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2.  Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera 
Molecular Cancer  2013;12:142.
JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.
PMCID: PMC4225507  PMID: 24252366
Polycythemia vera; Essential thrombocythemia; HSP70; 2D-DIGE/MS
3.  The mzQuantML Data Standard for Mass Spectrometry–based Quantitative Studies in Proteomics* 
The range of heterogeneous approaches available for quantifying protein abundance via mass spectrometry (MS)1 leads to considerable challenges in modeling, archiving, exchanging, or submitting experimental data sets as supplemental material to journals. To date, there has been no widely accepted format for capturing the evidence trail of how quantitative analysis has been performed by software, for transferring data between software packages, or for submitting to public databases. In the context of the Proteomics Standards Initiative, we have developed the mzQuantML data standard. The standard can represent quantitative data about regions in two-dimensional retention time versus mass/charge space (called features), peptides, and proteins and protein groups (where there is ambiguity regarding peptide-to-protein inference), and it offers limited support for small molecule (metabolomic) data. The format has structures for representing replicate MS runs, grouping of replicates (for example, as study variables), and capturing the parameters used by software packages to arrive at these values. The format has the capability to reference other standards such as mzML and mzIdentML, and thus the evidence trail for the MS workflow as a whole can now be described. Several software implementations are available, and we encourage other bioinformatics groups to use mzQuantML as an input, internal, or output format for quantitative software and for structuring local repositories. All project resources are available in the public domain from the HUPO Proteomics Standards Initiative
PMCID: PMC3734589  PMID: 23599424
4.  Contribution of crenarchaeal autotrophic ammonia oxidizers to the dark primary production in Tyrrhenian deep waters (Central Mediterranean Sea) 
The ISME Journal  2011;5(6):945-961.
Mesophilic Crenarchaeota have recently been thought to be significant contributors to nitrogen (N) and carbon (C) cycling. In this study, we examined the vertical distribution of ammonia-oxidizing Crenarchaeota at offshore site in Southern Tyrrhenian Sea. The median value of the crenachaeal cell to amoA gene ratio was close to one suggesting that virtually all deep-sea Crenarchaeota possess the capacity to oxidize ammonia. Crenarchaea-specific genes, nirK and ureC, for nitrite reductase and urease were identified and their affiliation demonstrated the presence of ‘deep-sea' clades distinct from ‘shallow' representatives. Measured deep-sea dark CO2 fixation estimates were comparable to the median value of photosynthetic biomass production calculated for this area of Tyrrhenian Sea, pointing to the significance of this process in the C cycle of aphotic marine ecosystems. To elucidate the pivotal organisms in this process, we targeted known marine crenarchaeal autotrophy-related genes, coding for acetyl-CoA carboxylase (accA) and 4-hydroxybutyryl-CoA dehydratase (4-hbd). As in case of nirK and ureC, these genes are grouped with deep-sea sequences being distantly related to those retrieved from the epipelagic zone. To pair the molecular data with specific functional attributes we performed [14C]HCO3 incorporation experiments followed by analyses of radiolabeled proteins using shotgun proteomics approach. More than 100 oligopeptides were attributed to 40 marine crenarchaeal-specific proteins that are involved in 10 different metabolic processes, including autotrophy. Obtained results provided a clear proof of chemolithoautotrophic physiology of bathypelagic crenarchaeota and indicated that this numerically predominant group of microorganisms facilitate a hitherto unrecognized sink for inorganic C of a global importance.
PMCID: PMC3131861  PMID: 21209665
crenarchaeal accA, amoA, nirK, ureC genes; autotrophic Crenarchaeota; Tyrrhenian Sea; dark ocean primary production; shotgun proteomics
5.  Diacylglycerol kinase ζ controls diacylglycerol metabolism at the immunological synapse 
Molecular Biology of the Cell  2011;22(22):4406-4414.
DGKα and DGKζ negatively regulate the DAG/RasGRP1/Ras pathway in T cells. Study of the specific contribution of each isoform to DAG metabolism during immune synapse formation by use of a combination of RNAi and videomicroscopy techniques identifies DGKζ as mainly responsible for DAG consumption at the immunological synapse.
Diacylglycerol (DAG) generation at the T cell immunological synapse (IS) determines the correct activation of antigen-specific immune responses. DAG kinases (DGKs) α and ζ act as negative regulators of DAG-mediated signals by catalyzing DAG conversion to phosphatidic acid (PA). Nonetheless, the specific input of each enzyme and their spatial regulation during IS formation remain uncharacterized. Here we report recruitment of endogenous DGKα and DGKζ to the T cell receptor (TCR) complex following TCR/CD28 engagement. Specific DGK gene silencing shows that PA production at the activated complex depends mainly on DGKζ, indicating functional differences between these proteins. DGKζ kinase activity at the TCR is enhanced by phorbol-12-myristate-13-acetate cotreatment, suggesting DAG-mediated regulation of DGKζ responsiveness. We used GFP-DGKζ and -DGKα chimeras to assess translocation dynamics during IS formation. Only GFP-DGKζ translocated rapidly to the plasma membrane at early stages of IS formation, independent of enzyme activity. Finally, use of a fluorescent DAG sensor confirmed rapid, sustained DAG accumulation at the IS and allowed us to directly correlate membrane translocation of active DGKζ with DAG consumption at the IS. This study highlights a DGKζ-specific function for local DAG metabolism at the IS and offers new clues to its mode of regulation.
PMCID: PMC3216665  PMID: 21937721
6.  ProteoRed Multicenter Experiment for Long-term Quality Control Evaluation of Proteomics Core Facilities 
Quality control (QC) is becoming increasingly important in proteomic experiments in order to guarantee the quality of research results. Deployment of QC metrics helps in monitoring stability, overall performance and reproducibility of analytical techniques. In an attempt to dispel some of the notions that LC-MS-based proteomics is poorly reproducible, the proteomics community has demonstrated increasingly concerns about the quality of proteomics data made publicly available. Here we describe the ProteoRed Multicenter Experiment for Quality Control (PMEQC), a longitudinal QC multicenter study involving 12 institutions, to assess the repeatability of LC-MS/MS proteomics data within a specific site, the reproducibility across multiple sites and across multiple platforms. Our experimental design also provided a unique opportunity to assess the repeatability of protein sample preparation within a specific site.
The main study was divided into 2 sub studies (Study A and B) that evaluate separately inter- and intra-laboratory variability. Each participant received two sample vials of trypsin-digested yeast proteins (Study A) and the same undigested protein sample (Study B). All participants were requested to follow a strict LC-MS/MS guideline for sample injection amounts and LC gradient. To enable inter-laboratory comparisons, data analysis was centralized and performed under standard procedures using a common workflow that includes well-known software tools for proteomics analysis such as msconvert.exe, X!Tandem, PeptideProphet, OpenMS and R programming language.
Here, we summarize the key findings of the PMEQC project and provide technical insights to better understand and pinpoint the main sources of variability and other issues faced by proteomics core facilities. Our study reveals that the overall performance regarding reproducibility, sensitivity, dynamic range, among other metrics, is directly related to laboratory staff expertise, and less dependent on instrumentation. Furthermore, the present study provides a rich data set of metrics against which other laboratories can benchmark their performance.
PMCID: PMC3630681
7.  The Gel Electrophoresis Markup Language (GelML) from the Proteomics Standards Initiative 
Proteomics  2010;10(17):3073-3081.
The Human Proteome Organisation’s Proteomics Standards Initiative (HUPO-PSI) has developed the GelML data exchange format for representing gel electrophoresis experiments performed in proteomics investigations. The format closely follows the reporting guidelines for gel electrophoresis, which are part of the Minimum Information About a Proteomics Experiment (MIAPE) set of modules. GelML supports the capture of metadata (such as experimental protocols) and data (such as gel images) resulting from gel electrophoresis so that laboratories can be compliant with the MIAPE Gel Electrophoresis guidelines, while allowing such data sets to be exchanged or downloaded from public repositories. The format is sufficiently flexible to capture data from a broad range of experimental processes, and complements other PSI formats for mass spectrometry data and the results of protein and peptide identifications to capture entire gel-based proteome workflows. GelML has resulted from the open standardisation process of PSI consisting of both public consultation and anonymous review of the specifications.
PMCID: PMC3193076  PMID: 20677327
data standard; gel electrophoresis; database; ontology
8.  A DIGE study on the effects of salbutamol on the rat muscle proteome - an exemplar of best practice for data sharing in proteomics 
BMC Research Notes  2011;4:86.
Proteomic techniques allow researchers to perform detailed analyses of cellular states and many studies are published each year, which highlight large numbers of proteins quantified in different samples. However, currently few data sets make it into public databases with sufficient metadata to allow other groups to verify findings, perform data mining or integrate different data sets. The Proteomics Standards Initiative has released a series of "Minimum Information About a Proteomics Experiment" guideline documents (MIAPE modules) and accompanying data exchange formats. This article focuses on proteomic studies based on gel electrophoresis and demonstrates how the corresponding MIAPE modules can be fulfilled and data deposited in public databases, using a new experimental data set as an example.
We have performed a study of the effects of an anabolic agent (salbutamol) at two different time points on the protein complement of rat skeletal muscle cells, quantified by difference gel electrophoresis. In the DIGE study, a total of 31 non-redundant proteins were identified as being potentially modulated at 24 h post treatment and 110 non redundant proteins at 96 h post-treatment. Several categories of function have been highlighted as strongly enriched, providing candidate proteins for further study. We also use the study as an example of best practice for data deposition.
We have deposited all data sets from this study in public databases for further analysis by the community. We also describe more generally how gel-based protein identification data sets can now be deposited in the PRoteomics IDEntifications database (PRIDE), using a new software tool, the PRIDESpotMapper, which we developed to work in conjunction with the PRIDE Converter application. We also demonstrate how the ProteoRed MIAPE generator tool can be used to create and share a complete and compliant set of MIAPE reports for this experiment and others.
PMCID: PMC3080311  PMID: 21443781
10.  Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement 
The Journal of Cell Biology  2007;179(7):1539-1553.
Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes.
PMCID: PMC2373511  PMID: 18158329
11.  The Pseudomonas putida Crc Global Regulator Controls the Expression of Genes from Several Chromosomal Catabolic Pathways for Aromatic Compounds 
Journal of Bacteriology  2004;186(5):1337-1344.
The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic P. putida strains containing either a wild-type or an inactivated crc allele was compared. The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P. putida. It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P. putida.
PMCID: PMC344427  PMID: 14973036
12.  Evidence of Multiple Regulatory Functions for the PtsN (IIANtr) Protein of Pseudomonas putida 
Journal of Bacteriology  2001;183(3):1032-1037.
The ptsN gene of Pseudomonas putida encodes IIANtr, a protein of the phosphoenol pyruvate:sugar phosphotransferase (PTS) system which is required for the C source inhibition of the ς54-dependent promoter Pu of the TOL (toluate degradation) plasmid pWW0. Using two-dimensional gel electrophoresis, we have examined the effect of ptsN disruption on the general expression pattern of P. putida. To this end, cells were grown in the presence or absence of glucose, and a 1,117-spot subset of the P. putida proteome was used as a reference for comparisons. Among all gene products whose expression was lowered by this carbon source (247 spots [about 22%]), only 6 behaved as Pu (i.e., were depressed in the ptsN background). This evidenced only a minor role for IIANtr in the extensive inhibition of gene expression in P. putida caused by glucose. However, the same experiments revealed a large incidence of glucose-independent effects brought about by the ptsN mutation. As many as 108 spots (ca. 9% of the cell products analyzed) were influenced, positively or negatively, by the loss of IIANtr. By matching this pattern with that of an rpoN::ΩKm strain of P. putida, which lacks the ς54 protein, we judge that most proteins whose expression was affected by ptsN were unrelated to the alternative sigma factor. These data suggest a role of IIANtr as a general regulator, independent of the presence of repressive carbon sources and not limited to ς54-dependent genes.
PMCID: PMC94971  PMID: 11208802
13.  A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect 
Nature Communications  2014;5:3608.
Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis.
Defective proteins or functional proteins that are no longer needed can be degraded in the endoplasmic reticulum. In this study, Lopez-Serra et al. show that DERL3, which is involved in protein degradation in the endoplasmic reticulum, is aberrantly silenced in cancer, leading to activation of a glucose transporter and dysregulated glycolysis.
PMCID: PMC3988805  PMID: 24699711

Results 1-13 (13)