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PubMed2006

Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery.

Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways. Through a three-enzyme (E1-E2-E3) cascade, the attachment of ubiquitin to proteins is catalysed by the E3 ubiquitin ligase, which is best represented by the superfamily of the cullin-RING complexes. Conserved from yeast to human, the DDB1-CUL4-ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair, DNA replication and transcription, and can also be subverted by pathogenic viruses to benefit viral infection. Lacking a canonical SKP1-like cullin adaptor and a defined substrate recruitment module, how the DDB1-CUL4-ROC1 E3 apparatus is assembled for ubiquitinating various substrates remains unclear. Here we present crystallographic analyses of the virally hijacked form of the human DDB1-CUL4A-ROC1 machinery, which show that DDB1 uses one beta-propeller domain for cullin scaffold binding and a variably attached separate double-beta-propeller fold for substrate presentation. Through tandem-affinity purification of human DDB1 and CUL4A complexes followed by mass spectrometry analysis, we then identify a novel family of WD40-repeat proteins, which directly bind to the double-propeller fold of DDB1 and serve as the substrate-recruiting module of the E3. Together, our structural and proteomic results reveal the structural mechanisms and molecular logic underlying the assembly and versatility of a new family of cullin-RING E3 complexes.

Amino Acid Motifs↗
PubMed2004

A proteomic study of SUMO-2 target proteins.

The SUMO family in vertebrates includes at least three distinct proteins (SUMO-1, -2, and -3) that are added as post-translational modifications to target proteins. A considerable number of SUMO-1 target proteins have been identified, but little is known about SUMO-2. A stable HeLa cell line expressing His6-tagged SUMO-2 was established and used to label and purify novel endogenous SUMO-2 target proteins. Tagged forms of SUMO-2 were functional and localized predominantly in the nucleus. His6-tagged SUMO-2 conjugates were affinity-purified from nuclear fractions and identified by mass spectrometry. Eight novel potential SUMO-2 target proteins were identified by at least two peptides. Three of these proteins, SART1, heterogeneous nuclear ribonucleoprotein (RNP) M, and the U5 small nuclear RNP 200-kDa helicase, play a role in RNA metabolism. SART1 and heterogeneous nuclear RNP M were both shown to be genuine SUMO targets, confirming the validity of the approach.

Antigens, Neoplasm↗
PubMed2005

Influences of blood sample processing on low-molecular-weight proteome identified by surface-enhanced laser desorption/ionization mass spectrometry.

BACKGROUND: Profiling approaches in proteomics, such as surface-enhanced laser desorption/ionization (SELDI) mass spectrometry, are used in disease marker discovery. The aim of this study was to investigate the potential influence of selected preanalytical factors on the results obtained. METHODS: Plasma samples anticoagulated with EDTA, citrate, or heparin, and serum samples from healthy volunteers were profiled by SELDI on CM10, immobilized metal affinity capture (IMAC) array with copper, and H50 chip surfaces. Using linear mixed-effects models, we examined the influence of elapsed time between venipuncture and sample separation (immediate to 24 h) and the type of serum tube used (Greiner Vacuette activator, gel serum separator, or plain tubes). We analyzed purified platelets, as well as platelet-poor and platelet-rich plasma samples treated with calcium and/or thrombin to determine the platelet contribution, directly or via the clotting process, to the profiles generated. We then used cluster analysis to identify samples with similar peak profiles. RESULTS: Different plasma types and sera could be distinguished on the basis of cluster analyses of their spectral profiles. Elapsed time between venipuncture and separation of plasma and serum from blood samples altered the profiles obtained, particularly for serum samples and particularly on IMAC chips. The type of serum collection tube also affected the profiles because of differences in clotting time. In vitro manipulation of platelets revealed that specific peaks in IMAC profiles of serum appeared to be derived directly from platelets. Several other peaks, including some of those exhibiting time-dependent changes, arose during the clotting process. CONCLUSION: Preanalytical variables, such as sample handling, can markedly influence results.

Adult↗
PubMed2006

Detection of proteins based on amino acid sequences by multiple aptamers against tripeptides.

A number of different ligands have been tested in the course of the development of protein array technology. The most extensively studied example of protein ligands has been based on antibody-antigen interaction. Other examples include protein-protein, protein-nucleic acid, and protein-small molecule interactions. All these ligands can recognize and specifically bind to protein epitopes. In this study, we have developed a novel technology using DNA-based aptamers to detect proteins based on their amino acid sequences. Mouse cathepsin D was used for the proof of principle experiment. Four tripeptides, Leu-Ala-Ser, Asp-Gly-Ile, Gly-Glu-Leu, and Lys-Ala-Ile, were selected based on the published amino acid sequence of mouse cathepsin D. DNA aptamers against the tripeptides were isolated using the systematic evolution of ligands of exponential enrichment method. We have demonstrated that the aptamers specifically interacted with mouse cathepsin D using the structure-switch method. We further performed a proximity-dependent ligation assay to demonstrate that multiple aptamers could specifically detect the protein from cell extracts. In principle, one library containing 8000 aptamers should be enough to detect almost all proteins in the whole proteome in all organisms. This technology could be applied to generate a new generation of protein arrays.

Amino Acid Sequence↗
PubMed2005

Human epidermal cell protein responses to arsenite treatment in culture.

Study of the responses of target cells in culture is anticipated to help understand the mechanisms by which inorganic arsenic causes pathological effects in vivo. Treatment of human epidermal cells with arsenic has been shown to produce a myriad of changes in gene transcription. Present work focused on finding the extent of arsenite-induced changes in the protein pattern and whether global effects on protein sulfhydryls were evident. First, examining the profile of protein expression by two-dimensional gel electrophoresis indicated that approximately 40% of the 300 distinct protein spots that were monitored changed by at least two-fold in amount all through a 9-day exposure period. Second, examining soluble extracts of the treated cells by Activated Thiol Sepharose column chromatography gave little indication of change in the overall protein thiol content. Finally, among the 10 proteins identified that showed prominent changes in amount as a result of treatment for 1 or 4 days, enzymes of the glycolytic pathway were seen to be substantially elevated as a result of treatment, suggesting decreased utilization by the cells of oxidative phosphorylation. Since these changes were more conspicuous at the protein level than in previous transcriptional studies, the results emphasize the importance of proteomic analysis to complement transcriptional analysis of cell responses to perturbation by arsenic.

3T3 Cells↗
PubMed2003

Protein production in Escherichia coli for structural studies by X-ray crystallography.

The arrival of genomic sequences to the database has provided a seemingly unlimited supply of targets for protein structure determination and the possibility of solving the structure of an entire proteome. Based on our experience with the proteomes of Pyrobaculum aerophilum and Mycobacterium tuberculosis, we have developed a simple strategy for the production of proteins for structural studies by X-ray crystallography. Our scheme demonstrates a strong protein target commitment and includes the expression of genes from these organisms in Escherichia coli. These proteins are expressed with affinity tags and purified for characterization and crystallization. We have identified protein solubility and crystallization as the two major bottlenecks in the process toward the determination of protein structures by X-ray diffraction. Strategies to overcome these bottlenecks are discussed.

Cloning, Molecular↗
PubMed2001

Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome.

The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.

Amino Acid Sequence↗
PubMed2004

Photoaptamer arrays applied to multiplexed proteomic analysis.

Multiplexed photoaptamer-based arrays that allow for the simultaneous measurement of multiple proteins of interest in serum samples are described. Since photoaptamers covalently bind to their target analytes before fluorescent signal detection, the arrays can be vigorously washed to remove background proteins, providing the potential for superior signal-to-noise ratios and lower limits of quantification in biological matrices. Data are presented here for a 17-plex photoaptamer array exhibiting limits of detection below 10 fM for several analytes including interleukin-16, vascular endothelial growth factor, and endostatin and able to measure proteins in 10% serum samples. The assays are simple, scalable, and reproducible. Affinity of the capture reagent is shown to be directly correlated to the limit of detection for the analyte on the array.

Antibodies↗
PubMed2005

T-cell-stimulating protein A elicits immune responses during meningococcal carriage and human disease.

In recognition of the need for immunological memory-inducing components for future Neisseria meningitidis group B vaccines, we previously searched the proteome of N. meningitidis and identified T-cell-stimulating protein A (TspA). This study was designed to confirm the immunogencity of TspA and to examine the subset of T-helper cell responses to the protein in patients and nasopharyngeal carriers. The tspA gene was reconstructed, cloned, and expressed in Escherichia coli, and the recombinant TspA (rTspA) protein was affinity purified. T-cell proliferative responses to rTspA were detected in the peripheral blood mononuclear cells (PBMCs) of convalescent patients and carriers, confirming that TspA-specific T-cell responses were stimulated by invasive disease and nasopharyngeal colonization. Following stimulation of PBMCs with meningococcal lysate, increased frequencies of both Th1 and Th2 cells were observed, indicating that, as during carriage, invasive meningococcal disease induced an unbiased T-helper subset response. A similar unbiased T-helper response was also detected against rTspA in the PBMCs of convalescent patients. The response of PBMCs from the carriers to TspA stimulation, however, was very weak, and the frequencies of cytokine-positive CD4 cells were not significantly greater than the frequencies in unstimulated control cultures. All of the patients and carriers responded with serum antimeningococcal immunoglobulin G (IgG) antibodies, while four of six samples from patients and 5 of 14 samples from carriers contained detectable anti-rTspA IgG antibodies. Taken together, the results of this study confirmed the immunogenicity of TspA in humans during natural meningococcal infection, and therefore, TspA is worthy of further investigation as a possible T-cell stimulating component of future vaccines.

Antigens, Bacterial↗
PubMed2000

Towards a human repertoire of monocytic lysosomal proteins.

The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well-known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins.

Amino Acid Sequence↗
PubMed1998

Two-dimensional map of basic proteins of Haemophilus influenzae.

Haemophilus influenzae is a bacterium of medical interest of which the entire genome has been sequenced. The proteome of the microorganism has been analyzed by two-dimensional gel electrophoresis, during which immobilized pH 3-10 gradient strips were used and approximately 300 proteins were identified. In order to detect additional, basic proteins, we analyzed the soluble protein fraction of H. influenzae and the proteins of fractions collected from affinity chromatography on heparin, by two-dimensional gel electrophoresis, using for the first-dimensional separation immobilized pH gradient strips comprising the pH region of 6-11. The protein spots were analyzed by matrix-assisted laser desorption ionization-mass spectrometry. One hundred and two proteins were identified, of which 58 were identified for the first time. A large percentage of the basic proteins represent nucleic acid binding and, in particular, ribosomal proteins. The locations of the identified basic proteins of H. influenzae are indicated in a two-dimensional map.

Bacterial Proteins↗
PubMed2005

Effective depletion of albumin using a new peptide-based affinity medium.

Blood plasma and serum are very useful samples for the detection, identification and quantitation of proteins associated with both health and disease. However, analysis of plasma and serum is a challenge because traces of interesting polypeptides and proteins can be dominated by the very high concentration of albumin present. Albumin may be depleted by adsorption to immunoaffinity columns or to columns containing dyes such as Cibacron Blue, or by ultrafiltration, but these methods are far from ideal. We describe a new peptide-based affinity medium which is effective for removing albumin and is very specific. The albumin-binding capacity is at least 14 mg per mL of gel. The material may be reused hundreds of times after a simple regeneration step involving NaOH, with full retention of specificity and capacity. The material was tested with human and monkey plasma and serum and rat serum, and has been used to deplete litre volumes of human plasma. The development of other peptide-based affinity media to deplete abundant proteins is briefly discussed.

Albumins↗
PubMedDate not supplied

Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks.

Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC-ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics.

Chromatography, Liquid↗
PubMed2006

Proteomic study of plasma proteins in pregnant women with asthma.

OBJECTIVE AND BACKGROUND: The course of asthma may be altered during pregnancy with at least one-third of women experiencing a worsening of asthma and 20% having an exacerbation during pregnancy. This study used the novel proteomic technique, surface-enhanced laser desorption ionization-time of flight mass spectrometry to determine if the presence of asthma during pregnancy was associated with alterations in plasma proteins. METHODS: Plasma collected from healthy (n = 23) and asthmatic (n = 27) pregnant women at 18 and 30 weeks gestation was applied to strong anion exchange (SAX2), weak cation exchange (WCX2) and immobilized metal affinity capture (IMAC-Cu(2+)) chips. Mass analysis was conducted using Ciphergen Protein Biology System IIc and significant differences in individual peak intensities between groups determined. RESULTS: At 18 weeks gestation, 91 peaks were significantly different between pregnant women with and without asthma, representing 28% of the total peaks identified. At 30 weeks gestation, 51 peaks were significantly different. There were two peaks that were significantly different between groups at both 18 and 30 weeks gestation and expressed at a similar level at both time points. One was increased in asthmatics (MW = 6444 Da) whereas the other decreased in asthmatics compared with non-asthmatic women (MW = 1846 Da). CONCLUSIONS: This study demonstrated that there are differences in protein patterns between pregnant women with and without asthma. Other techniques are needed to define the molecular species and classify pathophysiological significance. Surface-enhanced laser desorption ionization-time of flight mass spectrometry has potential as a tool to monitor disease progression in situations such as pregnancy.

Adult↗
PubMed2006

Structural, functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögren's syndrome.

Ro52, one of the major autoantigens in the rheumatic disease Sjögren's syndrome (SS), belongs to the tripartite motif (TRIM) or RING-B-box-coiled-coil (RBCC) protein family, thus comprising an N-terminal RING, followed by a B-box and a coiled-coil region. Several different proteomic functions have been suggested for Ro52, including DNA binding, protein interactions and Zn(2+)-binding. To analyze the presence and/or absence of these functions and, in particular, map those to different subregions, the modular composition of the Ro52 protein was experimentally characterized. Two structured parts of Ro52 were identified, corresponding to the RING-B-box and the coiled-coil regions, respectively. Secondary structure analysis by circular dichroism (CD) spectroscopy indicated that the two subregions are independently structured. The entire RING-B-box region displayed Zn(2+)-dependent stabilization against proteolysis in the presence of Zn2+, indicating functional Zn(2+)-binding sites in both the RING and the B-box. However, no stabilization with DNA was detected, irrespective of Zn(2+), thus suggesting that the RING-B-box region does not bind DNA. Oligomerization of the coiled-coil was investigated by analytical ultracentrifugation and in a mammalian two-hybrid system. Both methods show weak homodimer affinity, in parity with other coiled-coil domains involved in regulatory interactions. The C-terminal B30.2 region was rapidly degraded both during cellular expression and refolding, indicating a less stable structure. Immunologic analysis of the stable protein regions with sera from patients with Sjögren's syndrome shows that immunodominant epitopes to a large extent are localized in the structurally stable parts of Ro52. The results form a basis for further Ro52 functional studies on the proteome level.

Autoantigens↗
PubMed2005

Towards a proteome-scale map of the human protein-protein interaction network.

Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

Cloning, Molecular↗
PubMed2005

Proteomic analysis of TRPC5- and TRPC6-binding partners reveals interaction with the plasmalemmal Na(+)/K(+)-ATPase.

Mammalian transient receptor potential canonical (TRPC) genes encode a family of nonselective cation channels that are activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. In Drosophila photoreceptor cells, TRP channels are found in large, multimolecular signaling complexes in association with the PDZ-containing scaffolding protein, INAD. A similar mammalian TRPC "signalplex" has been proposed, but has yet to be defined. In the present study, affinity-purified polyclonal antibodies against TRPC5 and TRPC6 were used to immunoprecipitate signalplex components from rat brain lysates. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and sequenced by mass spectrometry. Proteins identified in the immunoprecipitates included cytoskeletal proteins spectrin, myosin, actin, drebrin, tubulin, and neurabin; endocytic vesicle-associated proteins clathrin, dynamin and AP-2; and the plasmalemmal Na(+)/K(+)-ATPase (NKA) pump. Several of these interactions were confirmed by reciprocal immunoprecipitation followed by Western blot analysis. In lysates from rat kidney, TRPC6, but not TRPC3, was found to coimmunoprecipitate with the NKA pump. Likewise, TRPC6, stably expressed in human embryonic kidney (HEK) cells, coimmunoprecipitated with endogenous NKA and colocalized with the pump to the plasmalemma when examined by immunofluorescence microscopy. Cell surface biotinylation experiments in intact HEK cells, confirmed that both the Na(+) pump and TRPC6 were present in the surface membrane and appeared to interact. Lastly, TRPC6 coimmunoprecipitated with the NKA pump when the proteins were coexpressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. These observations suggest that TRPC6 and the Na(+) pump are part of a functional complex that may be involved in ion transport and homeostasis in both the brain and kidney.

Animals↗
PubMed2005

How evolutionary pressure against protein aggregation shaped chaperone specificity.

As protein aggregation is potentially lethal, control of protein conformation by molecular chaperones is essential for cellular organisms. This is especially important during protein expression and translocation, since proteins are then unfolded and therefore most susceptible to form non-native interactions. Using TANGO, a statistical mechanics algorithm to predict protein aggregation, we here analyse the aggregation propensities of 28 complete proteomes. Our results show that between 10% and 20% of the residues in these proteomes are within aggregating protein segments and that this represents a lower limit for the aggregation tendency of globular proteins. Further, we show that not only evolution strongly pressurizes aggregation downwards by minimizing the amount of strongly aggregating sequences but also by selectively capping strongly aggregating hydrophobic protein sequences with arginine, lysine and proline. These residues are strongly favoured at these positions as they function as gatekeepers that are most efficient at opposing aggregation. Finally, we demonstrate that the substrate specificity of different unrelated chaperone families is geared by these gatekeepers. Chaperones face the difficulty of having to combine substrate affinity for a broad range of hydrophobic sequences and selectivity for those hydrophobic sequences that aggregate most strongly. We show that chaperones achieve these requirements by preferentially binding hydrophobic sequences that are capped by positively charged gatekeeper residues. In other words, targeting evolutionarily selected gatekeepers allows chaperones to prioritize substrate recognition according to aggregation propensity.

Algorithms↗