Glycolytic Enzyme Enolase Research Articles

Molecular recognition between Escherichia coli enolase and ribonuclease E.

In Escherichia coli and many other bacterial species, the glycolytic enzyme enolase is a component of the multi-enzyme RNA degradosome, an assembly that is involved in RNA processing and degradation. Enolase is recruited into the degradosome through interactions with a small recognition motif located within the degradosome-scaffolding domain of RNase E. Here, the crystal structure of enolase bound to its cognate site from RNase E (residues 823-850) at 1.9 A resolution is presented. The structure suggests that enolase may help to organize an adjacent conserved RNA-binding motif in RNase E.

The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex

In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram-positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein-protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA-like domains that are found in all DEAD box RNA helicases and a C-terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C-terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome.

NANETS Consensus Guidelines for the Diagnosis of Neuroendocrine Tumor

Neuroendocrine tumors (NETs) are rare, slow-growing neoplasms characterized by their ability to store and secrete different peptides and neuroamines. 1 Some of these substances cause specific clinical syndromes, 2 whereas other may have elevated plasma or urine levels that are not associated with specific syndromes or symptom complexes Unfortunately, there is no Bideal neuroendocrine tumor marker,[ 3 but according to the presentation, the sensitivity and specificity of each marker vary, and it is generally possible to choose those of greatest value for each clinical syndrome. The biochemical markers are those hormones or amines secreted by the neuroendocrine cells from which these tumors are derived. Some of these are not specific to any tumor, but are produced and secreted by most NETs, whereas other biochemical markers are more specific to the type of tumor and where their quantification can lead to the suspicion or confirmation of the presence of such a tumor. The annual incidence of NETs has risen to 40 to 50 cases per million, perhaps because of better diagnosis and the availability of highly specific and sensitive ways to measure these tumors’ products, improved immunohistochemistry, and enhanced techniques for tumor detection. Thus, the perceived increase in incidence may not be a real change in the incidence of the disease. There are a number of impediments to the diagnosis of these tumors. They are rare, comprising less than 2% of gastrointestinal (GI) malignancies, and are therefore not high on the list of causes of specific symptom complexes. Symptoms themselves are often nonspecific and do not lend themselves readily to identifying the specific underlying tumor. In addition, the manifestations are protean and mimic a variety of disorders. Tumors may be found incidentally on laparoscopy for abdominal pain or during the surgical removal of an appendix or even during a computerized tomographic scan of the abdomen for unexplained symptoms. Lung carcinoids may present with hemoptysis or asthma-like symptoms, and midgut carcinoids may be confused with irritable bowel syndrome (IBS). The natural history of this disease is invariably attended by a long history of vague abdominal symptoms, a series of visits to a primary care practitioner, and referral to a gastroenterologist, often with a misdiagnosis of IBS. These symptoms persist with a median latency to correct diagnosis of 9.2 years by which time the tumor has metastasized, causing symptoms such as flushing and diarrhea and progressing on its slow but relentless course until the patient dies. Clearly, a greater index of suspicion and a carcinoid tumor profile screen are warranted for all patients presenting with Btraditional IBS symptoms.[ Midgut carcinoids are associated with mesenteric fibrosis, which can compress mesenteric vessels and cause bowel ischemia and malabsorption, which may be found in the absence of an abdominal mass. The diagnosis of metastases to the liver is generally more obvious but often still takes place only after a delay of many years. Even then, an incorrect diagnosis is not uncommon. Unless biopsy material is examined for the secretory peptides chromogranin, synaptophysin, or neuron-specific enolase (NSE), tumors may be labeled erroneously as adenocarcinoma, with a negative impact on physician’s attitudes regarding management and underestimation of prospects for survival. 4 The common symptomatic manifestations of patients with carcinoid tumors are illustrated in Tables 1 and 2. Flushing

Hemolysis correction factor in the measurement of serum neuron-specific enolase

Neuron-specific enolase (NSE) is comprised of two isoforms, ag and gg, of the glycolytic enzyme enolase. It is present almost exclusively in neurons and neuroendocrine tissues (1–3). The protein is a valuable tumor marker for monitoring the outcome and disease course in patients with cancer of the neuroendocrine type, in particular small cell lung cancer and neuroblastoma (4, 5). In addition, the concentration of NSE in biological fluids might be a useful parameter for the assessment of neural brain damage as NSE was shown to be released into cerebrospinal fluid and blood following cerebral injury (5, 6). However, the ag-isoform was also reported to be present in circulating blood cells, especially in erythrocytes and platelets (3, 7). As a consequence, slight hemolysis – even undetectable to the naked eye (3) – can produce falsely increased degrees of immunoreactivity (1). Considering the role of NSE as an important biomarker, interpretation of NSE results is unreliable unless accompanied by an estimate of red cell disruption (3). In the case of hemolysis, it is common practice to include a comment on the laboratory report and not give the NSE value itself. We examined the possibility of using data correction to report a clinically more reliable result when measuring NSE in hemolysed serum, instead of reporting a comment that the assay could not be performed. NSE was measured using a sandwich immunoassay with the electrochemiluminescence (ECL) technology installed on a Modular E170 (Roche Diagnostics, Mannheim, Germany). Evaluation of the magnitude of hemolysis was achieved using the hemolytic index (H). The serum index was routinely measured with a Cobas 6000 analyzer (Roche Diagnostics, Mannheim, Germany) using absorbance values of two bichromatic readings (570 and 600 nm, and 660 and 700 nm). The absorbance values are converted into semi-

Abstract 1462: Triterpene extract of Ganoderma lucidum inhibits proliferation of premalignant human prostate cells by regulation of epithelial mesenchymal transition

Abstract Background: Prostate cancer is the leading cancer diagnosed in men. Ganoderma lucidum (GL), a well known medicinal fungus, has been widely used in the treatment and prevention of many diseases, including cancer, in Asian countries. The triterpene extract of GL, has been identified as an important active ingredient possessing anti-cancer activities, through, as yet an unknown mechanism of action. The aims of our current research were to characterise the inhibitory activity of triterpene extracts of GL in pre-malignant prostate cell lines (PINs) as a potential model of chemoprevention and to investigate the mechanism of action of triterpene. Materials and methods: Effects on growth of PIN cell lines were measured using an “in vitro” fluorometric cell viability assay and “in vivo” growth assays. Invasive behaviour of PIN was assessed “in vitro” by using a wound healing assay, and cell branching, in a matrigel invasion assay. The effect on angiogenesis was assessed by measuring formation of tubules by human endothelial cells. To identify the cellular targets of triterpene, two-dimensional gel electrophoresis was used, and proteins showing differential expression were identified by mass spectrometry. Effects on protein expression and gene regulation were further confirmed by western blotting, quantitative RT-PCR and luciferase reporter assay. Results: The triterpene extract of GL inhibited the “in vitro” proliferation of PINs with an IC50 ranging from 38.8μg/ml to 118.8μg/ml and inhibited the volume of PIN xenografts by 30% (p<0.05; 77mg/ml). In addition, triterpene (at the IC25) significantly suppressed angiogenesis (>95% inhibition, p<0.0001), migration (76% inhibition, p<0.001) and invasion of PIN (p<0.001). From proteomic analysis, two potential target proteins identified included the epithelial-mesenchymal transition (EMT) marker vimentin and the glycolytic enzyme enolase α. The down-regulation of vimentin and up-regulation of enolase α were further confirmed by western blotting. The possible role of triterpene in regulation of EMT markers including vimentin, E-cadherin, N-cadherin and slug was also demonstrated by quantitative RT-PCR of PIN cells collected from a cell branching assay. Additionally, triterpene was found to down-regulate the protooncogene c-myc, a downstream target of enolase α, which plays an important role in the cell cycle, cell proliferation and apoptosis. Conclusion: These findings suggest that the triterpene extract of GL could be a promising new agent in prevention of prostate cancer by regulation of EMT and c-myc. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1462.

Selection of RNA aptamers imported into yeast and human mitochondria

In the yeast Saccharomyces cerevisiae, nuclear DNA-encoded is partially imported into mitochondria. We previously found that the synthetic transcripts of yeast tRNA(Lys) and a number of their mutant versions could be specifically internalized by isolated yeast and human mitochondria. The mitochondrial targeting of tRNA(Lys) in yeast was shown to depend on the cytosolic precursor of mitochondrial lysyl-tRNA synthetase and the glycolytic enzyme enolase. Here we applied the approach of in vitro selection (SELEX) to broaden the spectrum of importable tRNA-derived molecules. We found that RNAs selected for their import into isolated yeast mitochondria have lost the potential to acquire a classical tRNA-shape. Analysis of conformational rearrangements in the importable RNAs by in-gel fluorescence resonance energy transfer (FRET) approach permitted us to suggest that protein factor binding and subsequent import require formation of an alternative structure, different from a classic L-form tRNA model. We show that in the complex with targeting protein factor, enolase 2, tRK1 adopts a particular conformation characterized by bringing together the 3'-end and the TPsiC loop. This is a first evidence for implication of RNA secondary structure rearrangement in the mechanism of mitochondrial import selectivity. Based on these data, a set of small RNA molecules with significantly improved efficiency of import into yeast and human mitochondria was constructed, opening the possibility of creating a new mitochondrial vector system able to target therapeutic oligoribonucleotides into deficient human mitochondria.

Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

Aberrant protein expression is associated with decreased developmental potential in porcine cumulus–oocyte complexes

Oocyte developmental competence is progressively obtained during pubertal development in females. Poor developmental potential in oocytes derived from prepubertal females suggests that essential processes required for oocyte development have not been fulfilled. The objective of this experiment was to analyze the protein profiles of porcine cumulus-oocyte complexes (COC) derived from cyclic and prepubertal females to identify alterations in protein abundance that correlate with developmental potential. COC complexes, aspirated from prepubertal and cyclic ovaries, were pooled into three replicates of 400 COCs each per treatment in approximately 100 microl SOF-HEPES medium. Protein samples were extracted and analyzed by two-dimensional differential in gel electrophoresis (2D-DIGE). Over 1,600 proteins were resolved on each of the three replicate gels. Sixteen protein spots were identified by mass spectrometry, representing 14 unique, differentially expressed proteins (volume ratio greater than 1.3). Glutathione-S-transferase and pyruvate kinase 3 were more abundant in COCs derived from cyclic females, whereas soluble epoxide hydrolase and transferrin were more abundant in prepubertal derived COCs. Abundance of several glycolytic enzymes (enolase 1, pyruvate kinase 3, and phosphoglycerate kinase) was increased in COCs derived from cyclic females, suggesting glucose metabolism is decreased in prepubertal derived COCs. We conclude that the abundance of proteins involved in metabolism and oxidative stress regulation is significantly altered in prepubertal derived COCs and may play a role in the mechanisms resulting in developmental competence.

Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host

The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.

Acclimatory responses of the Daphnia pulex proteome to environmental changes. I. Chronic exposure to hypoxia affects the oxygen transport system and carbohydrate metabolism

BackgroundFreshwater planktonic crustaceans of the genus Daphnia show a remarkable plasticity to cope with environmental changes in oxygen concentration and temperature. One of the key proteins of adaptive gene control in Daphnia pulex under hypoxia is hemoglobin (Hb), which increases in hemolymph concentration by an order of magnitude and shows an enhanced oxygen affinity due to changes in subunit composition. To explore the full spectrum of adaptive protein expression in response to low-oxygen conditions, two-dimensional gel electrophoresis and mass spectrometry were used to analyze the proteome composition of animals acclimated to normoxia (oxygen partial pressure [Po2]: 20 kPa) and hypoxia (Po2: 3 kPa), respectively.ResultsThe comparative proteome analysis showed an up-regulation of more than 50 protein spots under hypoxia. Identification of a major share of these spots revealed acclimatory changes for Hb, glycolytic enzymes (enolase), and enzymes involved in the degradation of storage and structural carbohydrates (e.g. cellubiohydrolase). Proteolytic enzymes remained constitutively expressed on a high level.ConclusionAcclimatory adjustments of the D. pulex proteome to hypoxia included a strong induction of Hb and carbohydrate-degrading enzymes. The scenario of adaptive protein expression under environmental hypoxia can be interpreted as a process to improve oxygen transport and carbohydrate provision for the maintenance of ATP production, even during short episodes of tissue hypoxia requiring support from anaerobic metabolism.

Novel Activities of Glycolytic Enzymes in Bacillus subtilis

Glycolysis is one of the most important metabolic pathways in heterotrophic organisms. Several genes encoding glycolytic enzymes are essential in many bacteria even under conditions when neither glycolytic nor gluconeogenic activities are required. In this study, a screening for in vivo interaction partners of glycolytic enzymes of the soil bacterium Bacillus subtilis was used to provide a rationale for essentiality of glycolytic enzymes. Glycolytic enzymes proved to be in close contact with several other proteins, among them a high proportion of essential proteins. Among these essential interaction partners, other glycolytic enzymes were most prominent. Two-hybrid studies confirmed interactions of phosphofructokinase with phosphoglyceromutase and enolase. Such a complex of glycolytic enzymes might allow direct substrate channeling of glycolytic intermediates. Moreover we found associations of glycolytic enzymes with several proteins known or suspected to be involved in RNA processing and degradation. One of these proteins, Rny (YmdA), which has so far not been functionally characterized, is required for the processing of the mRNA of the glycolytic gapA operon. Two-hybrid analyses confirmed the interactions between the glycolytic enzymes phosphofructokinase and enolase and the enzymes involved in RNA processing, RNase J1, Rny, and polynucleotide phosphorylase. Moreover RNase J1 interacts with its homologue RNase J2. We suggest that this complex of mRNA processing and glycolytic enzymes is the B. subtilis equivalent of the RNA degradosome. Our findings suggest that the functional interaction of glycolytic enzymes with essential proteins may be the reason why they are indispensable.

The global repressor SugR controls expression of genes of glycolysis and of the L-lactate dehydrogenase LdhA in Corynebacterium glutamicum.

The transcriptional regulator SugR from Corynebacterium glutamicum represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of sugR not only perturbed the growth of C. glutamicum on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (pfkA), fructose-1,6-bisphosphate aldolase (fba), enolase (eno), pyruvate kinase (pyk), and NAD-dependent L-lactate dehydrogenase (ldhA). The deletion of sugR resulted in increased mRNA levels of eno, pyk, and ldhA in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of sugR led to increased LdhA activity under aerobic and under oxygen deprivation conditions, L-lactate production by C. glutamicum was determined. The overexpression of sugR reduced L-lactate production by about 25%, and sugR deletion increased L-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative L-lactate dehydrogenase in C. glutamicum.

Alternate AChE-R variants facilitate cellular metabolic activity and resistance to genotoxic stress through enolase and RACK1 interactions

Tumorogenic transformation is a multifaceted cellular process involving combinatorial protein–protein interactions that modulate different cellular functions. Here, we report apparent involvement in two independent tumorogenic processes by distinct partner protein interactions of the stress-induced acetylcholinesterase AChE-R and N-AChE-R variants. Human testicular tumors showed elevated levels of N-terminally extended N-AChE-R compared with healthy tissue, indicating alternate promoter usage in the transformed cells. Two-hybrid screens demonstrate that the C-terminus common to both N-AChE-R and AChE-R interacts either with the glycolytic enzyme enolase or with the scaffold protein RACK1. In vitro, the AChE-R C-terminal peptide ARP elevated enolase's activity by 12%, suggesting physiological relevance for this interaction. Correspondingly, CHO cells expressing either human AChE-R or N-AChE-R but not AChE-S showed a 25% increase in cellular ATP levels, indicating metabolic significance for this upregulation of enolase activity. ATP levels could be reduced by AChE-targeted siRNA in CHO cells expressing AChE-R but not AChE-S, attributing this elevation to the AChE-R C-terminus. Additionally, transfected CHO cells expressing AChE-R but not N-AChE-R showed resistance to up to 60 μM of the common chemotherapeutic agent, cis-platinum, indicating AChE-R involvement in another molecular pathway. cis-Platinum elevates the expression of the apoptosis-regulator p53-like protein, p73, which is inactivated by interaction with the scaffold protein RACK1. In co-transfected cells, AChE-R competed with endogenous RACK1 for p73 interaction. Moreover, AChE-R-transfected CHO cells presented higher levels than control cells of the pro-apoptotic TAp73 as well as the anti-apoptotic dominant negative ΔNp73 protein, leading to an overall decrease in the proportion of pro-apoptotic p73. Together, these findings are compatible with the hypothesis that in cancer cells, both AChE-R and N-AChE-R elevate cellular ATP levels and that AChE-R modifies p73 gene expression by facilitating two independent cellular pathways, thus conferring both a selective metabolic advantage and a genotoxic resistance.

A Novel Mouse Enolase 1-like Gene is Expressed Specifically in Spermatogenic Cells and Encodes a Protein Present in the Principal Piece Region of the Sperm Flagellum.

The main source of ATP for sperm motility in the mouse is glycolysis and several spermatogenic cell-specific glycolytic enzymes are confined to the principal piece region of the flagellum. During a proteomics study using MALDI/MS to analyze non-ionic detergent-soluble sperm proteins, peptide sequences similar to those present in the glycolytic enzyme enolase 1 (alpha enolase) were identified. Other investigators reported that an enolase sperm-specific isozyme (ENOS) is present in the flagellum of spermatozoa of several mammalian species that is unlike other enolase family members (ENO1, ENO2 and ENO3) in electrophoretic mobility and enzymatic properties. However, the peptide and nucleotide sequences of this novel enolase have not been reported. Database mining identified multiple Eno1-like sequences in the mouse genome, but EST expression profiles and deduced protein sequence analyses strongly suggested that a gene located on chromosome 19 is the Enos gene. The use of PCR and 5 RACE determined that there are two Enos transcript variants expressed in mouse testis (Enos_v1 and Enos_v2), each with a 5 sequence different from those present in transcripts of Eno1 or of other enolase family members. Northern analysis using a probe from the 3′ region of the Enos transcript recognized mRNA only from testis. Northern analysis of RNA from testes of 10- to 35-day-old juvenile mice first detected Enos transcripts on day 14, corresponding to when pachytene spermatocytes appear in the first wave of spermatogenesis. In immunoblotting assays, a pan-enolase antibody reacted with a protein in 1 % Triton X-100, 1 % NP-40 and 0.1 % SDS lysates of testes and sperm, while enolase 2 or 3 were not detected in lysates of testis and sperm with antibodies specific to these enzymes. Immunofluorescence microscopy with this antibody recognized a protein localized to the principal piece region of sperm flagellum, strongly suggesting that the protein recognized by the pan-enolase antibody is ENOS. Extraction of sperm with NP-40 and then 6M urea and immunoblotting assays determined that ENOS is partially extracted by NP-40 and the remainder is extracted by 6M urea. These results suggest that ENOS is not an integral protein of the fibrous sheath like GAPDHS, AKAP4 and GSTM5, but is loosely associated like HK1S and PFKM. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

Microarray Analysis of the Overlap Between Genes Known to Be Induced by Hypoxia and/or Hypoxia-inducible Factor 1α (HIF-1α) and Genes Rapidly Induced by Estrogen in Rat Uterine Epithelial Cells.

Estrogen is the master regulator of gene transcription in the uterine endometrium, acting primarily through its α receptor (ERα). Recent work in our laboratory identified hypoxia-inducible factor 1α (HIF-1α) as a transcription factor which, along with ERα, is necessary for 17β-estradiol (E2)-induced vascular endothelial growth factor (VEGF) expression in the uterus. VEGF causes increased microvascular permeability and edema in the endometrium, which is necessary for subsequent cell proliferation, angiogenesis, and implantation. HIF-1α is normally present in the uterus at low levels and is recruited to the VEGF promoter by E2 via activation of the PI3K/Akt pathway. HIF-1α is also known to regulate the transcription of over 50 other genes in various cell types, many of which are also induced by E2 in the uterus, including most of the glycolytic enzymes, Glut-1, erythropoietin, adrenomedulin, and leptin, in addition to VEGF. Therefore, we hypothesize that E2-induced expression of many genes will involve HIF-1α. To better understand the extent of the overlap between E2- and hypoxia/HIF-1-regulated genes, we used a microarray specific for genes known to be induced by hypoxia and/or to be regulated by HIF-1α (SuperArray Rat Hypoxia Signaling Pathway Oligo GEArray) to determine how many are also induced by E2 in the uterus. Immature female rats were treated with E2 for 0, 1, or 4 h, and the luminal epithelial (LE) cells isolated. Total RNA was extracted and amplified. A biotin tag was attached, and the samples were then hybridized to the array. Of the 114 genes represented, 11 were significantly up-regulated by E2 by 1 and/or 4 h. There was little or no hybridization with the majority of the genes, however, including VEGF, which RT-PCR analysis showed was strongly induced by E2 in the same samples. This indicated that the methodology may be too insensitive to detect many of the target genes, even after E2 stimulation. Nevertheless, increases in the expression of several genes known to be induced by E2 in the uterus were detected, including fos, the glycolytic enzymes enolase, aldolase, and lactate dehyrogenase A, and interleukin 6. In addition, several other genes not previously reported to be controlled by E2 in the uterus were significantly induced. Two of these the transcriptional regulators retinoic acid receptor α (RARα) and inhibitor of DNA binding 1 (Id-1) are of particular interest. RARα was the most strongly up-regulated gene (22-fold). This suggests that it may play an important role in the regulation of gene transcription by E2. Inhibitor of DNA binding 1 (Id-1), which was recently shown to play a role in angiogenesis, was up-regulated 5- and 7-fold by 1 and 4 h, respectively. RT-PCR analysis of both E2-treated rat LE cells and human endometrial cancer (ECC-1) cells was done to confirm these results. These findings support our hypothesis that many of the genes that play a role in cellular adaptations to hypoxia are also required for E2-induced endometrial growth. Future studies will determine exactly how ERα and HIF-1α interact to regulate the expression of this cohort of genes. This work was supported by the NIH/NHLBI-sponsored Institutional Training Grant in Cardiac and Vascular Cell Biology (HL72751) and the NICHD U54 Cooperative Centers Program (HD36207).

Déjà vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins

After reading many 2-DE-based articles featuring lists of the differentially expressed proteins, one starts experiencing a disturbing déjà vu. The same proteins seem to predominate regardless of the experiment, tissue or species. To quantify the occurrence of individual differentially expressed proteins in 2-DE experiment reports, we compiled the identities of differentially expressed proteins identified in human, mouse, and rat tissues published in three recent volumes of Proteomics and calculated the appearance of the most predominant proteins in the dataset. The most frequently identified protein is a highly abundant glycolytic enzyme enolase 1, differentially expressed in nearly every third experiment on both human and rodent tissues. Heat-shock protein 27 (HSP27) and heat-shock protein 60 (HSP60) were differentially expressed in about 30 percent of human and rodent samples, respectively. Considering protein families as units, keratins and peroxiredoxins are the most frequently identified molecules, with at least one member of the group being differentially expressed in about 40 percent of all experiments. We suggest that the frequent identification of these proteins must be considered in the interpretation of any 2-DE studies. We consider if these commonly observed changes represent common cellular stress responses or are a reflection of the technical limitations of 2-DE.

Detection of protein alterations in male breast cancer using two dimensional gel electrophoresis and mass spectrometry: The involvement of several pathways in tumorigenesis

Background Little emphasis has been placed today on the elucidation of protein alterations in male breast carcinogenesis. Methods Protein extracts were subjected to both isoelectric focusing (IEF) and non-equilibrium pH gradient electrophoretic (NEPHGE) analyses. Differentially expressed proteins in tumor tissues were identified by matrix assisted laser desorption /ionization time of flight (MALDI–TOF) mass spectrometry and database search. Results Some of the alterations involve variations in the expression of cytokeratins 8, 18 and 19. More interestingly, tropomyosin1, a protein known to play a role in suppression of the malignant phenotype, was found to be under-expressed in cancer tissues, implicating a possible pivotal role for this protein in male breast carcinogenesis. Co-upregulation of molecular chaperones (heat shock protein HSP27 and protein disulfide isomerase), stress related proteins (peroxiredoxin 1 and peptidylprolyl isomerase A) and glycolytic enzymes (enolase 1) occurred also in male breast tumors. Some of the remaining alterations include proteins involved in invasion and metastasis, such as galectin 1 and cathepsin D. Conclusions The present study represents a first proteomic investigation of protein alterations in infiltrating ductal carcinomas (IDCA) of the male breast. A number of protein alterations in tumor tissues have been characterised thus, providing new insights into the molecular mechanisms underlying this disease.

Comparative Proteomics of Excretory-Secretory Proteins Released by the Liver Fluke Fasciola hepatica in Sheep Host Bile and during in Vitro Culture ex Host

Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates.

Identification of Actin as a 15-Deoxy-Δ12,14-prostaglandin J2Target in Neuroblastoma Cells: Mass Spectrometric, Computational, and Functional Approaches To Investigate the Effect on Cytoskeletal Derangement

A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.

DARBEPOETIN TREATMENT AFTER ETHANOL TOXICITY : AN EXPERIMENTAL STUDY IN RATS

Neuron-specific enolase(NSE) is a dimeric isoenzyme of the glycolytic enzyme enolase. S100 beta is a small, acidic, calcium binding protein which is highest in concentration in the vertebrate nervous system. S-100B is found in high concentrations in astroglial and Schwann cells. 44 Wistar-albino adult rats were divided into four groups: Saline-treated group(S) (n=10), Saline-Darbepoetin-treated group (SD)(n=10), Ethanol- treated group (ET) (n=12), Ethanol-Darbepoetin-treated group (ETD) (n=12). 20% ethanol solution prepared with sterile saline was administered to rats ip at a dosage of 2.5 g/kg for two times with a 2-h interval. Darbepoetin was administered ip just after the second dose of ethanol at a dosage of 10 μg /kg. Twenty-four hours after the first dose of ethanol, the animals were decapitated under Ketamine (5–10ml/kg). In this study, the effect of darbepoetin administration in ethanol toxicity on brain tissue’s oxidant-antioxidant parameters which are Malonyl dialdehyde(MDA), Cu-Zn Superoxide dismutase(Cu-Zn SOD), Glutathione(GSH) and Catalase(CAT); neuronal damage markers which are S-100Beta (CanAg S100 EIA REF 708-10, Sweden) and Neuron Specific Enolase(NSE) (CanAg NSE EIA REF 420-10) was evaluated. Ethanol increases significantly S100 levels(p<0.05) in ET. Darbepoetin administration decreases significantly NSE levels in ETD(p<0.01). Darbepoetin might be protect against ethanol induced neurodegenerative effect.