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PubMed2006

Comparative proteomic analysis reveals a function of the novel death receptor-associated protein BRE in the regulation of prohibitin and p53 expression and proliferation.

The brain and reproductive organ expressed (BRE) gene encodes a highly conserved stress-modulating protein. To gain further insight into the function of this gene, we used comparative proteomics to investigate the protein profiles of C2C12 and D122 cells resulting from small interfering RNA (siRNA)-mediated silencing as well as overexpression of BRE. Silencing of BRE in C2C12 cells, using siRNA, resulted in up-regulated Akt-3 and carbonic anhydrase III expression, while the 26S proteasome regulatory subunit S14 and prohibitin were down-regulated. Prohibitin is a potential tumour suppressor gene, which can directly interact with p53. We found that cell proliferation was significantly increased after knockdown of BRE, concomitant with reduced p53 and prohibitin expression. In contrast, we observed decreased proliferation and up-regulation of p53 and prohibitin when BRE was overexpressed in the D122 cell line. In total, five proteins were found to be up-regulated after BRE over-expression. The majority of these proteins can target or crosstalk with NF-kappaB, which plays a central role in regulating cell proliferation, differentiation and survival. Our results establish a crucial role for BRE in the regulation of key proteins of the cellular stress-response machinery and provide an explanation for the multifunctional nature of BRE.

Animals↗
PubMed2004

Comparative proteomics of primitive hematopoietic cell populations reveals differences in expression of proteins regulating motility.

Lineage-marker depleted (Lin(-)) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin(-)Sca(+)Kit(+) or Lin(-)Sca(+)Kit(-) cells. Lin(-)Sca(+)Kit(-) cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP(3)), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3- and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin micro-filament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1.

Acetylation↗
PubMed2004

Comparative proteome analysis of cellular proteins extracted from highly virulent Francisella tularensis ssp. tularensis and less virulent F. tularensis ssp. holarctica and F. tularensis ssp. mediaasiatica.

Francisella tularensis is the causative agent of the zoonotic disease tularemia. Four subspecies of this pathogen, namely ssp. tularensis, mediaasiatica, holarctica, and novicida are spread throughout the northern hemisphere. Although there are marked variations in their virulence to mammals, the subspecies are difficult to identify as they are closely genetically related. We carried out the comparative proteome analysis of cellular extracts from isolates representing the highly virulent subspecies tularensis, and the less virulent subspecies mediaasiatica and holarctica in order to identify new diagnostic markers and putative factors of virulence. We identified 27 protein spots that were either specifically present or at significantly higher abundance in ssp. tularensis strains, 22 proteins in ssp. mediaasiatica strains, and 26 proteins in ssp. holarctica strains. Subspecies tularensis-specific proteins might represent putative virulence factors. Of 27 identified tularensis-specific spots 17 represented charge and mass variants of proteins occurring in other subspecies, 7 spots were found to be present at higher abundance, and 3 spots were specifically present in tularensis strains. Amongst them, PilP protein, as a component necessary for the biogenesis of the type IV pilus, virulence and adhesion factor for many human pathogen, was identified. Furthermore, the identification of additional 27 proteins common for ssp. tularensis and mediaasiatica, and 19 proteins shared by ssp. mediaasiatica and holarctica documented apparent closer genetic similarity between ssp. tularensis and mediaasiatica.

Bacterial Proteins↗
PubMed2006

Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei.

Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.

Animals↗
PubMed2007

Comparative proteomic analysis of chronic myelogenous leukemia cells: inside the mechanism of imatinib resistance.

Imatinib is the first molecular targeted therapy that has shown clinical success, but imatinib acquired resistance, although a rare event, is critical during the therapy of chronic myelogenous leukaemia (CML). With the aim of better understanding the molecular mechanisms accompanying acquisition of resistance to this drug, a comparative proteomic approach was undertaken on CML cell lines LAMA 84 S (imatinib sensitive) and LAMA 84 R (imatinib resistant). Forty-four differentially expressed proteins were identified and categorized into five main functional classes: (I) heat shock proteins and chaperones; (II) nucleic acid interacting proteins (binding/synthesis/stability); (III) structural proteins, (IV) cell signaling, and (V) metabolic enzymes. Several heat shock proteins known to complex Bcr-Abl were overexpressed in imatinib resistant cells, showing a possible involvement of these proteins in the mechanism of resistance. HnRNPs also resulted in being up-regulated in imatinib resistant cells. These proteins have been shown to be strongly and directly related to Bcr-Abl activity. To our knowledge, this is the first direct proteomic comparison of imatinib sensitive/resistant CML cell lines.

Antineoplastic Agents↗
PubMed2006

Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice.

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.

Animals↗
PubMed2004

["Using model organisms and phylogenetic conservation, the DrosoMan project combines high tech hand-made science and industrial know-how to target the Achilles heel of cancer cells. A comparative proteomic project that associates Academia and Biotech"].

The post-genomic era offers a huge challenge for scientists used to hand-made tailored approaches that can deal with deep insight into a small number of objects. Reciprocally, industry is used to undergo massively parallel approaches, dealing with large numbers but rather shallow insight. The complementation is obviously tantalizing, and Institut Curie and Hybrigenics (HGX) have signed an alliance in the field of cancer and signal transduction. A comparative proteomic approach was undertaken, where ortholog baits from Homo sapiens and flies were used to screen extremely complex two-hybrid cDNA libraries. New partners of "old" proteins have been identified, new networks have emerged, and unexpected connectors have been shown to link biological niches supposed to be independent.

Animals↗
PubMed2005

Comparative proteome analysis revealed up-regulation of transthyretin in rat brain under chronic clozapine treatment.

Long-term administration of antipsychotic drugs can induce differential expression of various proteins in brain cells, which may underscore the molecular mechanism of their clinical efficacy and/or side effects. We used two-dimensional gel electrophoresis in combination with mass spectrometry to screen differentially expressed proteins in the brain cells of rats under chronic clozapine treatment. We identified a protein, transthyretin, which was significantly up-regulated in the hippocampus of rats after four weeks' treatment of clozapine (20mg/kg) as compared with control animals. Western blot analysis further supported this finding. Besides, we also observed increased transcription of transthyretin mRNA in the cerebral cortex of rats under chronic treatment of clozapine (20mg/kg) or olanzapine (2mg/kg), but not haloperidol (1mg/kg), using real-time quantitative PCR. Transthyretin is a retinol carrier protein, our findings suggest that antipsychotic drugs treatment may affect the retinoid signaling cascade in the brain. Besides, transthyretin is also an indicator of nutritional status; increased transthyretin may be associated with the body weight gain of taking atypical antipsychotic drugs. This study also demonstrates that comparative proteome analysis is a feasible method to study the molecular mechanism of chronic antipsychotic drugs treatment.

Animals↗
PubMed2004

Comparative proteomic analysis of de-N-glycosylated serum from hepatitis B carriers reveals polypeptides that correlate with disease status.

Analysis of the polypeptide profile in tissues, cells, and sera by high-resolution two-dimensional (2-D) gel electrophoresis offers promise in the identification of biomarkers that correlate with disease. However, sera contain many polypeptides bearing N-linked glycosylation that can complicate interpretation. Therefore, we tested the possibility that de-N-glycosylation of the polypeptides present in human serum would result in a simplification of serum proteome profiles. Briefly, polypeptides present in human serum were left untreated or subjected to de-N-glycosylation by incubation with PNGase F and resolved by high-resolution 2-D gel electrophoresis. De-N-glycosylation reduced the number of glycoform variants, enhanced the resolution of many polypeptides and allowed other polypeptides to become visible. As an initial test of concept, clinically relevant serum samples from individuals with or without diagnosis of hepatocellular carcinoma were compared. Several polypeptides, apparent only after de-N-glycosylation, were shown to correlate with disease. Although the results are preliminary and the identities of all the putative biomarkers not yet known, the data suggest that de-N-glycosylation offers a method to enhance the resolution of serum polypeptide profiles and has value in comparative proteomic studies.

Amyloid↗
PubMed2001

Comparative proteomics to establish genetic relationships in the Brassicaceae family.

The comparisons of the same proteome (aerial part of etiolated seedlings) in different species and genus of the plant family Brassicaceae (cabbages, mustards, rapes, radishes and Arabidopsis) was undertaken to establish genetic proximities between them. Genetic distances were calculated on the basis of common and distinct spots. The construction of phenetic trees and factorial correspondence analysis that were performed are in very good agreement with our actual knowledge of the taxonomy of the Brassicaceae. The position of the tetraploids, for instance, is in between the positions of their constitutive genome donor representatives. Comparative proteomics may be helpful to shorten the transfer between model and agronomic target species.

Brassicaceae↗
PubMed2006

Comparative proteomic profiles and the potential markers between Burkholderia pseudomallei and Burkholderia thailandensis.

Burkholderia pseudomallei is a bacterial pathogen causing the melioidosis disease, which is predominantly found in tropical areas of Southeast Asia and Northern Australia. Burkholderia thailandensis is a closely related species to B. pseudomallei but it is non-pathogenic species. In this study, we have constructed a proteome reference map of B. pseudomallei at the stationary phase of growth by using two-dimensional gel electrophoresis with a pH 4-7 immobilized pH gradient combined with matrix-assisted laser desorption ionization time of flight mass spectrometry. Approximately 550 spots could be detected by Coomassie brilliant blue G-250 staining, and 88 spots representing 77 unique proteins were identified. Eleven of the gene products were found in multiple spots indicating as isoforms. In attempt to detect distinctive expressed proteins between a virulent and a non-virulent species, the use of comparative proteomic profiles under the same condition were performed. We could identify more than 20 different spots. Twelve out of 14 spots are detected in B. pseudomallei and six proteins have been identified and indicated that they are involved in virulent characters of bacteria. Two hypothetical proteins were expressed and found only in B. pseudomallei. These proteins are potential markers to distinguish between these two species. Our study also provides a useful information of global intracellular protein expression and is a valuable starting point for analyzing a proteomic pathogenicity of the bacterial pathogen.

Amino Acid Sequence↗
PubMed2006

Comparative proteomics of pulmonary tumors with neuroendocrine differentiation.

We aimed to evaluate neuroendocrine pulmonary tumors (NEPT) by a novel method involving map tree construction by comparing all of the protein spots. We performed a proteomics analysis to assess the similarities in protein expression between neuroendocrine pulmonary tumors (NEPT), including typical carcinoids (TC), atypical carcinoids (AC), large cell neuroendocrine carcinomas (LCNEC) and small cell carcinomas (SCLC). Total protein lysates were obtained from seven histologically confirmed frozen NEPT tissues, including 1TC, 2 SCLC, and 4 cases ranging from AC to LCNEC. 2-DE demonstrated that TC was similar to normal lung. AC, LCNEC, and SCLC were similar to each other, forming a group separate from TC, however, SCLC at an early stage showed a similarity to TC. MALDI analysis detected 9 surrogate endpoint biomarkers, including eIF5A1, GST M3, cytokeratin 18 (CK 18), FK506-binding protein p59, p63, MAGE-D2, mitochondrial short-chain enoyl-coenzyme A hydratase 1, tranferrin and poly (rC) binding protein 1. Immunohistochemical staining revealed a gradual decrease in expression rate of p63 and CK 18 with poor differentiation of NEPT. Our results demonstrate that (1) the comparative proteomics of NEPT match the WHO classification except for AC and LCNEC; (2) SCLC show differences in their proteomics according to tumor stage; and (3) CK 18 and p63 may be useful as diagnostically and prognostically available markers.

Carcinoma, Large Cell↗
PubMed2002

Comparative proteomics based on stable isotope labeling and affinity selection.

Disease, external stimuli (such as drugs and toxins), and mutations cause changes in the rate of protein synthesis, post-translational modification, inter-compartmental transport, and degradation of proteins in living systems. Recognizing and identifying the small number of proteins involved is complicated by the complexity of biological extracts and the fact that post-translational alterations of proteins can occur at many sites in multiple ways. It is shown here that a variety of new tools and methods based on internal standard technology are now being developed to code globally all peptides in control and experimental samples for quantification. The great advantage of these stable isotope-labeling strategies is that mass spectrometers can rapidly target those proteins that have changed in concentration for further analysis. When coupled to stable isotope quantification, targeting can be further focused through chromatographic selection of peptide classes on the basis of specific structural features. Targeting structural features is particularly useful when they are unique to types of regulation or disease. Differential displays of targeted peptides show that stimulus-specific markers are relatively easy to identify and will probably be diagnostically valuable tools.

Animals↗
PubMed2006

Proteomic analysis of acidic chaperones, and stress proteins in extreme halophile Halobacterium NRC-1: a comparative proteomic approach to study heat shock response.

BACKGROUND: Halobacterium sp. NRC-1 is an extremely halophilic archaeon and has adapted to optimal growth under conditions of extremely high salinity. Its proteome is highly acidic with a median pI of 4.9, a unique characteristic which helps the organism to adapt high saline environment. In the natural growth environment, Halobacterium NRC-1 encounters a number of stressful conditions including high temperature and intense solar radiation, oxidative and cold stress. Heat shock proteins and chaperones play indispensable roles in an organism's survival under many stress conditions. The aim of this study was to develop an improved method of 2-D gel electrophoresis with enhanced resolution of the acidic proteome, and to identify proteins with diverse cellular functions using in-gel digestion and LC-MS/MS and MALDI-TOF approach. RESULTS: A modified 2-D gel electrophoretic procedure, employing IPG strips in the range of pH 3-6, enabled improved separation of acidic proteins relative to previous techniques. Combining experimental data from 2-D gel electrophoresis with available genomic information, allowed the identification of at least 30 cellular proteins involved in many cellular functions: stress response and protein folding (CctB, PpiA, DpsA, and MsrA), DNA replication and repair (DNA polymerase A alpha subunit, Orc4/CDC6, and UvrC), transcriptional regulation (Trh5 and ElfA), translation (ribosomal proteins Rps27ae and Rphs6 of the 30 S ribosomal subunit; Rpl31eand Rpl18e of the 50 S ribosomal subunit), transport (YufN), chemotaxis (CheC2), and housekeeping (ThiC, ThiD, FumC, ImD2, GapB, TpiA, and PurE). In addition, four gene products with undetermined function were also identified: Vng1807H, Vng0683C, Vng1300H, and Vng6254. To study the heat shock response of Halobacterium NRC-1, growth conditions for heat shock were determined and the proteomic profiles under normal (42 degrees C), and heat shock (49 degrees C) conditions, were compared. Using a differential proteomic approach in combination with available genomic information, bioinformatic analysis revealed five putative heat shock proteins that were upregulated in cells subjected to heat stress at 49 degrees C, namely DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2. CONCLUSION: The modified 2-D gel electrophoresis markedly enhanced the resolution of the extremely acidic proteome of Halobacterium NRC-1. Constitutive expression of stress proteins and chaperones help the organism to adapt and survive under extreme salinity and other stress conditions. The upregulated expression pattern of putative chaperones DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2 under elevated temperature clearly suggests that Halobacterium NRC-1 has a sophisticated defense mechanism to survive in extreme environments.

Journal Article↗
PubMed2004

Comparative proteome analysis of Saccharomyces cerevisiae grown in chemostat cultures limited for glucose or ethanol.

The use of chemostat culturing enables investigation of steady-state physiological characteristics and adaptations to nutrient-limited growth, while all other relevant growth conditions are kept constant. We examined and compared the proteomic response of wild-type Saccharomyces cerevisiae CEN.PK113-7D to growth in aerobic chemostat cultures limited for carbon sources being either glucose or ethanol. To obtain a global overview of changes in the proteome, we performed triplicate analyses using two-dimensional gel electrophoresis and identified proteins of interest using MS. Relative quantities of about 400 proteins were obtained and analyzed statistically to determine which protein steady-state expression levels changed significantly under glucose- or ethanol-limited conditions. Interestingly, only enzymes involved in central carbon metabolism showed a significant change in steady-state expression, whereas expression was only detected in one of both carbon source-limiting conditions for 15 of these enzymes. Side effects that were previously reported for batch cultivation conditions, such as responses to continuous variation of specific growth rate, to carbon-catabolite repression, and to accumulation of toxic substrates, were not observed. Moreover, by comparing our proteome data with corresponding mRNA data, we were able to unravel which processes in the central carbon metabolism were regulated at the level of the proteome, and which processes at the level of transcriptome. Importantly, we show here that the combined approach of chemostat cultivation and comprehensive proteome analysis allowed us to study the primary effect of single limiting conditions on the yeast proteome.

Aerobiosis↗
PubMed2006

High light response of the thylakoid proteome in arabidopsis wild type and the ascorbate-deficient mutant vtc2-2. A comparative proteomics study.

The thylakoid proteome of chloroplasts contains multiple proteins involved in antioxidative defense, protein folding, and repair. To understand this functional protein network, we analyzed the quantitative response of the thylakoid-associated proteome of Arabidopsis (Arabidopsis thaliana) wild type and the ascorbate-deficient mutant vtc2-2 after transition to high light (HL; 1,000 micromol photons m(-2) s(-1)). The soluble thylakoid proteomes of wild type and vtc2-2 were compared after 0, 1, 3, and 5 d of HL using two-dimensional gels with three independent experiments, followed by a multivariant statistical analysis and tandem mass spectrometry. After 5 d of HL, both wild-type and vtc2-2 plants accumulated anthocyanins, increased their total ascorbate content, and lost 10% of photosystem II efficiency, but showed no bleaching. Anthocyanin and total ascorbate concentrations in vtc2-2 were respectively 34% and 20% of wild type, potentially leading to enhanced oxidative stress in vtc2-2. Forty-five protein spots significantly changed as a consequence of genotype, light treatment, or both. Independent confirmation was obtained from western blots. The most significant response was the up-regulation of thylakoid YCF37 likely involved in photosystem I assembly, and specific fibrillins, a flavin reductase-like protein, and an aldolase, each located in thylakoid-associated plastoglobules. Fe-superoxide dismutase was down-regulated in vtc2-2, while Cu,Zn-superoxide dismutase was up-regulated. vtc2-2 also showed a systematic up-regulation of a steroid dehydrogenase-like protein. A number of other stress-related proteins, several thylakoid proteases, and lumenal isomerases did not change, while PsbS increased in wild type upon light stress. These findings are discussed in terms of plastid metabolism and oxidative stress defense, and emphasize that understanding of the chloroplast stress-response network must include the enzymatic role of plastoglobules.

Arabidopsis↗
PubMed2005

Comparative proteomic analysis of B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis.

OBJECTIVE: To compare the protein difference between B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis. METHOD: Protein samples were prepared by vorterx, ultrasonic treatment, and centrifugation. Protein concentrations were determined by Bradford method. Protein difference was compared by the first IEF and the second SDS-polyacrylamide gel electrophoresis. RESULTS: Protein concentrations in samples of Bartonella henselae Houston and Bartonella henselae Marseille were 2.117 microg/microL and 2.200 microg/microL respectively. Sample protein of 40 microg for IPG strips loading was perfect. The results of 2-DE in pH 4 to 7 IPG strips showed that the total protein spots of Bartonella henselae Houston and Bartonella henselae Marseille were 375 and 379 respectively, 95% of the spots were the same between the two strains of Bartonella henselae. CONCLUSION: The procedure of 2-DE may prove successful for the proteomic analysis of Bartonella henselae. Bartonella henselae Houston and Bartonella henselae Marseille are different genotypes.

Bacterial Proteins↗
PubMed2005

Comparative proteomic analysis of mammalian animal tissues and body fluids: bovine proteome database.

Characterizing the complete proteome of multicellular organisms is a challenging task using the currently available technologies. With the increasing degree of genetic complexity, animals acquire a broader repertoire of options to meet environmental challenges. Mammalian cells from different tissues/body fluids express different thousands of proteins with a predicted dynamic range of up to five to six orders of magnitude, thus necessitating the whole arsenal of dedicated analytical strategies for a detailed proteome characterization. Nevertheless, 2D-E analysis of whole cellular lysates still remains the most used initial approach for the proteomic description of specialized cells. It enables to obtain an overview of the main soluble protein components of a specific tissue/body fluid, allowing comparison between different cellular types and molecular description of organ specialization. Massive proteomic investigations have been reported mainly in the case of human, mouse and rat, allowing comparative analysis. For this reason, a research project focused on the 2D-E characterization of tissues and biological fluids from other domestic mammals has been undertaken in our laboratory. A number of high-resolution reference electrophoretic maps have been established for liver, kidney, muscle, plasma and red blood cells samples from Holstein Friesian bovine female individuals. Among the 1863 distinct protein features detected, 534 species were identified and associated to 209 different genes by a combination of MALDI-TOF mass fingerprint, capillary LC-ESI-IT-MS-MS and image gel matching procedures. Identified polypeptide species and differences in expression profiles between various tissues/fluids clearly reflected organ biochemical specialization. This experimental output allowed establishing a 2D-E bovine database accessible at the URL address for image comparison.

Animals↗