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At least 343 records · Page 19Linked to original sources
PubMed2002

Rapid phosphorylation of heterogeneous nuclear ribonucleoprotein C1/C2 in response to physiologic levels of hydrogen peroxide in human endothelial cells.

Hydrogen peroxide (H(2)O(2)) has been implicated as a signaling agent in numerous signal transduction pathways in mammalian cells. However, to date, no sensor for low concentrations (<10 microm) of H(2)O(2) has been identified. Using a functional proteomic approach, nuclear extracts from human umbilical vein endothelial cells were analyzed by two-dimensional PAGE with or without prior treatment with a low concentration of H(2)O(2). A protein doublet with a molecular mass of 39-41 kDa and a pI of approximately 5.0 was observed to be consistently altered by the treatment. Using proteolytic peptide mass fingerprinting, the protein was identified as heterogeneous nuclear ribonucleoprotein C1/C2, a nuclear restricted, pre-mRNA-binding protein. Upon two-dimensional PAGE, each heterogeneous nuclear ribonucleoprotein-C splice form was present as multiple spots because of differing levels of phosphorylation. Upon treatment with H(2)O(2), there was an increase in phosphorylation at 10-20 min, which partially reversed by 30 min. Subsequently, at 60 min after treatment, a population of unphosphorylated protein was transiently present. The effects were observed with as little as 1 microm H(2)O(2) and were maximal with 5-8 microm H(2)O(2). The H(2)O(2)-stimulated phosphorylation was inhibited by catalase, but not by the transcriptional inhibitor actinomycin D.

Amino Acid Sequence↗
PubMed2003

Receptor/transporter-independent targeting of functional peptides across the plasma membrane.

Targeting of peptides, proteins, and other functional cargo into living cells is contingent upon efficient transport across the plasma membrane barrier. We have harnessed the signal sequence hydrophobic region (SSHR) to deliver functional cargoes to cultured cells and to experimental animals. We now report evidence that two chirally distinct forms of SSHR composed of all l or all d amino acids showed similar membrane-translocating activity as assessed by confocal microscopy, flow cytometry, and direct fluorescence measurement. An attached nuclear localization sequence ferried by the SSHR enantiomers displayed similar intracellular function by inhibiting inducible nuclear import of transcription factor nuclear factor kappa B and suppressing nuclear factor kappa B-dependent gene expression of cytokines. A nuclear localization sequence comprised of a positively charged cluster of amino acids was rapidly translocated by SSHR enantiomers to the interior of unilamellar phospholipid vesicles. These findings indicate that the SSHR translocates functional peptides directly through the plasma membrane phospholipid bilayer without involving chirally specific receptor/transporter mechanisms. This mechanism of SSHR translocation is suitable for facile delivery of biologically active peptides for cell-based and animal-based functional proteomic studies.

Amino Acid Sequence↗
PubMed2004

Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase.

Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 x g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes.

Adipocytes↗
PubMed2004

ERK1/2 regulates intracellular ATP levels through alpha-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation.

Extracellular signal-regulated kinase 1/2 (ERK1/2) is known to function in cell survival in response to various stresses; however, the mechanism of cell survival by ERK1/2 remains poorly elucidated in ischemic heart. Here we applied functional proteomics by two-dimensional electrophoresis to identify a cellular target of ERK1/2 in response to ischemic hypoxia. Approximately 1500 spots were detected by Coomassie Brilliant Blue staining of a sample from unstimulated cells. The staining intensities of at least 50 spots increased at 6-h reoxygenation after 2-h ischemic hypoxia. Of the 50 spots that increased, at least 4 spots were inhibited in the presence of PD98059, a MEK inhibitor. A protein with a molecular mass of 52 kDa that is strongly induced by ERK1/2 activation in response to ischemic hypoxia and reoxygenation was identified as alpha-enolase, a rate-limiting enzyme in the glycolytic pathway, by liquid chromatography-mass spectrometry and amino acid sequencing. The expressions of the alpha-enolase mRNA and protein are inhibited during reoxygenation after ischemic hypoxia in the cells containing a dominant negative mutant of MEK1 and treated with a MEK inhibitor, PD98059, leading to a decrease in ATP levels. alpha-Enolase expression is also observed in rat heart subjected to ischemia-reperfusion. The induction of alpha-enolase by ERK1/2 appears to be mediated by c-Myc. The introduction of the alpha-enolase protein into the cells restores ATP levels and prevents cell death during ischemic hypoxia and reoxygenation in these cells. These results show that alpha-enolase expression by ERK1/2 participates in the production of ATP during reoxygenation after ischemic hypoxia, and a decrease in ATP induces apoptotic cell death. Furthermore, alpha-enolase improves the contractility of cardiomyocytes impaired by ischemic hypoxia. Our results reveal that ERK1/2 plays a role in the contractility of cardiomyocytes and cell survival through alpha-enolase expression during ischemic hypoxia and reoxygenation.

Adenosine Triphosphate↗
PubMed2006

Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta-hydrolase 4 in this pathway.

N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.

AlkB Homolog 4, Lysine Demethylase↗
PubMed2006

A second fatty acid amide hydrolase with variable distribution among placental mammals.

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.

Amidohydrolases↗
PubMed2003

The power and the limitations of cross-species protein identification by mass spectrometry-driven sequence similarity searches.

Mass spectrometry-driven BLAST (MS BLAST) is a database search protocol for identifying unknown proteins by sequence similarity to homologous proteins available in a database. MS BLAST utilizes redundant, degenerate, and partially inaccurate peptide sequence data obtained by de novo interpretation of tandem mass spectra and has become a powerful tool in functional proteomic research. Using computational modeling, we evaluated the potential of MS BLAST for proteome-wide identification of unknown proteins. We determined how the success rate of protein identification depends on the full-length sequence identity between the queried protein and its closest homologue in a database. We also estimated phylogenetic distances between organisms under study and related reference organisms with completely sequenced genomes that allow substantial coverage of unknown proteomes.

Animals↗
PubMed2006

Serum peptidome patterns that distinguish metastatic thyroid carcinoma from cancer-free controls are unbiased by gender and age.

Serum peptidomics is a special form of functional proteomics. The small number of blood proteins that are the source of most prominent peptides in human serum serve as a substrate pool for commonly occurring and/or cancer-derived proteases. Exoprotease activities in particular, when superimposed on the ex vivo coagulation and complement degradation pathways, contribute to generation of not only cancer-specific but also "cancer type"-specific serum peptides. Following development of a unique, semiautomated serum peptide profiling platform and after completing investigations to eliminate common experimental bias, we have now studied possible effects of gender and age on serum peptidomes of 200 healthy men and women, ages 20-80, and of 60 patients (30 men and 30 women) with metastatic thyroid carcinomas. Extensive MALDI-TOF MS and data analysis suggested negligible contributions of both age and gender to the serum peptidome patterns except that healthy men and women under 35 years, but not older individuals, could be distinguished with approximately 70% accuracy. Considering the more advanced age of most patients, this finding is unlikely to interfere with peptidomics analysis of most cancers. By examining patient samples and age/gender-matched controls followed by variability analysis of either demographic or disease (versus control) groups, we could conclusively rule out demographic bias. An optimized, 12-peptide ion thyroid cancer signature was then developed, enabling classification of an independent validation set with 95% sensitivity and 95% specificity (binomial confidence intervals, 75.1-99.9%). Ten of these peptides had previously been assigned to signature patterns of other solid tumor cancers. One of the two newly discovered peptides was dehydro-Ala(3)-fibrinopeptide A. As we expand this study to include hundreds of thyroid cancer patients, the peptide signature will be adjusted, further validated, and then evaluated in a clinical setting used either independently or in combination with existing markers.

Adult↗
PubMed2003

Gastrointestinal cancer: towards a tailored tumour therapy.

Major progress has been made in the treatment of metastatic colorectal cancer, but conventional chemotherapy is unlikely to lead to a major breakthrough in the treatment of gastrointestinal tumours. Prognosticators, novel means of early diagnosis of cancer which at the same time indicate prognosis, as well as novel, tumour-specific therapeutic strategies, are urgently needed. There is an increasing amount of promising data from array technology and functional proteomics suggesting that this goal could be achieved in the near future. Research into the mechanisms of cancer signal transduction over the past 20 years has enabled the identification of numerous novel targets for tumour therapy and subsequently the development of various novel drugs termed magic bullets. The in vitro results with these drugs are promising; many drugs lost their magic after the first clinical trials. The state of novel diagnostics and therapeutics in gastrointestinal cancer and the implications for the future treatment of these tumours are discussed in this review.

Antineoplastic Agents↗
PubMed2002

The role of hydrogen peroxide in endothelial proliferative responses.

Hydrogen peroxide (H2O2) is a recently recognized second messenger regulating proliferation in mammalian cells. Endothelial cells possess NADPH oxidases, which produce the H202 precursor superoxide (.O2-) in response to receptor-mediated signaling. Multiple physiologic agents have been shown to stimulate endothelial cells to produce .O2-/H2O2, including growth factors, such as vascular endothelial growth factor and transforming growth factor-beta1, and alterations in biomechanical forces, such as shear stress and cyclic strain. Downstream effects of these stimuli can often be inhibited by scavenging H2O2. Low concentrations of H2O2 stimulate proliferation or enhanced survival in a wide variety of cell types. Also, low concentrations of H2O2 stimulate endothelial migration as well as tube formation in an in vitro model of angiogenesis. Although low concentrations of H2O2 have been shown to be involved in numerous signal transduction pathways and to independently stimulate mitogenesis, there has been little information presented on precisely how mammalian cells respond biochemically to these low concentrations of H2O2. Recently a functional proteomics approach has been utilized to identify proteins responsive to low concentrations of H2O2 in human endothelial cells.

Animals↗
PubMed2000

Mutation-tolerant protein identification by mass spectrometry.

Database search in tandem mass spectrometry is a powerful tool for protein identification. High-throughput spectral acquisition raises the problem of dealing with genetic variation and peptide modifications within a population of related proteins. A method that cross-correlates and clusters related spectra in large collections of uncharacterized spectra (i.e., from normal and diseased individuals) would be very valuable in functional proteomics. This problem is far from being simple since very similar peptides may have very different spectra. We introduce a new notion of spectral similarity that allows one to identify related spectra even if the corresponding peptides have multiple modifications/mutations. Based on this notion, we developed a new algorithm for mutation-tolerant database search as well as a method for cross-correlating related uncharacterized spectra.

Algorithms↗
PubMed2003

The pre-ribosomal network.

Recent achievements in yeast functional proteomics have significantly advanced our knowledge about ribosome biogenesis. Here, we present a program developed to integrate data from various proteome analyses with cell biological data on components present in the ribosome producing factories. This program allows users to attribute factors to certain complexes and to specific steps of ribosome biogenesis. Thus, it helps to gain novel insights into the complex network involved in maturation of ribosomal subunits. The database can be accessed at the URL http://www.pre-ribosome.de.

Biomarkers↗
PubMed2005

Farm animal genomics and informatics: an update.

Farm animal genomics is of interest to a wide audience of researchers because of the utility derived from understanding how genomics and proteomics function in various organisms. Applications such as xenotransplantation, increased livestock productivity, bioengineering new materials, products and even fabrics are several reasons for thriving farm animal genome activity. Currently mined in rapidly growing data warehouses, completed genomes of chicken, fish and cows are available but are largely stored in decentralized data repositories. In this paper, we provide an informatics primer on farm animal bioinformatics and genome project resources which drive attention to the most recent advances in the field. We hope to provide individuals in biotechnology and in the farming industry with information on resources and updates concerning farm animal genome projects.

Animals↗
PubMed2005

Evolution of alternative splicing after gene duplication.

Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evolve independently. In the early stage after gene duplication, young duplicates may take over a certain amount of protein function diversity that previously was carried out by the alternative splicing mechanism. In the late stage, the gain and loss of alternative splicing seem to be independent between duplicates.

Alternative Splicing↗
PubMed2000

Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins?

An obligatory step in cellulose degradation by anaerobic bacteria is the adhesion of the bacterium to the polysaccharide. In many anaerobic bacteria the adhesion protein, and the enzymes required for extensive polysaccharide hydrolysis, are organized into a complex and interesting structure called the cellulosome. The Gram-positive anaerobe Ruminococcus albus also produces a cellulosome-like complex, but the bacterium appears to possess other mechanism(s) for adhesion to plant surfaces and genes encoding functions relevant to growth on cellulose are conditionally expressed, as suggested by a combination of functional proteomics, differential display reverse-transcriptase PCR, and mutational analysis. A novel form of cellulose-binding protein has been identified and shown to belong to the Pil-protein family, being most similar to the type 4 fimbrial proteins of Gram-negative, pathogenic bacteria. These studies have provided new insights into the adhesion of bacteria to plant surfaces, and call attention to the likely existence of genetically analogous adhesion determinants in both pathogenic and non-pathogenic bacteria.

Adhesins, Bacterial↗
PubMed2003

Programmed adsorption and release of proteins in a microfluidic device.

A microfluidic device has been developed that can adsorb proteins from solution, hold them with negligible denaturation, and release them on command. The active element in the device is a 4-nanometer-thick polymer film that can be thermally switched between an antifouling hydrophilic state and a protein-adsorbing state that is more hydrophobic. This active polymer has been integrated into a microfluidic hot plate that can be programmed to adsorb and desorb protein monolayers in less than 1 second. The rapid response characteristics of the device can be manipulated for proteomic functions, including preconcentration and separation of soluble proteins on an integrated fluidics chip.

Acrylic Resins↗
PubMed2004

Bmx, a member of the Tec family of nonreceptor tyrosine kinases, is a novel participant in pharmacological cardioprotection.

Previous studies have indicated that PKC-epsilon is a central regulator of protective signal transduction in the heart. However, the signaling modules through which PKC-epsilon exerts its protective effects have only begun to be understood. We have identified a novel participant in the PKC-epsilon signaling system in cardioprotection, the nonreceptor tyrosine kinase Bmx. Functional proteomic analyses of PKC-epsilon signaling complexes identified Bmx as a member of these complexes. Subsequent studies in rabbits have indicated that Bmx is activated by nitric oxide (NO) in the heart, concomitant with the late phase of NO donor-induced protection, and provide the first analysis of Bmx expression/distribution in the setting of cardioprotection. In addition, increased expression of Bmx induced by NO donors was blocked by the same mechanism that blocked cardioprotection: inhibition of PKC with chelerythrine. These findings indicate that a novel type of PKC-tyrosine kinase module (involving Bmx) is formed in the heart and may be involved in pharmacological cardioprotection by NO donors.

Alkaloids↗
PubMed2002

Molecular conformation dictates signaling module formation: example of PKCepsilon and Src tyrosine kinase.

Our laboratory has conducted multiple functional proteomic analyses to characterize the components of protein kinase C (PKC)epsilon cardioprotective signaling complexes and found that activation of PKCepsilon induces dynamic modulation of these complexes. In addition, it is known that signal transduction within a complex involves the formation of modules, one of which has been shown to include PKCepsilon and Src tyrosine kinase in the rabbit heart. However, the cellular mechanisms that define the assembly of PKCepsilon modules remain largely unknown. To address this issue, the interactions between PKCepsilon and Src were studied. We used recombinant proteins of wild-type PKCepsilon (PKCepsilon-WT) and open conformation mutants of the kinase (PKCepsilon-AE5 and PKCepsilon-AN59), the regulatory and catalytic domains of PKCepsilon, along with glutathione-S-transferase (GST) fusion proteins of Src (GST-Src) and two domains of Src (GST-SH2 and GST-SH3). GST pulldown assays demonstrated that Src and PKCepsilon are binding partners and that the interaction between PKCepsilon and Src appears to involve multiple sites. This finding was supported for endogenous PKCepsilon and Src in the murine heart using immunofluorescence-based confocal microscopy and coimmunoprecipitation. Furthermore, PKCepsilon-WT and GST-Src interactions were significantly enhanced in the presence of phosphatidyl-L-serine, an activator of PKC, indicating that Src favors interaction with activated PKCepsilon. This finding was confirmed when the PKCepsilon-WT was replaced with PKCepsilon-AE5 or PKCepsilon-AN59, demonstrating that the conformation of PKCepsilon is a critical determinant of its interactions with Src. Together, these results illustrate that formation of a signaling module between PKCepsilon and Src involves specific domains within the two molecules and is governed by the molecular conformation of PKCepsilon.

Animals↗