Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.

Proteomic analysis of Porphyromonas gingivalis exposed to nicotine and cotinine

Cogo K, de Andrade A, Labate CA, Bergamaschi CC, Berto LA, Franco GCN, Goncalves RB, Groppo FC. Proteomic analysis of Porphyromonas gingivalis exposed to nicotine and cotinine. J Periodont Res 2012; 47: 766775. (c) 2012 John Wiley & Sons A/S Background and Objective: Smokers are more predisposed than nonsmokers to infection with Porphyromonas gingivalis, one of the most important pathogens involved in the onset and development of periodontitis. It has also been observed that tobacco, and tobacco derivatives such as nicotine and cotinine, can induce modifications to P. gingivalis virulence. However, the effect of the major compounds derived from cigarettes on expression of protein by P.gingivalis is poorly understood. Therefore, this study aimed to evaluate and compare the effects of nicotine and cotinine on the P.gingivalis proteomic profile. Material and Methods: Total proteins of P gingivalis exposed to nicotine and cotinine were extracted and separated by two-dimensional electrophoresis. Proteins differentially expressed were successfully identified through liquid chromatography-mass spectrometry and primary sequence databases using MASCOT search engine, and gene ontology was carried out using DAVID tools. Results: Of the approximately 410 protein spots that were reproducibly detected on each gel, 23 were differentially expressed in at least one of the treatments. A particular increase was seen in proteins involved in metabolism, virulence and acquisition of peptides, protein synthesis and folding, transcription and oxidative stress. Few proteins showed significant decreases in expression; those that did are involved in cell envelope biosynthesis and proteolysis and also in metabolism. Conclusion: Our results characterized the changes in the proteome of P.gingivalis following exposure to nicotine and cotinine, suggesting that these substances may modulate, with minor changes, protein expression. The present study is, in part, a step toward understanding the potential smokepathogen interaction that may occur in smokers with periodontitis.

Quantitative Proteomic Analysis of the Effect of Fluoride on the Acquired Enamel Pellicle

The acquired enamel pellicle (AEP) is a thin film formed by the selective adsorption of salivary proteins onto the enamel surface of teeth. The AEP forms a critical interface between the mineral phase of teeth (hydroxyapatite) and the oral microbial biofilm. This biofilm is the key feature responsible for the development of dental caries. Fluoride on enamel surface is well known to reduce caries by reducing the solubility of enamel to acid. Information on the effects of fluoride on AEP formation is limited. This study aimed to investigate the effects of fluoride treatment on hydroxyapatite on the subsequent formation of AEP. In addition, this study pioneered the use of label-free quantitative proteomics to better understand the composition of AEP proteins. Hydroxyapatite discs were randomly divided in 4 groups (n = 10 per group). Each disc was exposed to distilled water (control) or sodium fluoride solution (1, 2 or 5%) for 2 hours. Discs were then washed and immersed in human saliva for an additional 2 hours. AEP from each disc was collected and subjected to liquid chromatography electrospray ionization mass spectrometry for protein identification, characterization and quantification. A total of 45 proteins were present in all four groups, 12 proteins were exclusively present in the control group and another 19 proteins were only present in the discs treated with 5% sodium fluoride. Relative proteomic quantification was carried out for the 45 proteins observed in all four groups. Notably, the concentration of important salivary proteins, such as statherin and histatin 1, decrease with increasing levels of fluoride. It suggests that these proteins are repulsed when hydroxyapatite surface is coated with fluoride. Our data demonstrated that treatment of hydroxyapatite with fluoride (at high concentration) qualitatively and quantitatively modulates AEP formation, effects which in turn will likely impact the formation of oral biofilms.

Differential Proteomic Analysis of Noncardia Gastric Cancer from Individuals of Northern Brazil

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets.

Proteomic analysis of papaya fruit ripening using 2DE-DIGE

Papayas have a very short green life as a result of their rapid pulp softening as well as their susceptibility to physical injury and mold growth. The ripening-related changes take place very quickly, and there is a continued interest in the reduction of postharvest losses. Proteins have a central role in biological processes, and differential proteomics enables the discrimination of proteins affected during papaya ripening. A comparative analysis of the proteomes of climacteric and pre-climacteric papayas was performed using 2DE-DIGE. Third seven proteins corresponding to spots with significant differences in abundance during ripening were submitted to MS analysis, and 27 proteins were identified and classified into six main categories related to the metabolic changes occurring during ripening. Proteins from the cell wall (alpha-galactosidase and invertase), ethylene biosynthesis (methionine synthase), climacteric respiratory burst, stress response, synthesis of carotenoid precursors (hydroxymethylbutenyl 4-diphosphate synthase, GcpE), and chromoplast differentiation (fibrillin) were identified. There was some correspondence between the identified proteins and the data from previous transcript profiling of papaya fruit, but new, accumulated proteins were identified, which reinforces the importance of differential proteomics as a tool to investigate ripening and provides potentially useful information for maintaining fruit quality and minimizing postharvest losses. (C) 2011 Elsevier B.V. All rights reserved.

Proteomic analysis of banana fruit reveals proteins that are differentially accumulated during ripening

Bananas (Musa spp.) are highly perishable fruit of notable economic and nutritional relevance. Because the identification of proteins involved in metabolic pathways could help to extend green-life and improve the quality of the fruit, this study aimed to compare the proteins of banana pulp at the pre-climacteric and climacteric stages. The use of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) revealed 50 differentially expressed proteins, and comparing those proteins to the Mass Spectrometry Protein Sequence Database (MSDB) identified 26 known proteins. Chitinases were the most abundant types of proteins in unripe bananas, and two isoforms in the ripe fruit have been implicated in the stress/defense response. In this regard, three heat shock proteins and isoflavone reductase were also abundant at the climacteric stage. Concerning fruit quality, pectate lyase, malate dehydrogenase, and starch phosphorylase accumulated during ripening. In addition to the ethylene formation enzyme amino cyclo carboxylic acid oxidase, the accumulation of S-adenosyl-L-homocysteine hydrolase was needed because of the increased ethylene synthesis and DNA methylation that occurred in ripening bananas. Differential analysis provided information on the ripening-associated changes that occurred in proteins involved in banana flavor, texture, defense, synthesis of ethylene, regulation of expression, and protein folding, and this analysis validated previous data on the transcripts during ripening. In this regard, the differential proteomics of fruit pulp enlarged our understanding of the process of banana ripening. (C) 2012 Elsevier B.V. All rights reserved.

A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts

Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

Analysis of the biofilm proteome of Xylella fastidiosa

Abstract Background Xylella fastidiosa is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of X. fastidiosa strain 9a5c, in comparison to planktonic growth condition. Results We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of X. fastidiosa. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments. Conclusions We observed overexpression of proteins related to quorum sensing, proving the existence of communication between cells, and thus the development of structuring the biofilm (mature biofilm) leading to obstruction of vessels and development of disease. This paper reports a first proteomic analysis of mature biofilm of X. fastidiosa, opening new perspectives for understanding the biochemistry of mature biofilm growth in a plant pathogen.

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Background: The unicellular parasite Trypanosoma cruzi is the causative agent of Chagas disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored. Methodology/Principal Findings: Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin. Conclusions/Significance: Herein it is shown, for the first time, that paraflagellar rod proteins and alpha-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

Prognostic significance of NDRG1 expression in oral and oropharyngeal squamous cell carcinoma

Human N-myc downstream-regulated gene 1 (NDRG1) is a metastasis suppressor gene with several potential functions, including cell differentiation, cell cycle regulation and response to hormones, nickel and stress. The purpose of this study was to investigate the immunoexpression of NDRG1 in oral and oropharyngeal squamous cell carcinomas searching for its role in the clinical course of these tumors. We investigated immunohistochemical expression of NDRG1 protein in 412 tissue microarray cores of tumor samples from 103 patients with oral and oropharyngeal squamous cell carcinomas and in 110 paraffin-embedded surgical margin sections. The results showed NDRG1 up-regulation in 101/103 (98.1 %) tumor samples, but no expression in any normal tissue sample. Western blot assays confirmed the immunohistochemical findings, suggesting that lower levels of NDRG1 are associated with a high mortality rate. NDRG1 overexpression was related to long-term specific survival (HR = 0.38; p = 0.009), whereas the presence of lymph-node metastasis showed the opposite association with survival (HR = 2.45; p = 0.013). Our findings reinforce the idea that NDRG1 plays a metastasis suppressor role in oral and oropharyngeal squamous cell carcinomas and may be a useful marker for these tumors.

BPIFB1 (LPLUNC1) is upregulated in cystic fibrosis lung disease

Although the biology the PLUNC (recently renamed BPI fold, BPIF) family of secreted proteins is poorly understood, multiple array based studies have suggested that some are differentially expressed in lung diseases. We have examined the expression of BPIFB1 (LPLUNC1), the prototypic two-domain containing family member, in lungs from CF patients and in mouse models of CF lung disease. BPIFB1 was localized in CF lung samples along with BPIFA1, MUC5AC, CD68 and NE and directly compared to histologically normal lung tissues and that of bacterial pneumonia. We generated novel antibodies to mouse BPIF proteins to conduct similar studies on ENaC transgenic (ENaC-Tg) mice, a model for CF-like lung disease. Small airways in CF demonstrated marked epithelial staining of BPIFB1 in goblet cells but staining was absent from alveolar regions. BPIFA1 and BPIFB1 were not co-localised in the diseased lungs. In ENaC-Tg mice there was strong staining of both proteins in the airways and luminal contents. This was most marked for BPIFB1 and was noted within 2 weeks of birth. The two proteins were present in distinct cells within epithelium. BPIFB1 was readily detected in BAL from ENaC-Tg mice but was absent from wild-type mice. Alterations in the expression of BPIF proteins is associated with CF lung disease in humans and mice. It is unclear if this elevation of protein production, which results from phenotypic alteration of the cells within the diseased epithelium, plays a role in the pathogenesis of the disease.

SET protein accumulates in HNSCC and contributes to cell survival: Antioxidant defense, Akt phosphorylation and AVOs acidification

Objectives: Determination of the SET protein levels in head and neck squamous cell carcinoma (HNSCC) tissue samples and the SET role in cell survival and response to oxidative stress in HNSCC cell lineages. Materials and Methods: SET protein was analyzed in 372 HNSCC tissue samples by immunohistochemistry using tissue microarray and HNSCC cell lineages. Oxidative stress was induced with the pro-oxidant tert-butylhydroperoxide (50 and 250 mu M) in the HNSCC HN13 cell lineage either with (siSET) or without (siNC) SET knockdown. Cell viability was evaluated by trypan blue exclusion and annexin V/propidium iodide assays. It was assessed caspase-3 and -9, PARP-1, DNA fragmentation, NM23-H1, SET, Akt and phosphorylated Akt (p-Akt) status. Acidic vesicular organelles (AVOs) were assessed by the acridine orange assay. Glutathione levels and transcripts of antioxidant genes were assayed by fluorometry and real time PCR, respectively. Results: SET levels were up-regulated in 97% tumor tissue samples and in HNSCC cell lineages. SiSET in HN13 cells (i) promoted cell death but did not induced caspases, PARP-1 cleavage or DNA fragmentation, and (ii) decreased resistance to death induced by oxidative stress, indicating SET involvement through caspase-independent mechanism. The red fluorescence induced by siSET in HN13 cells in the acridine orange assay suggests SET-dependent prevention of AVOs acidification. NM23-H1 protein was restricted to the cytoplasm of siSET/siNC HN13 cells under oxidative stress, in association with decrease of cleaved SET levels. In the presence of oxidative stress, siNC HN13 cells showed lower GSH antioxidant defense (GSH/GSSG ratio) but higher expression of the antioxidant genes PRDX6, SOD2 and TXN compared to siSET HN13 cells. Still under oxidative stress, p-Akt levels were increased in siNC HN13 cells but not in siSET HN13, indicating its involvement in HN13 cell survival. Similar results for the main SET effects were observed in HN12 and CAL 27 cell lineages, except that HN13 cells were more resistant to death. Conclusion: SET is potential (i) marker for HNSCC associated with cancer cell resistance and (ii) new target in cancer therapy. (C) 2012 Elsevier Ltd. All rights reserved.

Different approaches, one target: understanding cellular mechanisms of Parkinson's and Alzheimer's diseases

Neurodegenerative disorders are undoubtedly an increasing problem in the health sciences, given the increase of life expectancy and occasional vicious life style. Despite the fact that the mechanisms of such diseases are far from being completely understood, a large number of studies; that derive from both the basic science and clinical approaches have contributed substantial data in that direction. In this review, it is discussed several frontiers of basic research on Parkinson's and Alzheimer's diseases, in which research groups from three departments of the Institute of Biomedical Sciences of the University of Sao Paulo have been involved in a multidisciplinary effort. The main focus of the review involves the animal models that have been developed to study cellular and molecular aspects of those neurodegenerative diseases, including oxidative stress, insulin signaling and proteomic analyses, among others. We anticipate that this review will help the group determine future directions of joint research in the field and, more importantly, set the level of cooperation we plan to develop in collaboration with colleagues of the Nucleus for Applied Neuroscience Research that are mostly involved with clinical research in the same field.

Identification of epsilon PKC Targets During Cardiac Ischemic Injury

Background: Epsilon-protein kinase C (epsilon PKC) protects the heart from ischemic injury. However, the mechanism(s) of epsilon PKC cardioprotection is still unclear. Identification of the epsilon PKC targets may aid in elucidating the epsilon PKC-mediated cardioprotective mechanisms. Previous studies, using epsilon PKC transgenic mice and difference in gel electrophoresis, identified proteins involved in glucose metabolism, the expression of which was modified by epsilon PKC. Those studies were accompanied by metabolomic analysis, suggesting that increased glucose oxidation may be responsible for the cardioprotective effect of epsilon PKC. Whether these epsilon PKC-mediated alterations were because of differences in protein expression or phosphorylation was not determined. Methods and Results: In the present study, we used an epsilon PKC -specific activator peptide, psi epsilon RACK, combined with phosphoproteomics, to find epsilon PKC targets, and identified that the proteins whose phosphorylation was altered by selective activation of epsilon PKC were mostly mitochondrial proteins. Analysis of the mitochondrial phosphoproteome led to the identification of 55 spots, corresponding to 37 individual proteins, exclusively phosphorylated, in the presence of psi epsilon RACK. The majority of the proteins identified were involved in glucose and lipid metabolism, components of the respiratory chain as well as mitochondrial heat shock proteins. Conclusions: The protective effect of epsilon PKC during ischemia involves phosphorylation of several mitochondrial proteins involved in glucose and lipid metabolism and oxidative phosphorylation. Regulation of these metabolic pathways by epsilon PKC phosphorylation may lead to epsilon PKC-mediated cardioprotection induced by psi epsilon RACK. (Circ J 2012; 76: 1476-1485)