Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis.

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

Dual and Opposite Costimulatory Targeting with a Novel Human Fusion Recombinant Protein Effectively Prevents Renal Warm Ischemia Reperfusion Injury and Allograft Rejection in Murine Models.

Many studies have shown both the CD28-D80/86 costimulatory pathway and the PD-1-PD-L1/L2 coinhibitory pathway to be important signals in modulating or decreasing the inflammatory profile in ischemia-reperfusion injury (IRI) or in a solid organ transplant setting. The importance of these two opposing pathways and their potential synergistic effect led our group to design a human fusion recombinant protein with CTLA4 and PD-L2 domains named HYBRI. The objective of our study was to determine the HYBRI binding to the postulated ligands of CTLA4 (CD80) and PD-L2 (PD-1) using the Surface Plasmon Resonance technique and to evaluate the in vivo HYBRI effects on two representative kidney inflammatory models-rat renal IRI and allogeneic kidney transplant. The Surface Plasmon Resonance assay demonstrated the avidity and binding of HYBRI to its targets. HYBRI treatment in the models exerted a high functional and morphological improvement. HYBRI produced a significant amelioration of renal function on day one and two after bilateral warm ischemia and on days seven and nine after transplant, clearly prolonging the animal survival in a life-sustaining renal allograft model. In both models, a significant reduction in histological damage and CD3 and CD68 infiltrating cells was observed. HYBRI decreased the circulating inflammatory cytokines and enriched the FoxP3 peripheral circulating, apart from reducing renal inflammation. In conclusion, the dual and opposite costimulatory targeting with that novel protein offers a good microenvironment profile to protect the ischemic process in the kidney and to prevent the kidney rejection, increasing the animal's chances of survival. HYBRI largely prevents the progression of inflammation in these rat models.

SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.

Background: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. Results: We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. Conclusions: We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.

SIRT6-dependent cysteine monoubiquitination in the PRE-SET domain of Suv39h1 regulates the NF-κB pathway.

Sirtuins are NAD+-dependent deacetylases that facilitate cellular stress response. They include SirT6, which protects genome stability and regulates metabolic homeostasis through gene silencing, and whose loss induces an accelerated aging phenotype directly linked to hyperactivation of the NF-κB pathway. Here we show that SirT6 binds to the H3K9me3-specific histone methyltransferase Suv39h1 and induces monoubiquitination of conserved cysteines in the PRE-SET domain of Suv39h1. Following activation of NF-κB signaling Suv39h1 is released from the IκBα locus, subsequently repressing the NF-κB pathway. We propose that SirT6 attenuates the NF-κB pathway through IκBα upregulation via cysteine monoubiquitination and chromatin eviction of Suv39h1. We suggest a mechanism based on SirT6-mediated enhancement of a negative feedback loop that restricts the NF-κB pathway.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

KAT6B Is a Tumor Suppressor Histone H3 Lysine 23 Acetyltransferase Undergoing Genomic Loss in Small Cell Lung Cancer.

Recent efforts to sequence human cancer genomes have highlighted that point mutations in genes involved in the epigenetic setting occur in tumor cells. Small cell lung cancer (SCLC) is an aggressive tumor with poor prognosis, where little is known about the genetic events related to its development. Herein, we have identified the presence of homozygous deletions of the candidate histone acetyltransferase KAT6B, and the loss of the corresponding transcript, in SCLC cell lines and primary tumors. Furthermore, we show, in vitro and in vivo, that the depletion of KAT6B expression enhances cancer growth, while its restoration induces tumor suppressor-like features. Most importantly, we demonstrate that KAT6B exerts its tumor-inhibitory role through a newly defined type of histone H3 Lys23 acetyltransferase activity.

The proteomic 2D-DIGE approach reveals the protein voltage-dependent anion channel 2 as a potential therapeutic target in epithelial thyroid tumours.

We investigated the role of VDAC2 in human epithelial thyroid tumours using proteomic 2D-DIGE analysis and qRT-PCR. We found a significant up-regulation of VDAC2 in thyroid tumours and in thyroid tumour cell lines (TPC-1 and CAL-62). We did not detect overexpression of VDAC2 in a normal thyroid cell line (Nthy-ori 3-1). Silico analysis revealed that two proteins, BAK1 and BAX, had a strong relationship with VDAC2. BAK1 gene expression showed down-regulation in thyroid tumours (follicular and papillary tumours) and in TPC-1 and CAL-62 cell lines. Transient knockdown of VDAC2 in TPC-1 and CAL-62 promoted upregulation of the BAK1 gene and protein expression, and increased susceptibility to sorafenib treatment. Overexpression of the BAK1 gene in CAL-62 showed lower sorafenib sensitivity than VDAC2 knockdown cells. We propose the VDAC2 gene as a novel therapeutic target in these tumours.

Integrative analysis reveals novel pathways mediating the interaction between adipose tissue and pancreatic islets in obesity in rats.

AIMS/HYPOTHESIS: Comprehensive characterisation of the interrelation between the peripancreatic adipose tissue and the pancreatic islets promises novel insights into the mechanisms that regulate beta cell adaptation to obesity. Here, we sought to determine the main pathways and key molecules mediating the crosstalk between these two tissues during adaptation to obesity by the way of an integrated inter-tissue, multi-platform analysis. METHODS: Wistar rats were fed a standard or cafeteria diet for 30 days. Transcriptomic variations by diet in islets and peripancreatic adipose tissue were examined through microarray analysis. The secretome from peripancreatic adipose tissue was subjected to a non-targeted metabolomic and proteomic analysis. Gene expression variations in islets were integrated with changes in peripancreatic adipose tissue gene expression and protein and metabolite secretion using an integrated inter-tissue pathway and network analysis. RESULTS: The highest level of data integration, linking genes differentially expressed in both tissues with secretome variations, allowed the identification of significantly enriched canonical pathways, such as the activation of liver/retinoid X receptors, triacylglycerol degradation, and regulation of inflammatory and immune responses, and underscored interaction network hubs, such as cholesterol and the fatty acid binding protein 4, which were unpredicted through single-tissue analysis and have not been previously implicated in the peripancreatic adipose tissue crosstalk with beta cells. CONCLUSIONS/INTERPRETATION: The integrated analysis reported here allowed the identification of novel mechanisms and key molecules involved in peripancreatic adipose tissue interrelation with beta cells during the development of obesity; this might help the development of novel strategies to prevent type 2 diabetes.

Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome.

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

Proteomics and gene expression analyses of squalene-supplemented mice identify microsomal thioredoxin domain-containing protein 5 changes associated with hepatic steatosis.

Squalene is an abundant hydrocarbon present in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE-knock-out mice. To study its effects on microsomal proteins, 1g/kg/day of squalene was administered to those mice. After 10 weeks, hepatic fat content was assessed and protein extracts of microsomal enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by peptide fingerprinting and MSMS analysis. Squalene administration modified the expression of thirty-one proteins involved in different metabolic functions and increased the levels of those involved in vesicle transport, protein folding and redox status. Only mRNA levels of 9 genes (Arg1, Atp5b, Cat, Hyou1, Nipsnap1, Pcca, Pcx, Pyroxd2, and Txndc5) paralleled these findings. No such mRNA changes were observed in wild-type mice receiving squalene. Thioredoxin domain-containing protein 5 (TXNDC5) protein and mRNA levels were significantly associated with hepatic fat content in apoE-ko mice. These results suggest that squalene action may be executed through a complex regulation of microsomal proteins, both at the mRNA and post-transcriptional levels and the presence of apoE may change the outcome. Txndc5 reflects the anti-steatotic properties of squalene and the sensitivity to lipid accumulation.

Anti-obesity sodium tungstate treatment triggers axonal and glial plasticity in hypothalamic feeding centers.

OBJECTIVE: This study aims at exploring the effects of sodium tungstate treatment on hypothalamic plasticity, which is known to have an important role in the control of energy metabolism. METHODS: Adult lean and high-fat diet-induced obese mice were orally treated with sodium tungstate. Arcuate and paraventricular nuclei and lateral hypothalamus were separated and subjected to proteomic analysis by DIGE and mass spectrometry. Immunohistochemistry and in vivo magnetic resonance imaging were also performed. RESULTS: Sodium tungstate treatment reduced body weight gain, food intake, and blood glucose and triglyceride levels. These effects were associated with transcriptional and functional changes in the hypothalamus. Proteomic analysis revealed that sodium tungstate modified the expression levels of proteins involved in cell morphology, axonal growth, and tissue remodeling, such as actin, CRMP2 and neurofilaments, and of proteins related to energy metabolism. Moreover, immunohistochemistry studies confirmed results for some targets and further revealed tungstate-dependent regulation of SNAP25 and HPC-1 proteins, suggesting an effect on synaptogenesis as well. Functional test for cell activity based on c-fos-positive cell counting also suggested that sodium tungstate modified hypothalamic basal activity. Finally, in vivo magnetic resonance imaging showed that tungstate treatment can affect neuronal organization in the hypothalamus. CONCLUSIONS: Altogether, these results suggest that sodium tungstate regulates proteins involved in axonal and glial plasticity. The fact that sodium tungstate could modulate hypothalamic plasticity and networks in adulthood makes it a possible and interesting therapeutic strategy not only for obesity management, but also for other neurodegenerative illnesses like Alzheimer's disease.

Proteomics and gene expression analyses of mitochondria from squalene-treated apoE-deficient mice identify short-chain specific acyl-CoA dehydrogenase changes associated with fatty liver amelioration.

Squalene, a hydrocarbon involved in cholesterol biosynthesis, is an abundant component in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE knock-out mice. To study the effect of squalene on mitochondrial proteins in fatty liver, 1 g/kg/day of this isoprenoid was administered to those mice. After 10 weeks, hepatic fat was assessed and protein extracts from mitochondria enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by MS analysis. Squalene administration modified the expression of eighteen proteins involved in different metabolic processes, 12 associated with hepatic fat content. Methionine adenosyltransferase I alpha (Mat1a) and short-chain specific acyl-CoA dehydrogenase (Acads) showed significant increased and decreased transcripts, respectively, consistent with their protein changes. These mRNAs were also studied in wild-type mice receiving squalene, where Mat1a was found increased and Acads decreased. However, this mRNA was significantly increased in the absence of apolipoprotein E. These results suggest that squalene action may be executed through a complex regulation of mitochondrial protein expression, including changes in Mat1a and Acads levels. Indeed, Mat1a is a target of squalene administration while Acads reflects the anti-steatotic properties of squalene.

Expression and purification of recombinant apolipoprotein A-I Zaragoza (L144R) and formation of reconstituted HDL particles.

Apolipoprotein A-I Zaragoza (L144R) (apo A-I Z), has been associated with severe hypoalphalipoproteinemia and an enhanced effect of high density lipoprotein (HDL) reverse cholesterol transport. In order to perform further studies with this protein we have optimized an expression and purification method of recombinant wild-type apo A-I and apo A-I Z and produced mimetic HDL particles with each protein. An pET-45 expression system was used to produce N-terminal His-tagged apo A-I, wild-type or mutant, in Escherichia coli BL21 (DE3) which was subsequently purified by affinity chromatography in non-denaturing conditions. HDL particles were generated via a modified sodium cholate method. Expression and purification of both proteins was verified by SDS-PAGE, MALDI-TOF MS and immunochemical procedures. Yield was 30mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wild-type apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies.

Proteomics in obesity research.

Obesity has emerged as one of the major global epidemics of the 21st century and is now reaching alarming proportions. Obese subjects have an increased morbidity and mortality, decreased quality of life and a major risk of developing pathologies such as diabetes mellitus, insulin resistance and cardiovascular disease. Obesity is a complex disease characterised by an increase in body fat mass resulting from an imbalance between energy intake and expenditure. Signal integration between adipose tissue, other peripheral organs and the CNS seems to regulate energy homeostasis. Proteomics may be useful in unravelling the pathogenesis of obesity, since a combination of genetic predisposition and environmental factors account for its development. Most of the proteomic studies performed to date have focused on protein profiling of adipose tissue in different models of experimental obesity and the study of the adipocyte differentiation process. Another issue that has recently attracted attention is the characterisation of the adipocyte secretome, which may be important in signalling to other organs and in regulating energy balance. Target identification of potential therapies has also been investigated by proteomics. This review focuses on the contributions of proteomics to understanding the molecular mechanisms of obesity and their potential therapies.

Integration of DIGE and bioinformatics analyses reveals a role of the antiobesity agent tungstate in redox and energy homeostasis pathways in brown adipose tissue.

Our previous results demonstrated that tungstate decreased weight gain and adiposity in obese rats through increased thermogenesis and lipid oxidation, suggesting that brown adipose tissue was one of the targets of its antiobesity effect. To identify potential targets of tungstate, we used DIGE to compare brown adipose tissue protein extracts from the following experimental groups: untreated lean, tungstate-treated lean, untreated obese, and tungstate-treated obese rats. To distinguish direct targets of tungstate action from those that are secondary to body weight loss, we also included in the analysis an additional group consisting of obese rats that lose weight by caloric restriction. Hierarchical clustering of analysis of variance and t test contrasts clearly separated the different experimental groups. DIGE analysis identified 20 proteins as tungstate obesity direct targets involved in Krebs cycle, glycolysis, lipolysis and fatty acid oxidation, electron transport, and redox. Protein oxidation was decreased by tungstate treatment, confirming a role in redox processes; however, palmitate oxidation, as a measure of fatty acid beta-oxidation, was not altered by tungstate, thus questioning its putative function in fatty acid oxidation. Protein network analyses using Ingenuity Pathways Analysis highlighted peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) as a potential target. We confirmed by real time PCR that indeed tungstate up-regulates PGC-1alpha, and its major target, uncoupling protein 1, was also increased as shown by Western blot. These results illustrate the utility of proteomics and bioinformatics approaches to identify targets of obesity therapies and suggest that in brown adipose tissue tungstate modulates redox processes and increases energy dissipation through uncoupling and PGC-1alpha up-regulation, thus contributing to its overall antiobesity effect.

Phosphorylation events implicating p38 and PI3K mediate tungstate-effects in MIN6 beta cells.

Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. Several lines of evidence indicate the pancreatic beta cell as one of the targets of tungstate action. Here, we examined the molecular mechanism by which this compound exerts its effects on the beta cell line MIN6. Tungstate treatment induced phosphorylation and subsequent activation of p38 and PI3K which in turn are implicated in tungstate PDX-1 nuclear localization and activation. Although no effect was observed in glucose-induced insulin secretion we found that tungstate activates basal insulin release, a process driven, at least in part, by activation of p38. These results show a direct involvement of p38 and PI3K phosphorylation in the mechanism of action of tungstate in the beta cell.

Target identification of the novel antiobesity agent tungstate in adipose tissue from obese rats.

Adipose tissue plays an active role in the development of obesity, and thus characterization of the molecular changes related to obesity in this tissue is a priority. Recently, we identified tungstate as a potent body weight reducing agent in obese animals, adipose tissue being one of the targets of its action. In this study a proteomics approach combining 2-DE and MS was used to identify proteins associated with obesity and targets of tungstate in white adipose tissue. Twenty-nine proteins were found differentially expressed between lean and diet-induced obese rats. Expression changes in transferrin, vimentin, vinculin, peroxiredoxins, Rho-GTP dissociation inhibitor, grifin, guanine deaminase and 3-phosphoglycerate dehydrogenase were associated here for the first time with obesity. Furthermore, tungstate treatment of obese rats reverted expression changes of 70% of the proteins modulated by obesity and another ten proteins were regulated by tungstate independently of the body weight reduction. The results suggest that the tungstate antiobesity effect can be mediated by the modulation of cellular structure, metabolism, redox state and signalling processes in adipose tissue. These findings open new avenues for the study of the aetiology of obesity and its treatment.

Tungstate decreases weight gain and adiposity in obese rats through increased thermogenesis and lipid oxidation.

The increasing worldwide incidence of obesity and the limitations of current treatments raise the need for finding novel therapeutic approaches to treat this disease. The purpose of the current study was first to investigate the effects of tungstate on body weight and insulin sensitivity in a rat model of diet-induced obesity. Second, we aimed to gain insight into the molecular mechanisms underlying its action. Oral administration of tungstate significantly decreased body weight gain and adiposity without modifying caloric intake, intestinal fat absorption, or growth rate in obese rats. Moreover, the treatment ameliorated dislipemia and insulin resistance of obese rats. These effects were mediated by an increase in whole-body energy dissipation and by changes in the expression of genes involved in the oxidation of fatty acids and mitochondrial uncoupling in adipose tissue. Furthermore, treatment increased the number of small adipocytes with a concomitant induction of apoptosis. Our results indicate that tungstate treatment may provide the basis for a promising novel therapy for obesity.

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.