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At least 181 records · Page 10Linked to original sources
PubMed2006

Proteomic methods applied to the analysis of immobilized biocatalysts.

Methods adapted from proteomics can directly characterize proteins present in immobilized biocatalysts. Complete hydrolysis followed by HPLC analysis of Tyr and Phe estimates total protein bound, and is preferable to conventional difference methods, as tested with subtilisin Carlsberg on silica. This new method shows that various treatments give quantitative desorption of proteins immobilized by adsorption. Intact desorbed proteins may be analyzed by electrospray mass spectrometry. The Candida antarctica lipase B from Novozyme 435 was shown to be heavily glycosylated, while the lipase from Lipozyme RM IM was a mixture of four N-terminally truncated forms. Peptides from selective cleavage were analyzed by tandem mass spectrometry, leading to automatic identification of proteins present. A second major protein present in Lipozyme RM IM was thus found to be alpha-amylase from Aspergillus oryzae. These methods should be valuable complements to activity measurements in understanding immobilized enzyme activity and stability.

Adsorption↗
PubMed2005

Proteomic analysis of progressive factors in uterine cervical cancer.

Human papillomavirus (HPV) infections play a crucial role in the progress of cervical cancer. The high-risk HPV types are frequently associated with the development of malignant lesions. Some of the latest studies have demonstrated that the high-risk HPV 16 and 18 are predominantly detected in the more aggressive cancers. In the present study, we aimed to establish the proteomic profiles and characterization of the tumor related proteins by using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). For proteomic analysis, patients infected by HPV 16 or 18 were included in this study. We compared nuclear protein and cytoplasmic protein, separately by using the subcellular fraction. Differential protein spots between cervical cancer with high-risk HPV, HPV 16 or HPV 18, and HaCaT cell lines were characterized by 2-DE. Those proteins analyzed by peptide mass fingerprinting based on MALDI-TOF MS and database searching were the products of oncogenes or proto-oncogenes, and the others were involved in the regulation of cell cycle, for general genomic stability, telomerase activation, and cell immortalization. However, there was no difference in protein characterization for cervical cancer between HPV 16 and HPV 18 infection. Nonetheless, these data are valuable for the mass identification of differentially expressed proteins involved in human uterine cervical cancer. Moreover, the data has enormous value for establishing the human uterine cervical cancer proteome database that can be used in screening a molecular marker for the further study of human uterine cervical cancer, and also for studying any correlation among the cancers induced by HPV.

Adult↗
PubMed2007

Quantitative proteomics profiling of sarcomere associated proteins in limb and extraocular muscle allotypes.

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.

Animals↗
PubMed2006

From gene to HSQC in under five hours: high-throughput NMR proteomics.

A simple, rapid, in vitro cell-free protein expression system, Expressway NMR, is introduced and used to express the small ubiquitin-related modifier protein SUMO-1. This 12 kDa molecule is challenging for NMR as it has limited solubility and requires relatively high salt (200 mM) for stability in solution. Starting with the gene, the cell-free system, and milligram amounts of nitrogen-15 isotopically enriched amino acids, sufficient protein is produced in 4 h to obtain a high-resolution 2D HSQC spectrum of the protein in 40 min. This time would be closer to 10 min with the aid of a higher sensitivity salt-tolerant cryogenic NMR probe. With all protein purification steps included, and aggressive data processing using the filter diagonalization method (FDM), it is but 6 h from gene to heteronuclear single quantum coherence (HSQC). As the cell-free system is nearly background-free, it is also possible to work with the crude reaction mixture, in which case only a total of 5 h is required. Sample stability over time, whether crude extract or purified, was notable, with no significant change in the 15N-1H HSQC spectrum over 6 months at 4 degrees C (300 muM, pH 6.1, capped NMR tube). The combination of a turnkey, high-yield, protease-free in vitro protein expression system, an optimized sensitivity-enhanced HSQC pulse sequence, and FDM processing makes this scheme an attractive first step to rapidly assess the suitability of proteins for complete solution structure determination.

Journal Article↗
PubMed2006

Predicting Shine-Dalgarno sequence locations exposes genome annotation errors.

In prokaryotes, Shine-Dalgarno (SD) sequences, nucleotides upstream from start codons on messenger RNAs (mRNAs) that are complementary to ribosomal RNA (rRNA), facilitate the initiation of protein synthesis. The location of SD sequences relative to start codons and the stability of the hybridization between the mRNA and the rRNA correlate with the rate of synthesis. Thus, accurate characterization of SD sequences enhances our understanding of how an organism's transcriptome relates to its cellular proteome. We implemented the Individual Nearest Neighbor Hydrogen Bond model for oligo-oligo hybridization and created a new metric, relative spacing (RS), to identify both the location and the hybridization potential of SD sequences by simulating the binding between mRNAs and single-stranded 16S rRNA 3' tails. In 18 prokaryote genomes, we identified 2,420 genes out of 58,550 where the strongest binding in the translation initiation region included the start codon, deviating from the expected location for the SD sequence of five to ten bases upstream. We designated these as RS+1 genes. Additional analysis uncovered an unusual bias of the start codon in that the majority of the RS+1 genes used GUG, not AUG. Furthermore, of the 624 RS+1 genes whose SD sequence was associated with a free energy release of less than -8.4 kcal/mol (strong RS+1 genes), 384 were within 12 nucleotides upstream of in-frame initiation codons. The most likely explanation for the unexpected location of the SD sequence for these 384 genes is mis-annotation of the start codon. In this way, the new RS metric provides an improved method for gene sequence annotation. The remaining strong RS+1 genes appear to have their SD sequences in an unexpected location that includes the start codon. Thus, our RS metric provides a new way to explore the role of rRNA-mRNA nucleotide hybridization in translation initiation.

Base Sequence↗
PubMed2026

PRDX1 facilitates USP7-dependent stabilization of SCD1 and promotes bladder cancer progression.

Bladder cancer is characterized by redox adaptation and metabolic plasticity, but the mechanisms linking these processes remain incompletely understood. Integrating bulk, single-cell, and spatial transcriptomic analyses, we identified PRDX1 as a malignant epithelial cell-associated factor linked to adverse outcome. Genetic gain- and loss-of-function studies showed that PRDX1 promoted proliferation, motility, and xenograft growth while limiting reactive oxygen species accumulation and mitochondrial apoptosis. Proteomic and biochemical analyses identified an association between PRDX1 and SCD1. PRDX1 prolonged the SCD1 protein half-life without detectably altering SCD1 transcript abundance and increased USP7-SCD1 co-precipitation. USP7 removed K48-linked polyubiquitin chains from SCD1 and prevented its proteasomal degradation, whereas catalytically inactive USP7 failed to deubiquitinate SCD1. Deletion of PRDX1 residues 157-199 weakened its association with SCD1 and reduced USP7-SCD1 co-precipitation. Depletion of SCD1 or USP7 suppressed PRDX1-dependent growth in vitro and in xenografts. These findings support a model in which PRDX1 facilitates USP7-dependent stabilization of SCD1 and promotes bladder cancer progression.

Bladder cancer↗
PubMed2006

Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics.

One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.

Antibodies↗
PubMed2005

Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant innate immunity.

Plant innate immunity against invasive biotrophic pathogens depends on the intracellular defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). We show here that Arabidopsis thaliana EDS1 interacts in vivo with another protein, SENESCENCE-ASSOCIATED GENE101 (SAG101), discovered through a proteomic approach to identify new EDS1 pathway components. Together with PHYTOALEXIN-DEFICIENT4 (PAD4), a known EDS1 interactor, SAG101 contributes intrinsic and indispensable signaling activity to EDS1-dependent resistance. The combined activities of SAG101 and PAD4 are necessary for programmed cell death triggered by the Toll-Interleukin-1 Receptor type of nucleotide binding/leucine-rich repeat immune receptor in response to avirulent pathogen isolates and in restricting the growth of normally virulent pathogens. We further demonstrate by a combination of cell fractionation, coimmunoprecipitation, and fluorescence resonance energy transfer experiments the existence of an EDS1-SAG101 complex inside the nucleus that is molecularly and spatially distinct from EDS1-PAD4 associations in the nucleus and cytoplasm. By contrast, EDS1 homomeric interactions were detected in the cytoplasm but not inside the nucleus. These data, combined with evidence for coregulation between individual EDS1 complexes, suggest that dynamic interactions of EDS1 and its signaling partners in multiple cell compartments are important for plant defense signal relay.

Amino Acid Sequence↗
PubMed2001

Physical markers for landmarking fluorescently stained gels that facilitate automated spot-picking.

The quantitative comparison of spot patterns relies heavily on protein stains that do provide an appropriate dynamic range. Unfortunately most spot picking robot devices are still limited to nonfluorescent protein stains and the appropriate equipment is still quite expensive. These problems are solved by the application of a newly developed "GelMarker" that combines a spot picking robot device and a UV scanner. The "GelMarkers" are detectable in both the visible and UV range of light and permit the comparison of gel pictures taken by such different devices. The application of these "GelMarkers" together with the transformation of spot coordinates by using a "spot matching" procedure allows the automated excision of selected protein spots. The obtained picking accuracies are as good as those obtainable from visible stained gels due to the shape stability of the gels even over a longer time period.

Automation↗
PubMed2004

Numerous posttranslational modifications provide opportunities for the intricate regulation of metabolic enzymes at multiple levels.

The metabolic plasticity displayed by plants during normal development, and in response to environmental fluctuations and stressors, is essential for their growth and survival. The capacity to regulate metabolic enzymes intricately arises in part from posttranslational modifications that can affect enzymatic activity, intracellular localization, protein-protein interactions, and stability. Protein phosphorylation and thiol/disulfide redox modulation are important modifications in plants, and it is likely that O-glycosylation and S-nitrosylation will also emerge as important mechanisms. Recent advances in the field of proteomics, in particular the development of novel and specific chemistries for the detection of a diverse number of modifications, are rapidly expanding our awareness of possible modifications and our understanding of the enzymes whose functions are likely to be regulated posttranslationally.

Arabidopsis↗
PubMed2005

SELEX: just another separation?

The potential for quickly isolating high affinity, highly selective ligands generated much excitement when SELEX was first described. Fifteen years later, SELEX has still not achieved widespread acceptance due to limitations in aptamer affinity, stability and throughput. Recent research is beginning to address these limitations though and SELEX is quickly regaining its standing as a hot topic in areas as diverse as drug discovery, chemical analysis, and even genomics and proteomics.

Animals↗
PubMed2005

14-3-3 proteins: regulators of numerous eukaryotic proteins.

14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.

14-3-3 Proteins↗
PubMed2002

Mechanisms and future directions for angiogenesis-based cancer therapies.

Targeting angiogenesis represents a new strategy for the development of anticancer therapies. New targets derived from proliferating endothelial cells may be useful in developing anticancer drugs that prolong or stabilize the progression of tumors with minimal systemic toxicities. These drugs may also be used as novel imaging and radiommunotherapeutic agents in cancer therapy. In this review, the mechanisms and control of angiogenesis are discussed. Genetic and proteomic approaches to defining new potential targets on tumor vasculature are then summarized, followed by discussion of possible antiangiogenic treatments that may be derived from these targets and current clinical trials. Such strategies involve the use of endogenous antiangiogenic agents, chemotherapy, gene therapy, antiangiogenic radioligands, immunotherapy, and endothelial cell-based therapies. The potential biologic end points, toxicities, and resistance mechanisms to antiangiogenic agents must be considered as these therapies enter clinical trials.

Angiogenesis Inhibitors↗
PubMed2006

Solid phase proteomics: dramatic reinforcement of very weak protein-protein interactions.

Very weak protein-protein interactions may play a critical role in cell physiology but they are not easily detectable in "in vitro" experiments. To detect these weak interactions, we have developed a strategy that included: (a) design of a rapid and very effective crosslinking of protein-protein complexes with poly-functional reagents; (b) selective adsorption of very large proteins on lowly activated ionic exchangers, based on the need of a multipoint physical adsorption to incorporate the proteins into the matrix; (c) purification by selective adsorption of protein-protein complexes formed by strong protein-protein interactions, via selective adsorption of the complexes on lowly activated ionic exchangers via multi-protein physical adsorption and leaving the non-associated proteins in the solution; (d) reinforcement of very weak protein-protein interactions by selective adsorption of the complex on lowly activated ionic exchange supports via a synergetic cooperation of the weak protein-protein interaction plus the interactions of both proteins with the support enabling the almost full shifting of the equilibrium towards the association position; (e) control of the aggregation state of proteins like BSA, formed by weak protein-protein interactions. In this last case, it seems that the interaction of the protein molecules placed on the borders of the aggregate with the groups on the support partially stabilizes the whole aggregate, although, some molecules of the aggregate cannot interact with the support. The size of the aggregates may be defined by controlling the concentration of ionised groups on the support: the less activated the supports are, the bigger the complexes. In this way, solid-phase proteomics could be a very interesting tool to detect weak protein-protein interactions.

Adsorption↗
PubMed2006

Subcellular pH and predicted pH-dependent features of proteins.

A characteristic of two-dimensional proteomics gels is a general bimodal distribution of isoelectric (pI) values. Discussion of this feature has focussed on the balance of acidic and basic ionisable residues, and potential relationships between pI distributions and organism classification or protein subcellular location. Electrostatics calculations on a set of protein structures with known subcellular location show that predicted folded state pI are similar to those calculated from sequence alone, but adjusted according to a general stabilising effect from interactions between ionisable groups. Bimodal distributions dominate both pI and the predicted pH of maximal stability. However, there are significant differences between these features. The average pH of maximal stability generally follows organelle pH. Average pI values are well removed from organelle pH in most subcellular environments, consistent with the view that proteins have evolved to carry (on average) net charge in a given subcellular location, and relevant to discussion of solubility in crowded environments. Correlation of the predicted pH of maximum stability with subcellular pH suggests an evolutionary pressure to adjust folded state interactions according to environment. Finally, our analysis of ionisable group contributions to stability suggests that Golgi proteins have the largest such term, although this dataset is small.

Cell Nucleus↗
PubMed2006

Proteomic identification of the wt-p53-regulated tumor cell secretome.

Tumor-stroma interactions play a major role in tumor development, maintenance and progression. Yet little is known on how the genetic alterations that underlie cell transformation elicit cell extrinsic changes modulating heterotypic cell interactions. We hypothesized that these events involve a modification in the complement of secreted proteins by the cell, acting as mediators of intercellular communication. To test this hypothesis, we examined the role of wt-p53, a major tumor suppressor, on the tumor microenvironment through its regulation of secreted factors. Using a combination of 2-DE and cICAT proteomic techniques, we found a total of 111 secreted proteins, 39 of which showed enhanced and 21 inhibited secretion in response to wt-p53 expression. The majority of these were not direct targets of p53 transcription factor activity, suggesting a novel role for wt-p53 in the control of intracellular protein trafficking and/or secreted protein stability. Evidence for p53-controlled post-translational modifications on nine secreted proteins was also found. These findings will enhance our understanding of wt-p53 modulated interactions of the tumor with its environment.

Cell Line, Tumor↗
PubMed2004

[Development of novel DDS technologies for pharmacoproteomic-based drug discovery and development].

With the success of the Human Genome Project, the focus of life science research has shifted to the functional and structural analyses of proteins, such as proteomics and structural genomics. These novel approaches to the analysis of proteins, including newly identified ones, are expected to help in the identification and development of protein therapies for various diseases. Thus pharmacoproteomic-based drug discovery currently has a very high profile. Nevertheless, the use of bioactive proteins in the clinical setting is not straightforward because in vivo these proteins have low stability and pleiotropic action. To promote pharmacoproteomic-based drug discovery and development, we have attempted to establish a system for creating functional mutant proteins (muteins) with the desired properties and to develop a site-specific bioconjugation system for further improving their therapeutic potency. These innovative protein-drug systems are discussed in this review.

Drug Delivery Systems↗
PubMed2003

Proteomic approach to coronary atherosclerosis shows ferritin light chain as a significant marker: evidence consistent with iron hypothesis in atherosclerosis.

Coronary artery disease (CAD) is the leading cause of mortality and morbidity in developed nations. We hypothesized that CAD is associated with distinct patterns of protein expression in the coronary arteries, and we have begun to employ proteomics to identify differentially expressed proteins in diseased coronary arteries. Two-dimensional (2-D) gel electrophoresis of proteins and subsequent mass spectrometric analysis identified the ferritin light chain as differentially expressed between 10 coronary arteries from patients with CAD and 7 coronary arteries from normal individuals. Western blot analysis indicated significantly increased expression of the ferritin light chain in the diseased coronary arteries (1.41 vs. 0.75; P = 0.01). Quantitative real-time PCR analysis showed that expression of ferritin light chain mRNA was decreased in diseased tissues (0.70 vs. 1.17; P = 0.013), suggesting that increased expression of ferritin light chain in CAD coronary arteries may be related to increased protein stability or upregulation of expression at the posttranscriptional level in the diseased tissues. Ferritin light chain protein mediates storage of iron in cells. We speculate that increased expression of the ferritin light chain may contribute to pathogenesis of CAD by modulating oxidation of lipids within the vessel wall through the generation of reactive oxygen species. Our results provide in situ proteomic evidence consistent with the "iron hypothesis," which proposes an association between excessive iron storage and a high risk of CAD. However, it is also possible that the increased ferritin expression in diseased coronary arteries is a consequence, rather than a cause, of CAD.

Adult↗