Loss of 4E-BPs prevents the hindlimb immobilization-induced decrease in protein synthesis in skeletal muscle.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression.NEW & NOTEWORTHY Basal rates of protein synthesis are elevated in skeletal muscle in the immobilized leg of mice lacking the translational repressors, 4E-BP1 and 4E-BP2 (knockout mice), compared with wild-type mice. However, disuse-induced muscle atrophy occurs to the same extent in both wild-type and knockout mice suggesting that compensatory mechanisms are induced that overcome the upregulation of muscle protein synthesis. Proteomic analysis revealed that mRNAs encoding several glycolytic enzymes are differentially translated in wild-type and knockout mice.

Enrichment of Newly Synthesized Proteins following treatment of C2C12 Myotubes with Endotoxin and Interferon-γ.

Inflammation in muscle induces the synthesis of mediators that can impair protein synthesis and enhance proteolysis, and when sustained lead to muscle atrophy. Furthermore, muscle-derived mediators that are secreted may participate in disrupting the function of other peripheral organs. Selective identification of newly synthesized proteins can provide insight on biological processes that depend on the continued synthesis of specific proteins to maintain homeostasis as well as those proteins that are up- or down-regulated in response to inflammation. We used puromycin-associated nascent chain proteomics (PUNCH-P) to characterize new protein synthesis in C2C12 myotubes and changes resulting from their exposure to the inflammatory mediators lipopolysaccharide (LPS) and interferon (IFN)-γ for either a short (4 h) or prolonged (16 h) time period. We identified sequences of nascent polypeptide chains belonging to a total of 1523 proteins and report their detection from three independent samples of each condition at each time point. The identified nascent proteins correspond to approximately 15% of presently known proteins in C2C12 myotubes and are enriched in specific cellular components and pathways. A subset of these proteins was identified only in treated samples and has functional characteristics consistent with the synthesis of specific new proteins in response to LPS/IFNγ. Thus, the identification of proteins from their nascent polypeptide chains provides a resource to analyze the role of new synthesis of proteins in both protein homeostasis and in proteome responses to stimuli in C2C12 myotubes. Our results reveal a profile of actively translating proteins for specific cellular components and biological processes in normal C2C12 myotubes and a different enrichment of proteins in response to LPS/IFNγ. Collectively, our data disclose a highly interconnected network that integrates the regulation of cellular proteostasis and reveal a diverse immune response to inflammation in muscle which may underlie the concomitantly observed atrophy and be important in inter-organ communication.

The Ubiquitin-Proteasome Pathway and Epigenetic Modifications in Cancer.

BACKGROUND: The ubiquitin-proteasome pathway is involved in almost all cellular processes (cell cycle, gene transcription and translation, cell survival and apoptosis, cell metabolism and protein quality control) mainly through the specific degradation of the majority of intracellular proteins (>80%) or partial processing of transcription factors (e.g., NF-κB). A growing amount of evidence now indicates that epigenetic changes are also regulated by the ubiquitin-proteasome pathway. Recent studies indicate that epigenetic regulations are equally crucial for almost all biological processes as well as for pathological conditions such as tumorigenesis, as compared to non-epigenetic control mechanisms (i.e., genetic alterations or classical signal transduction pathways). OBJECTIVE: Here, we reviewed the recent work highlighting the interaction of the ubiquitin-proteasome pathway components (e.g., ubiquitin, E1, E2 and E3 enzymes and 26S proteasome) with epigenetic regulators (histone deacetylases, histone acetyltransferases and DNA methyltransferases). RESULTS: Alterations in the regulation of the ubiquitin-proteasome pathway have been discovered in many pathological conditions. For example, a 2- to 32-fold increase in proteasomal activity and/or subunits has been noted in primary breast cancer cells. Although proteasome inhibitors have been successfully applied in the treatment of hematological malignancies (e.g., multiple myeloma), the clinical efficacy of the proteasomal inhibition is limited in solid cancers. Interestingly, recent studies show that the ubiquitin-proteasome and epigenetic pathways intersect in a number of ways through the regulation of epigenetic marks (i.e., acetylation, methylation and ubiquitylation). CONCLUSION: It is therefore believed that novel treatment strategies involving new generation ubiquitinproteasome pathway inhibitors combined with DNA methyltransferase, histone deacetylase or histone acetyltransferase inhibitors may produce more effective results with fewer adverse effects in cancer treatment as compared to standard chemotherapeutics in hematological as well as solid cancers.

Proteomic analysis of labial saliva of the generalist cabbage looper (Trichoplusia ni) and its role in interactions with host plants.

Insect saliva is one of the first secretions to come in contact with plants during feeding. The composition and role of caterpillar saliva has not been as thoroughly studied as that of sucking insects. This study focuses on characterizing the proteome of the cabbage looper (Trichoplusia ni) saliva using iTRAQ labeling and LC-MS/MS. We also measured how the saliva proteome changed when larvae were reared on different diets - cabbage, tomato, and an artificial pinto bean diet. We identified 254 proteins in the saliva out of which 63 were differentially expressed. A large percentage (56%) of the proteins identified function in protein metabolism, followed by proteins involved in vesicle transport (6%) and oxidoreductase activity (5%), among other categories. Several proteins identified are antioxidants or reactive oxygen species (ROS) scavengers. Among these ROS scavengers, we identified a catalase and further analyzed its gene expression and enzymatic activity. We also applied commercial, purified catalase on tomato and measured the activity of defensive proteins - trypsin proteinase inhibitor, polyphenol oxidase and peroxidase. Catalase gene expression was significantly higher in the salivary glands of larvae fed on tomato. Also, catalase suppressed the induction of tomato trypsin proteinase inhibitor levels, but not the induction of polyphenol oxidase or peroxidase. These results add to our understanding of proteomic plasticity in saliva and its role in herbivore offense against plant defenses.

Quantitative proteomic analysis of the fall armyworm saliva.

Lepidopteran larvae secrete saliva on plant tissues during feeding. Components in the saliva may aid in food digestion, whereas other components are recognized by plants as cues to elicit defense responses. Despite the ecological and economical importance of these plant-feeding insects, knowledge of their saliva composition is limited to a few species. In this study, we identified the salivary proteins of larvae of the fall armyworm (FAW), Spodoptera frugiperda; determined qualitative and quantitative differences in the salivary proteome of the two host races-corn and rice strains-of this insect; and identified changes in total protein concentration and relative protein abundance in the saliva of FAW larvae associated with different host plants. Quantitative proteomic analyses were performed using labeling with isobaric tags for relative and absolute quantification followed by liquid chromatography-tandem mass spectrometry. In total, 98 proteins were identified (>99% confidence) in the FAW saliva. These proteins were further categorized into five functional groups: proteins potentially involved in (1) plant defense regulation, (2) herbivore offense, (3) insect immunity, (4) detoxification, (5) digestion, and (6) other functions. Moreover, there were differences in the salivary proteome between the FAW strains that were identified by label-free proteomic analyses. Thirteen differentially identified proteins were present in each strain. There were also differences in the relative abundance of eleven salivary proteins between the two FAW host strains as well as differences within each strain associated with different diets. The total salivary protein concentration was also different for the two strains reared on different host plants. Based on these results, we conclude that the FAW saliva contains a complex mixture of proteins involved in different functions that are specific for each strain and its composition can change plastically in response to diet type.

Fall Armyworm-Associated Gut Bacteria Modulate Plant Defense Responses.

Mechanical damage caused by insect feeding along with components present in insect saliva and oral secretions are known to induce jasmonic acid-mediated defense responses in plants. This study investigated the effects of bacteria from oral secretions of the fall armyworm Spodoptera frugiperda on herbivore-induced defenses in tomato and maize plants. Using culture-dependent methods, we identified seven different bacterial isolates belonging to the family Enterobacteriacea from the oral secretions of field-collected caterpillars. Two isolates, Pantoea ananatis and Enterobacteriaceae-1, downregulated the activity of the plant defensive proteins polyphenol oxidase and trypsin proteinase inhibitors (trypsin PI) but upregulated peroxidase (POX) activity in tomato. A Raoultella sp. and a Klebsiella sp. downregulated POX but upregulated trypsin PI in this plant species. Conversely, all of these bacterial isolates upregulated the expression of the herbivore-induced maize proteinase inhibitor (mpi) gene in maize. Plant treatment with P. ananatis and Enterobacteriaceae-1 enhanced caterpillar growth on tomato but diminished their growth on maize plants. Our results highlight the importance of herbivore-associated microbes and their ability to mediate insect plant interactions differently in host plants fed on by the same herbivore.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1.

BACKGROUND: The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. RESULTS: Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type, but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. CONCLUSIONS: Our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.

Radiation-Induced Alteration of the Brain Proteome: Understanding the Role of the Sirtuin 2 Deacetylase in a Murine Model.

Whole brain radiotherapy (WBRT) produces unwanted sequelae, albeit via unknown mechanisms. A deacetylase expressed in the central nervous system, Sirtuin 2 (SIRT2), has been linked to neurodegeneration. Therefore, we sought to challenge the notion that a single disease pathway is responsible for radiation-induced brain injury in Sirt2 wild-type (WT) and knockout (KO) mice at the proteomic level. We utilized isobaric tag for relative and absolute quantitation to analyze brain homogenates from Sirt2 WT and KO mice with and without WBRT. Selected proteins were independently verified, followed by ingenuity pathway analysis. Canonical pathways for Huntington's, Parkinson's, and Alzheimer's were acutely affected by radiation within 72 h of treatment. Although loss of Sirt2 preferentially affected both Huntington's and Parkinson's pathways, WBRT most significantly affected Huntington's-related proteins in the absence of Sirt2. Identical protein expression patterns were identified in Mog following WBRT in both Sirt2 WT and KO mice, revealing a proteomic radiation signature; however, long-term radiation effects were found to be associated with altered levels of a small number of key neurodegeneration-related proteins, identified as Mapt, Mog, Snap25, and Dnm1. Together, these data demonstrate the principle that the presence of Sirt2 can have significant effects on the brain proteome and its response to ionizing radiation.

Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling.

Nitrogen starvation induces a global stress response in microalgae that results in the accumulation of lipids as a potential source of biofuel. Using GC-MS-based metabolite and iTRAQ-labeled protein profiling, we examined and correlated the metabolic and proteomic response of Chlamydomonas reinhardtii under nitrogen stress. Key amino acids and metabolites involved in nitrogen sparing pathways, methyl group transfer reactions, and energy production were decreased in abundance, whereas certain fatty acids, citric acid, methionine, citramalic acid, triethanolamine, nicotianamine, trehalose, and sorbitol were increased in abundance. Proteins involved in nitrogen assimilation, amino acid metabolism, oxidative phosphorylation, glycolysis, TCA cycle, starch, and lipid metabolism were elevated compared with nonstressed cultures. In contrast, the enzymes of the glyoxylate cycle, one carbon metabolism, pentose phosphate pathway, the Calvin cycle, photosynthetic and light harvesting complex, and ribosomes were reduced. A noteworthy observation was that citrate accumulated during nitrogen stress coordinate with alterations in the enzymes that produce or utilize this metabolite, demonstrating the value of comparing protein and metabolite profiles to understand complex patterns of metabolic flow. Thus, the current study provides unique insight into the global metabolic adjustments leading to lipid storage during N starvation for application toward advanced biofuel production technologies.

Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

BACKGROUND: Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media. RESULTS: We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to high glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction. CONCLUSIONS: Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.

Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers.

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.

Survival during long-term starvation: global proteomics analysis of Geobacter sulfurreducens under prolonged electron-acceptor limitation.

The bioavailability of terminal electron acceptors (TEAs) and other substrates affects the efficiency of subsurface bioremediation. While it is often argued that microorganisms exist under "feast or famine", in the laboratory most organisms are studied under "feast" conditions, whereas they typically encounter "famine" in nature. The work described here aims to understand the survival strategies of the anaerobe Geobacter sulfurreduces under TEA-starvation conditions. Cultures were starved for TEA and at various times sampled to perform global comparative proteomic analysis using iTRAQ to obtain insight into the dynamics of change in proteins/enzymes expression associated with change in nutrient availability/environmental stress. Proteins varying in abundance with a high level of statistical significance (p < 0.05) were identified to understand how cells change from midlog to (i) stationary phase and (ii) conditions of prolonged starvation (survival phase). The most highly represented and significantly up-regulated proteins in the survival phase cells are involved in energy metabolism, cell envelope, and transport and binding functional categories. The majority of the proteins were predicted to be localized in the cell membranes. These results document that changes in the outer and cytoplasmic membranes are needed for survival of Geobacter under starvation conditions. The cell shuts down anabolic processes and becomes poised, through changes in its membrane proteins, to sense nutrients in the environment, to transport nutrients into the cell, and to detect or utilize TEAs that are encountered. Under TEA-limiting conditions, the cells turned from translucent white to red in color, indicating higher heme content. The increase in heme content supported proteomics results showing an increase in the number of cytochromes involved in membrane electron transport during the survival phase. The cell is also highly reduced with minimal change in energy charge (ATP to total adenine nucleotide ratio). Nonetheless, these proteomic and biochemical results indicate that even under TEA starvation cells remain poised for bioremediation.

Proteomic changes induced by effective chemopreventive ratios of n-3:n-6 fatty acids and tamoxifen against MNU-induced mammary cancer in the rat.

We used a proteomic approach to gain insights into the mechanisms of protection at the protein level by a high n-3:n-6 ratio in the absence and presence of Tamoxifen. Four groups were treated with 1-methyl-1-nitrosourea (MNU) and fed the following diets with varied n-3:n-6 ratios; group 1 = 1:1; groups 2 and 3 = 10:1 and 25:1, respectively; group 4: (25:1) plus Tamoxifen (1 mg/kg diet). The plasma from six rats/group was pooled and analyzed with the isobaric tags for relative and absolute quantitation method; 148 proteins were identified with 95% confidence by ProteinPilot 4.0. In plasma of rats fed 10:1, 25:1 n-3:n-6, and 25:1 plus Tamoxifen, the number of proteins that met our criteria (P ≤ 0.05, error factor ≤ 2) were 10, 14, and 19 proteins, respectively. Selected proteins were further validated by Western blotting. Compared to 1:1, both 10:1 and 25:1 diets upregulated vitamin D binding protein, gelsolin, and 14-3-3 sigma, reported to have tumor suppressive effects, whereas alpha-1B-glycoprotein, which has been reported to be elevated in the serum of breast cancer patients was decreased. Compared to 25:1, the 25:1 plus Tamoxifen diet downregulated apolipoprotein E, haptoglobin, and inter-α-inhibitor H4 heavy chain. Ingenuity pathway analysis determined that the trends of specific proteins were related to lipid metabolism in the 25:1 n-3:n-6 group, whereas the 25:1 n-3:n-6 plus Tamoxifen group included proteins involved in cancer and inflammation. Our results show that several proteins were altered in a manner consistent with chemoprevention. Such proteins may serve as biomarkers to monitor efficacy of n-3 and Tamoxifen in future clinical chemoprevention trials.

Hyperglycemia mediates a shift from cap-dependent to cap-independent translation via a 4E-BP1-dependent mechanism.

Diabetes and its associated hyperglycemia induce multiple changes in liver function, yet we know little about the role played by translational control of gene expression in mediating the responses to these conditions. Here, we evaluate the hypothesis that hyperglycemia-induced O-GlcNAcylation of the translational regulatory protein 4E-BP1 alters hepatic gene expression through a process involving the selection of mRNA for translation. In both streptozotocin (STZ)-treated mice and cells in culture exposed to hyperglycemic conditions, expression of 4E-BP1 and its interaction with the mRNA cap-binding protein eIF4E were enhanced in conjunction with downregulation of cap-dependent and concomitant upregulation of cap-independent mRNA translation, as assessed by a bicistronic luciferase reporter assay. Phlorizin treatment of STZ-treated mice lowered blood glucose concentrations and reduced activity of the cap-independent reporter. Notably, the glucose-induced shift from cap-dependent to cap-independent mRNA translation did not occur in cells lacking 4E-BP1. The extensive nature of this shift in translational control of gene expression was revealed using pulsed stable isotope labeling by amino acids in cell culture to identify proteins that undergo altered rates of synthesis in response to hyperglycemia. Taken together, these data provide evidence for a novel mechanism whereby O-GlcNAcylation of 4E-BP1 mediates translational control of hepatic gene expression.

Changes in proteomic profiles in different prostate lobes of male rats throughout growth and development and aging stages of the life span.

BACKGROUND: Aging-related changes in important cellular pathways in the prostate may promote a permissive environment for an increased risk for prostatic disease development such as prostate cancer. Our objectives were to examine for such changes, by systematically determining the effects of growth and development and aging on proteomic profiles in different lobes of the rat prostate. METHODS: Prostate lobes (dorsolateral lobe, DL and ventral lobe, VL) were obtained from male Fisher rats of various ages representing young (4 months), mature (12 months), old (18 months), and very old (24 months). Differentially expressed proteins between age groups in each lobe were identified using a proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ). Select changes in the DL and VL were verified by immunoblot analysis. RESULTS: iTRAQ identified 317 proteins with high confidence. iTRAQ discovered 12 and 6 proteins significantly modulated in response to growth and development in the DL and VL, respectively, and 42 and 29 proteins significantly modulated in response to aging in the DL and VL, respectively. Proteins modulated during growth and development in the DL and VL are involved in a variety of biological processes including cell communication and development, whereas proteins modulated during aging were predominantly related to antioxidant activity and immunity. Immunoblot analysis verified age-related changes for α-1 antitrypsin, annexin A1, hypoxia up-regulated protein 1, and 78 kDa glucose-regulated protein. CONCLUSIONS: Aging results in changes in numerous prostatic proteins and pathways which are mainly linked to inflammation and may lead to prostatic disease development.

Comparative proteomic analysis of transition of saccharomyces cerevisiae from glucose-deficient medium to glucose-rich medium.

BACKGROUND: When glucose is added to Saccharomyces cerevisiae grown in non-fermentable carbon sources, genes encoding ribosomal, cell-cycle, and glycolytic proteins are induced. By contrast, genes involved in mitochondrial functions, gluconeogenesis, and the utilization of other carbon sources are repressed. Glucose also causes the activation of the plasma membrane ATPase and the inactivation of gluconeogenic enzymes and mitochondrial enzymes. The goals of this study were to use the iTRAQ-labeling mass spectrometry technique to identify proteins whose relative levels change in response to glucose re-feeding and to correlate changes in protein abundance with changes in transcription and enzymatic activities. We used an experimental condition that causes the degradation of gluconeogenic enzymes when glucose starved cells are replenished with glucose. Identification of these enzymes as being down-regulated by glucose served as an internal control. Furthermore, we sought to identify new proteins that were either up-regulated or down-regulated by glucose. RESULTS: We have identified new and known proteins that change their relative levels in cells that were transferred from medium containing low glucose to medium containing high glucose. Up-regulated proteins included ribosomal subunits, proteins involved in protein translation, and the plasma membrane ATPase. Down-regulated proteins included small heat shock proteins, mitochondrial proteins, glycolytic enzymes, and gluconeogenic enzymes. Ach1p is involved in acetate metabolism and is also down-regulated by glucose. CONCLUSIONS: We have identified known proteins that have previously been reported to be regulated by glucose as well as new glucose-regulated proteins. Up-regulation of ribosomal proteins and proteins involved in translation may lead to an increase in protein synthesis and in nutrient uptake. Down-regulation of glycolytic enzymes, gluconeogenic enzymes, and mitochondrial proteins may result in changes in glycolysis, gluconeogenesis, and mitochondrial functions. These changes may be beneficial for glucose-starved cells to adapt to the addition of glucose.

Functional proteomic analysis reveals sex-dependent differences in structural and energy-producing myocardial proteins in rat model of alcoholic cardiomyopathy.

Long-term ethanol exposure leads to a sexually dimorphic response in both the susceptibility to cardiac pathology (protective effect of the female heart) and the expression of selected myocardial proteins. The purpose of the present study was to use proteomics to examine the effect of chronic alcohol consumption on a broader array of cardiac proteins and how these were affected between the sexes. Male and female rats were maintained for 18 wk on a 40% ethanol-containing diet in which alcohol was provided in drinking water and agar blocks. Differences in the content of specific cardiac proteins in isopycnic centrifugal fractions were determined using mass spectrometry on iTRAQ-labeled tryptic fragments. A random effects model of meta-analysis was developed to combine the results from multiple iTRAQ experiments. Analysis of a network of proteins involved in cardiovascular system development and function showed that troponins were oppositely regulated by alcohol exposure in females (upregulated) vs. males (downregulated), and this effect was validated by Western blot analysis. Pathway analysis also revealed that alcohol-consuming males showed increased expression of proteins involved in various steps of oxidative phosphorylation including complexes I, III, IV, and V, whereas females showed no change or decreased content. One implication from these findings is that females may be protected from the toxic effects of alcohol due to their ability to maintain contractile function, maintain efficiency of force generation, and minimize oxidative stress. However, the alcohol-induced insult may lead to increased production of reactive oxygen species and structural abnormalities in male myocardium.

Proteomic profiling of human plasma by iTRAQ reveals down-regulation of ITI-HC3 and VDBP by cigarette smoking.

Biomarkers in noninvasive fluids indicative of cigarette smoke's effects are urgently needed. In this pilot study, we utilized the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to identify differentially expressed plasma proteins in healthy cigarette smokers compared to healthy nonsmokers; select proteins were further confirmed by immunoblot analysis. Significant, differentially expressed proteins identified in the plasma separated subjects based on their condition as smokers or nonsmokers. Several of the proteins identified in this study are associated with immunity and inflammatory responses and have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Proteins up-regulated in smokers included complement component 8 polypeptide chains α, ß, and γ, and mannose-binding protein C, and proteins down-regulated included inter-α-trypsin inhibitor heavy chain H3 (ITI-HC3) and vitamin D-binding protein (VDBP). In addition, gelsolin and vitronectin, known tissue leakage proteins, were up- and down-regulated, respectively. Our results demonstrate for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome. Proteins identified in this pilot study may serve as candidate biomarkers of diseases resulting from exposure to cigarette smoke in future molecular epidemiological studies.

Proteomics of cauda epididymal fluid from mature Holstein bulls.

The proteome of cauda epididymal fluid (CEF) from Holstein bulls was defined. Fluid was collected from the vas deferens, subjected to 2-D SDS-PAGE and spots identified by CapLC-MS/MS and MALDI-ToF/ToF. Because albumin accounted for 21.1% of all spot intensities in the gels examined by PDQuest, samples were subjected to albumin depletion and then analyzed again as before. Original CEF gels had 114 ± 3 spots, including as the most abundant: albumin, epididymal secretory protein E1, prostaglandin d-synthase and gelsolin. Epididymal fluid also expressed: clusterin, transferrin, N-acetyl-ß-glucosaminidase, cauxin, glutathione peroxidase, acidic seminal fluid protein (aSFP), aldehyde reductase, α-l-fucosidase, α-1-ß-glycoprotein, apolipoprotein A-1, ß actin, calmodulin, cathepsin D, cystatin E/M, enolase, galectin 3-binding protein, leucine amino-peptidase and nucleobindin. Albumin depletion decreased that very spot to 10% of its original intensity and the resulting gels had, on average, 137 ± 4 spots. Spots identified as dipeptidyl-peptidase 7, angiotensin-converting enzyme, arylsulfatase A, aspartylglucosaminidase, serine protease inhibitors, new isoforms of calmodulin, cystatin E/M and a 17-kDa nucleobindin appeared only in depleted maps. This study is the first to report nucleobindin and aSFP as epididymal components. We suggest that CEF proteins act to facilitate membrane remodeling, transport of lipophilic substances, protect sperm and prevent premature acrosome reaction.

Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane.

BACKGROUND: The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila. RESULTS: We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments. CONCLUSION: A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.