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PubMed2005

The hypoxic proteome is influenced by gene-specific changes in mRNA translation.

BACKGROUND AND PURPOSE: Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined. MATERIALS AND METHODS: DU145 prostate carcinoma cells were exposed to 4h of hypoxia (<0.02% O2). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR. RESULTS: One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity. CONCLUSIONS: Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia.

Cell Hypoxia↗
PubMedDate not supplied

Proteomics in developmental toxicology.

The objective of this presentation is to review the major proteomic technologies available to developmental toxicologists and, when possible, to provide examples of how various proteomic technologies have been used in developmental toxicology or toxicology in general. The field of proteomics is too broad for us to go into great depth about each technology, so we have attempted to provide brief overviews supplemented with many references that cover the subjects in more detail. Proteomics tools produce a global view of complex biological systems by examining complex protein mixtures using large-scale, high-throughput technologies. These technologies speed up the process of protein separation, quantification, and identification. As an important complement to genomics, proteomics allows for the examination of the entire complement of proteins in an organism, tissue, or cell-type. Current proteomics technologies not only identify protein expression, but also post-translational modifications and protein interactions. The field of proteomics is expanding rapidly to provide greater volume and quality of protein information to help understand the multifaceted nature of biological systems.

Animals↗
PubMed2005

New methodologies for measuring protein interactions in vivo and in vitro.

The identification and characterization of protein interactions is a key topic in current life science research; a huge variety of methodologies have been established in recent years to expedite research in this area. Generic methods have been established for monitoring protein interactions in vivo by protein fragment complementation and for screening protein interactions in vitro by highly parallel solid-phase techniques. Substantial progress has been made in identifying and characterizing interactions with and between membrane proteins. Studying protein interactions on the single-molecule level has become an important tool for understanding protein function in vivo and in vitro.

Fluorescence Resonance Energy Transfer↗
PubMed2006

Protein tyrosine kinase-substrate interactions.

Protein tyrosine kinases (PTKs) are enzymes that catalyze the phosphorylation of tyrosyl residues. They are important in physiological and pathophysiological processes. Protein substrates of PTKs are often difficult to discern, but recently reported methods have helped to identify targets and characterize their structural interactions with kinases. A mechanism-based bisubstrate analog strategy has given X-ray crystallographic insights into how several topical PTKs, including the insulin receptor, Abl and epidermal growth factor receptor, interact with tyrosine-containing peptide substrates. These PTK co-crystal structures reveal both conserved and specialized features of recognition that probably contribute to substrate selection and the individual functions of these key enzymes.

Amino Acid Sequence↗
PubMed2006

Immobilisation of proteins by atomic clusters on surfaces.

In this Opinion article, we describe a nanotechnology-based approach to immobilize and orient proteins onto surfaces using atomic clusters prepared by physical methods. This is relevant to future protein biochips where dilute arrays of protein binding sites, each designed to immobilize no more than one protein molecule, would be ideal. In the case of a surface consisting of size-selected atomic gold clusters, proteins containing free cysteine residues can chemisorb directly to the bare cluster surface, thus effecting oriented immobilisation. The selection of atomic gold clusters in the size range 1-100 atoms (<3nm in diameter) is intended to ensure that, typically, only one protein can bind directly to the cluster surface. These nanoclusters of a smaller size scale than that of the protein present minimal contact between the gold and the protein, and hence imply a reduced risk of protein denaturing compared with gold films or extended surfaces.

Binding Sites↗
PubMed2006

PDZ-domain arrays for identifying components of GPCR signaling complexes.

Many G-protein-coupled receptors (GPCRs) modulate the activity of multiple effectors. Yet, despite this apparent promiscuity, signaling in the context of differentiated cells is often highly specific. This specificity is attributable to the formation of cell-type-specific signaling complexes that are held together by scaffolding proteins, many of which contain one or more PDZ domains. Identifying the set of potential interactions among GPCRs, other signaling molecules and these scaffolding proteins is essential for understanding physiological signaling processes. A recent article describes an elegantly simple PDZ-domain array that can identify potential interacting partners of GPCRs and other signaling molecules.

Animals↗
PubMed2006

Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions.

Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.

Actins↗
PubMed2005

Biomarker discovery in biological fluids.

Discovery of novel protein biomarkers is essential for successful drug discovery and development. These novel protein biomarkers may aid accelerated drug efficacy, response, or toxicity decision making based on their enhanced sensitivity and/or specificity. These biomarkers, if necessary, could eventually be converted into novel diagnostic marker assays. Proteomic platforms developed over the past few years have given us the ability to rapidly identify novel protein biomarkers in various biological matrices from cell cultures (lysates, supernatants) to human clinical samples (serum, plasma, and urine). In this article, we delineate an approach to biomarker discovery. This approach is divided into three steps, (i) identification of markers, (ii) prioritization of identified markers, and (iii) preliminary validation (qualification) of prioritized markers. Using drug-induced idiosyncratic hepatotoxicity as a case study, the article elaborates methods and techniques utilized during the three steps of biomarker discovery process. The first step involves identification of markers using multi-dimensional protein identification technology. The second step involves prioritization of a subset of marker candidates based on several criteria such as availability of reagent set for assay development and literature association to disease biology. The last step of biomarker discovery involves development of preliminary assays to confirm the bio-analytical measurements from the first step, as well as qualify the marker(s) in pre-clinical models, to initiate future marker validation and development.

14-3-3 Proteins↗
PubMed2006

Using peptide arrays to define nuclear carrier binding sites on nucleoporins.

In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.

Active Transport, Cell Nucleus↗
PubMed2003

Optimizing antibody immobilization strategies for the construction of protein microarrays.

Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.

Animals↗
PubMed2003

Next-generation protein-handling method: puromycin analogue technology.

Puromycin is a well-known antibiotic that inhibits protein synthesis by competitive incorporation against an aminoacyl tRNA on the ribosome A site. Novel technology using this property of puromycin has been developed for convenient handling methods in protein research. Puromycin modified with another molecule is incorporated into a protein at the C-terminus, thus linking the desired molecule to the protein. Combination of in vitro translation with puromycin analogues has resulted in novel technologies such as display technology for screening, fluorescence labeling, affinity purification, and protein chip for proteomics.

Biotechnology↗
PubMed2003

Cytokine regulation by peroxisome proliferator-activated receptor gamma in human endometrial cells.

OBJECTIVE: To determine whether peroxisome proliferator-activated receptor (PPAR)-gamma ligands can affect the expression of interleukin-6 (IL-6) and cytokines related to the pathogenesis of endometriosis. DESIGN: In vitro study to determine whether PPARs are expressed in human endometrial cells and determine the effects of various PPAR-gamma ligands on IL-6 and other cytokine expression in these cells. SETTING: Academic medical center. PATIENT(S): Women presenting for infertility workup. INTERVENTION(S): Endometrial cell cultures were treated with PPAR-gamma ligands. MAIN OUTCOME MEASURE(S): Interleukin-6, IL-8, colony stimulating factor-1 (CSF-1) and macrophage chemotactic factor (MCP-1) protein secretion, messenger RNA expression of IL-6, PPAR-alpha, -beta, and -gamma. RESULT(S): Using a human endometrial cell line (EM42), as well as primary stromal and epithelial endometrial cells, we show the presence of PPAR-alpha, -beta, and -gamma by reverse transcription-polymerase chain reaction (RT-PCR) in these cells. PPAR-gamma ligands stimulated IL-6 secretion and induced enhancement of IL-6 mRNA levels. These ligands also stimulated the secretion of IL-8 and CSF-1. CONCLUSION(S): PPAR-gamma may play a role in the pathogenesis of endometriosis related to the production of IL-6 and some other cytokines.

Benzophenones↗
PubMed2003

Next generation of protein microarray support materials: evaluation for protein and antibody microarray applications.

The performance of protein and antibody microarrays is dependent on various factors, one of which is the use of an appropriate microarray surface for the immobilisation of either protein or antibody samples. We have investigated the properties of seven new surfaces in the context of both protein and antibody microarray technology. We have demonstrated the functionality of all new slide coatings and investigated the mean signal to spotted concentration ratio, determined detection limits and calculated coefficients of variation. Moreover, new concepts for slide coatings such as dendrimer and poly(ethylene glycol)-epoxy slides were evaluated and improved qualities of novel slide surfaces were observed. Optimal slide coatings for antibody and protein chips were proposed and the requirements for both technologies were discussed.

Antibodies↗
PubMed2003

Expression of soluble recombinant proteins in a cell-free system using a 96-well format.

For structural and functional genomics programs, new high-throughput methods to obtain well-expressing and highly soluble proteins are essential. Here, we describe a rapid procedure to express recombinant proteins in an Escherichia coli cell-free system using a 96-well format. The identification of soluble proteins is performed by the Dot Blot procedure using an anti-His tag antibody. The applications and the automation of this method are described.

Cell-Free System↗
PubMed2003

Isolating ligands specific for human vasculature using in vivo phage selection.

The endothelium lining blood vessels expresses molecules that are restricted in their expression to a particular tissue or organ. These molecules are attractive targets for therapy and diagnosis because they allow agents to be delivered specifically to the blood vessels supplying the desired tissue. However, it is difficult to identify these tissue-specific molecules because endothelium loses much of its tissue-specific nature when it is removed from the organ. This can be overcome by using in vivo phage selection - injecting libraries of phage bearing antibodies or peptides into an animal and isolating phage that bind to the relevant tissue. A variation on this approach, in which in vivo phage selection is performed in animals bearing human tissue xenografts, allows the isolation of peptides (and presumably other molecules) specific for human vasculature.

Animals↗
PubMed2003

Antibodies in proteomics II: screening, high-throughput characterization and downstream applications.

There are many ways in which the use of antibodies and antibody selection can be improved and developed for high-throughput characterization. Standard protocols, such as immunoprecipitation, western blotting and immunofluorescence, can be used with antibody fragments generated by display technologies. Together with novel approaches, such as antibody chips and intracellular immunization, these methods will yield useful proteomic data following adaptation of the protocols for increased reliability and robustness. To date, most work has focused on the use of standard, well-characterized commercial antibodies. Such protocols need to be adapted for broader use, for example, with antibody fragments or other binders generated by display technologies, because it is unlikely that traditional approaches will provide the required throughput.

Antibodies↗