than classic PDQuest analysis. Moreover, it allows the achievement of the best performances of the model in terms of prediction and provides therefore more robust and reliable results. From this point of view, the multivariate procedure applied can be considered a good alternative to standard differential analysis, also taking into account the interdependencies existing among the variables.

Effect of tannic acid on Lactobacillus plantarum wine strain during starvation: A proteomic study

The molecular mechanisms involved in tannic‐acid (TA)‐mediated cell growth retardation and viability prolongation of Lactobacillus plantarum VP08 strain were evaluated by a proteomic analysis of starved cells grown in the presence of TA or glucose as carbon source. The tannase activity and the cell growth retardation as well as viability prolongation were confirmed by enzymatic assay and growing tests, respectively. In order to gain information about the effect triggered at the molecular level by TA, total proteins (extracted from starved cells grown in 250 mg/L TA, or 2 g/L glucose) were analyzed by a 2D‐PAGE/MS approach to detect differentially expressed proteins. A total of 15 spots were found to be down‐regulated and 21 up‐regulated in TA‐grown cells. The results indicate an overall impact of TA on proteins involved in some cellular and metabolic pathways: glycolysis, amino acid metabolism, translation and protein folding. The modulation of specific proteins correlates with the positive effect of TA on the survival of tannase‐positive L. plantarum.

A proteomic approach for evaluating the cell response to a novel histone deacetylase inhibitor in colon cancer cells

Epigenetic inactivation of gene expression is a general phenomenon associated with malignant transformation. Recently, we have found that a novel series of histone deacetylases (HDAC) inhibitors exhibit a broad-spectrum inhibition profile characterized by a marked effect on acetylation of histone and non-histone proteins. RC307, a representative compound of this series, caused a growth-inhibitory effect in colon carcinoma cells HCT116 associated with G2 accumulation and induction of apoptosis. The present study was designed to investigate the effect of RC307 on protein expressions in the HCT116 cells following treatment with cytotoxic drug concentrations. HCT116 cells were cultured in the absence or presence of RC307 and total cell lysates, as well as nuclear proteins, were extracted. The protein samples were then subjected to two-dimensional polyacrylamide gel electrophoresis, and the 2D gel images were compared to discover the protein changes caused by RC307 treatment. A total of 48 and 46 different spots were found to be modulated by RC307 in total lysates and nuclear proteome of HCT116 cell line. The modulated proteins were identified by tandem mass spectrometry. We found that RC307 exposure modulates proteins that are involved in proliferation, cell cycle regulation, apoptosis, gene expression, as well as chromatin and cytoskeleton organization.

Female urinary proteomics: New insight into exogenous and physiological hormone-dependent changes†

Purpose: In the frame of a research study on possible urinary markers related to physiological hormones cycle, 33 volunteer, healthy, normotensive fertile women were selected. Clinical parameters and renin–angiotensin–aldosterone system (RAAS) components were also investigated, on the basis of the well‐known relation linking sex female hormones and renin and aldosterone levels.

Effects of mushroom and chicory extracts on the shape, physiology and proteome of the cariogenic bacterium Streptococcus mutans

BackgroundDental caries is an infectious disease which results from the acidic demineralisation of the tooth enamel and dentine as a consequence of the dental plaque (a microbial biofilm) accumulation. Research showed that several foods contain some components with antibacterial and antiplaque activity. Previous studies indicated antimicrobial and antiplaque activities in a low-molecular-mass (LMM) fraction of extracts from either an edible mushroom (Lentinus edodes) or from Italian red chicory (Cichorium intybus).MethodsWe have evaluated the antimicrobial mode of action of these fractions on Streptococcus mutans, the etiological agent of human dental caries. The effects on shape, macromolecular syntheses and cell proteome were analysed.ResultsThe best antimicrobial activity has been displayed by the LMM mushroom extract with a bacteriostatic effect. At the MIC of both extracts DNA synthesis was the main macromolecular synthesis inhibited, RNA synthesis was less inhibited than that of DNA and protein synthesis was inhibited only by roughly 50%. The partial inhibition of protein synthesis is compatible with the observed significant increase in cell mass. The increase in these parameters is linked to the morphological alteration with transition from cocci of the untreated control to elongated cells. Interestingly, these modifications were also observed at sub-MIC concentrations. Finally, membrane and cytosol proteome analysis was conducted under LMM mushroom extract treatment in comparison with untreated S. mutans cells. Significant changes were observed for 31 membrane proteins and 20 of the cytosol fractions. The possible role of the changed proteins is discussed.ConclusionsThis report has shown an antibiotic-like mode of action of mushroom and chicory extracts as demonstrated by induced morphogenetic effects and inhibition of specific macromolecular synthesis. This feature as well as the safe use of this extract as result of its natural origin render the LMM both mushroom and chicory extracts suitable for the formulation into products for daily oral hygiene such as mouthwashes or toothpastes.

2D immunomic approach for the study of IgG autoantibodies in the experimental model of multiple sclerosis.

2D-immunomics may be useful in the identification of autoantigens in neurological autoimmune diseases, but its application may be limited by denaturation of target proteins. Here we compared the capacity of a single or multiple antigens to elicit autoantibodies targeting multiple neural autoantigens by ELISA and 2D-immunomics. We induced experimental autoimmune encephalomyelitis (EAE) with MBP peptide(89-104), total MBP or spinal cord homogenate. Both techniques showed anti-MBP IgG only after immunization with total MBP. In addition, 2D-immunomics revealed the presence in EAE mice of autoantibodies targeting other neural proteins, some displaying partial sequence homology with MBP. The present finding by 2D-immunomics of multiple neural proteins targeted by autoantibodies generated by a single antigen may help to explain the complex autoimmune response observed in multiple sclerosis.