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At least 307 records · Page 17Linked to original sources
PubMed2006

Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines.

Ubiquitin specific proteases (USPs) regulate the production and recycling of ubiquitin and are thereby critically involved in the control of cell growth, differentiation, and apoptosis. Increasing evidence implicates deregulation of USPs in malignant transformation but there is very little information on the overall and specific activity of USPs in normal and tumor tissues. We have used a chemistry-based functional proteomics approach to profile the activities of individual USPs in biopsies of human papillomavirus (HPV) carrying cervical carcinoma and adjacent normal tissue. To assess the contribution of HPV proteins, USP activity was also compared in HPV positive and negative cervical carcinoma cell lines and HPV E6/E7 immortalized human keratinocytes. The activity of the C-terminal hydrolases UCH-L3 and UCH37 was upregulated in the majority of tumor tissues compared to the adjacent normal tissues. UCH-L1 activity was lower in a significant proportion of the tumors but to a less extent in advanced tumors. In accordance with the relatively low UCH-L1 activity in tumor biopsies, UCH-L1 was detected only in one out of eight cervical carcinoma lines. UCH-L1, UCH-L3, USP7, and USP9X activity was upregulated following HPV E6/E7 immortalization of keratinocytes, suggesting a role of these enzymes in growth transformation.

Biopsy↗
PubMed2006

Transfer and multiplex immunoblotting of a paraffin embedded tissue.

As we transition from genomics to the challenges of the functional proteome, new tools to explore the expression of proteins within tissue are essential. We have developed a method of transferring proteins from a formalin fixed, paraffin embedded tissues section to a stack of membranes which is then probed with antibodies for detection of individual epitopes. This method converts a traditional tissue section into a multiplex platform for expression profiling. A single tissue section can be transferred to up to ten membranes, each of which is probed with different antibodies, and detected with fluorescent secondary antibodies, and quantified by a microarray scanner. Total protein can be determined on each membrane, hence each antibody has its own normalization. This method works with phospho-specific antibodies as well as antibodies that do not readily work well with paraffin embedded tissue. This novel technique enables archival paraffin embedded tissue to be molecularly profiled in a rapid and quantifiable manner, and reduces the tissue microarray to a form of protein array. This method is a new tool for exploration of the vast archive of formalin fixed, paraffin embedded tissue, as well as a tool for translational medicine.

Antibodies, Phospho-Specific↗
PubMed2005

Cross-linking chemistry and biology: development of multifunctional photoaffinity probes.

An efficient method of photoaffinity labeling has been developed based on rationally designed multifunctional photoprobes. Photoaffinity techniques have been used to elucidate the protein structure at the interface of biomolecules by the photochemical labeling of interacting sites. However, the identification of labeled sites within target proteins is often difficult. Novel biotinyl bioprobes bearing a diazirine photophore have contributed significantly to the rapid elucidation of ligand binding sites within proteins, thereby extending conventional photoaffinity methods. This article discusses the synthesis and applications of various photoprobes bearing a biotin, including strategies using cleavable linkages between photophores. The combination of photoaffinity methods with chip technology is also described as a novel entry to rapid affinity-based screening of inhibitors. This review focuses on a rapid and reliable photoaffinity method utilizing diazirine-based multifunctional photoprobes with numerous potential applications in functional proteomics of biomolecular interactions.

Diazomethane↗
PubMed1998

Molecular alterations in bladder cancer.

In recent years, significant information has been accumulated on the molecular alterations that take place during development of transitional cell carcinoma (TCC). A number of studies aimed at defining loss of heterozygosity have shown a general chromosomal instability in TCC with loss of parts of chromosome 9 at early stages of papillomas, and of chromosomes 11, 13, 3, 4, 8, 17 and 18 during further development of the tumor. Oncogenes are activated, exemplified by mutations in the ras gene family and overexpression of the c-erbB-2 gene, in a minor fraction of tumors. Alterations of tumor suppressors (involved in control of the cell cycle, DNA quality control and activation of apoptosis) seem to be frequently involved. Among these p53 has a key role, and one p53 allele is frequently lost in TCC followed by mutation of the remaining allele.These alterations are correlated with survival, disease progression, invasion and recurrence. Also frequently lost are the cell cycle control genes p16 and p15. The predictive value of this has not yet been determined. Studies of glycosylation genes have shown downregulation of the ABO gene, followed by loss of ABO blood group structures and accumulation of the Lewis cell adhesion molecules in high grade tumors. Functional proteome analysis has furthermore identified biomarkers that are correlated with grade and stage. Molecular models for TCC development can now be built, and clinical testing of these is urgently needed.

Blood Group Antigens↗
PubMed2004

Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round.

Site-directed mutagenesis is a powerful tool to explore the structure-function relationship of proteins, but most traditional methods rely on the mutation of only one site at a time and efficiencies drop drastically when more than three sites are targeted simultaneously. Many applications in functional proteomics and genetic engineering, including codon optimization for heterologous expression, generation of cysteine-less proteins, and alanine-scanning mutagenesis, would greatly benefit from a multiple-site mutagenesis method with high efficiency. Here we describe the development of a simple and rapid method for site-directed mutagenesis of more than 10 sites simultaneously with up to 100% efficiency. The method uses two terminal tailed primers with a unique 25-nucleotide tail each that are simultaneously annealed to template DNA together with the set of mutagenic primers in between. Following synthesis of the mutant strand by primer extension and ligation with T4 DNA polymerase and ligase, the unique mutant strand-specific tails of the terminal primers are used as anchors to specifically amplify the mutant strand by high-fidelity polymerase chain reaction. We have employed this novel method to mutate simultaneously all 9 and 11 CUG leucine codons of the Hyg and Neo resistance genes, respectively, to the Candida albicans-friendly UUG leucine codon at 100% efficiency.

Amino Acid Substitution↗
PubMed2006

Cholesterol loading increases the translocation of ATP synthase beta chain into membrane caveolae in vascular endothelial cells.

Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs) and have been suggested to contribute to cell signaling and cholesterol trafficking. This study investigated the effect of cholesterol on the movement of caveolae-related proteins in human umbilical vein ECs with use of caveolae functional proteomics. After cholesterol exposure to ECs for 2 to 4 h, caveolae were isolated and separated on 2-D protein gels. Among 40 protein spots revealed in caveolae fractions, the ATP synthase beta subunit (ATPS-beta), one of the 3 proteins enriched by cholesterol in caveolae, was confirmed by western blotting and confocal microscopy. Further, cholesterol exposure increased the level of ATPS-beta, along with Cav-1 and cholesterol in caveolae. These effects could be blocked by cytochalasin B, an actin cytoskeleton disruptor. ATPS-beta was physically associated with Cav-1, as demonstrated by co-immunoprecipitation and GST-Cav-1 fusion protein pull-down assay. Cholesterol increased the extracellular ATP release mediated by ATPS-beta, since this action could be blocked by piceatannol or oligomycin, ATPS inhibitors. Thus, the ectopic localization of ATPS-beta may participate in the energy balance of cells in response to the change in intracellular cholesterol levels.

Caveolae↗
PubMedDate not supplied

High throughput cloning and expression strategies for protein production.

Traditionally, the production of a recombinant protein requires a preliminary cloning step of the target gene into an expression vector before evaluating its cellular expression. Among current methods, site-specific recombination cloning techniques, which eliminate the use of restriction endonucleases and ligase, offer several advantages in the context of high throughput (HT) procedures. Rapid and highly efficient, the recombinational cloning technology is largely used for structural genomics and functional proteomics. However, the correct expression of some genes requires further optimization steps that are time-consuming and carried out at relatively late stages in the cloning-expression process. An alternative strategy is described where expression is tested in vitro before cloning the target gene. This technology, amenable to automation for HT studies, makes the expression of several hundreds of genes possible from PCR products in cell-free transcription-translation systems. Once this preliminary step is achieved, the PCR product, which gives satisfying expression levels, is selected, and then cloned in a plasmid for its cellular expression and perpetuation.

Animals↗
PubMed2004

Synthesis and characterization of bifunctional probes for the specific labeling of fusion proteins.

Labeling proteins with synthetic probes is important for studying and characterizing protein function. We have recently introduced a general method for the specific in vivo and in vitro labeling of fusion proteins that is based on the reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives. Here we report two complementary routes for the synthesis of O6-benzylguanine derivatives, which allow for the labeling of AGT fusion proteins with bifunctional synthetic probes and demonstrate the specific labeling of AGT fusion proteins with these probes. These molecules should become useful tools for various applications in functional proteomics.

Cloning, Molecular↗
PubMed2004

Protein microarray detection strategies: focus on direct detection technologies.

Protein microarrays are being utilized for functional proteomic analysis, providing information not obtainable by gene arrays. Microarray technology is applicable for studying protein-protein, protein-ligand, kinase activity and posttranslational modifications of proteins. A precise and sensitive protein microarray, the direct detection or reverse-phase microarray, has been applied to ongoing clinical trials at the National Cancer Institute for studying phosphorylation events in EGF-receptor-mediated cell signaling pathways. The variety of microarray applications allows for multiple, creative microarray designs and detection strategies. Herein, we discuss detection strategies and challenges for protein microarray technology, focusing on direct detection of protein microarrays.

Animals↗
PubMed2006

Checking and fixing the cellular nanomachinery: towards medical nanoscopy.

Most diseases, regardless of their diverse etiologies, manifest themselves as defects of cellular proteins. Cellular proteins have been recently shown to form specific complexes exerting their functions as if they were nanoscopic machines. Such nanoscopic protein machines cooperate in functional modules, yielding extended, highly compartmentalized networks. The classical resolution limits of fluorescence microscopy have also been recently overcome, opening the nanometer domain to live-cell imaging. Together, progress in functional proteomics and live-cell imaging provide novel possibilities for directly analyzing and modifying nanoscopic protein machines in living cells and tissues.

Animals↗
PubMed2005

Making the most of affinity tags.

Proteins do not naturally lend themselves to high-throughput analysis because of their diverse physiochemical properties. Consequently, affinity tags have become indispensable tools for structural and functional proteomics initiatives. Although originally developed to facilitate the detection and purification of recombinant proteins, in recent years it has become clear that affinity tags can have a positive impact on the yield, solubility and even the folding of their fusion partners. However, no single affinity tag is optimal with respect to all of these parameters; each has its strengths and weaknesses. Therefore, combinatorial tagging might be the only way to harness the full potential of affinity tags in a high-throughput setting.

Animals↗
PubMed2003

A microscope-based screening platform for large-scale functional protein analysis in intact cells.

A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects.

Automation↗
PubMed2001

Parasite genome initiatives.

During 1993-1994, scientists from developing and developed countries planned and initiated a number of parasite genome projects and several consortiums for the mapping and sequencing of these medium-sized genomes were established, often based on already ongoing scientific collaborations. Financial and other support came from WHO/TDR, Wellcome Trust and other funding agencies. Thus, the genomes of Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, Leishmania major, Trypanosoma brucei, Brugia malayi and other pathogenic nematodes are now under study. From an initial phase of network formation, mapping efforts and resource building (EST, GSS, phage, cosmid, BAC and YAC library constructions), sequencing was initiated in gene discovery projects but soon also on a small chromosome, and now on a fully fledged genome scale. Proteomics, functional analysis, genetic manipulation and microarray analysis are ongoing to different degrees in the respective genome initiatives, and as the funding for the whole genome sequencing becomes secured, most of the participating laboratories, apart from larger sequencing centres, become oriented to post-genomics. Bioinformatics networks are being expanded, including in developing countries, for data mining, annotation and in-depth analysis.

Animals↗
PubMed2002

Theories on malarial pigment formation and quinoline action.

Haeme metabolism remains a vulnerable problem for the intraerythrocytic Plasmodium which catabolises haemoglobin as a source of amino acids in an acidic, oxygen-rich lysosome-like digestive vacuole. Haeme monomer, capable of generating oxygen radicals, transforms into an inert crystal named malarial pigment or haemozoin by forming unique dimers that then crystalise. Laveran first described pigmented bodies in humans to define a protozoan as the aetiologic agent of malaria. The trail of malaria pigment enabled Ross to implicate the mosquito in the life cycle of Plasmodium. In 1991, Slater and Cerami postulated a unique iron-carboxylate bond between two haemes in haemozoin crystals based on infrared and X-ray spectroscopy data. Additionally, parasite extracts were shown to possess a 'haeme polymerase' enzymatic activity as the process of crystal formation was then termed. Importantly, the quinolines, such as choloroquine, inhibit haemozoin formation. A Plasmodium falciparum derived histidine-rich protein II, which binds haeme and initiates haemozoin formation, is present in the digestive vacuole. Pfhistidine-rich protein II and Pfhistidine-rich protein III are sufficient, but not necessary for haemozoin formation as a laboratory clone lacking both still makes the haeme crystals. The reduvid bug, and the Schistosoma and Haemoproteus genera also make haemozoin. Recently, Bohle and coworkers used X-ray diffraction to document the iron-carboxylate bond in intact desiccated parasites and to show that a Fe1-O41 head to tail haeme dimer is the unit building block of haemozoin. The role of the Plasmodium histidine-rich protein family members, lipids or potential novel proteins in the exact molecular assembly of the large molecular weight haeme crystals in the protein rich digestive vacuole needs to be solved. Accurate experimental determination of the role of haemozoin formation and inhibition as the target of chloroquine is fundamental to determination of the mechanism of quinoline drug action and resistance. The enhanced understanding of the biosynthetic pathway leading to haemozoin formation using functional proteomic tools and the mechanisms through which existing antimalarial drugs affect Plasmodium haeme chemistry will help design improved chaemotherapeutic agents.

Animals↗
PubMed2003

Multiprotein signaling complexes and regulation of cardiac phenotype.

There is increasing evidence across disciplines for multiprotein complexes as a mechanism for signal transduction in various cell types. These multiprotein complexes appear to be altered as a direct mechanism to confer signaling responses and to alter phenotype. Although classical experimental techniques are effective to delineate the signaling role of one or a few molecules, they are limited in their ability to thoroughly characterize multiprotein complexes. In this review, the contribution of functional proteomics to a protein complex-based portrait of cardiac cell signaling will be examined along with a discussion and brief evaluation of the state-of-the-art proteomic approaches for multiprotein complex analysis.

Animals↗
PubMed2005

Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium.

In this chapter we describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.

Chromatography, Gel↗
PubMed2002

Analysis by systematic deletion of amino acids of the action of the ribotoxin restrictocin.

A series of contiguous deletions were made in a cDNA encoding the ribonuclease restrictocin with the purpose of identifying the amino acids that are essential for the cleavage of the phosphodiester bond on the 3' side of G4325 in the alpha-sarcin/ricin domain of mammalian (rat) 28S rRNA. In all 93 of 149 amino acids, 62% of the residues in restrictocin, were not essential for the action of the toxin. Of the five residues that have been proposed to constitute the active site, three could be deleted without loss of activity if they were part of a deletion of three or five amino acids but not if they were removed singly. It is likely that the loss of these three residues is compensated for by a neighboring residue that occupies the structural space created by the larger amino acid deletions. This was demonstrated to be the case for the active site residue Glu95 which in the deletion mutant Delta91-95 is replaced by Asp90. Systematic deletion of amino acids is a rapid, cost effective method for identifying the residues in a protein likely to contribute directly to function and, hence, deserving of closer scrutiny. Moreover, a semiquantitative estimate of the contribution of the residue to function can be made. For this reason the method may be useful for functional proteomics.

Allergens↗