Activation-associated secreted proteins are the most abundant antigens in a host protective fraction from Ostertagia ostertagi.
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Amino Acid Sequence↗
Biomedical subjects
K Gevaert
Publications and source records attributed to K Gevaert.
A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid.
A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondria derived from liver-specific Bcl-2-transgenic mice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.
Adenylate Kinase↗
The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity.
Proteome analysis of supernatant of isolated mitochondria exposed to recombinant tBid, a proapoptotic Bcl-2 member, revealed the presence of the serine protease Omi, also called HtrA2. This release was prevented in mitochondria derived from Bcl-2-transgenic mice. Release of Omi under apoptotic conditions was confirmed in vivo in livers from mice injected with agonistic anti-Fas antibodies and was prevented in livers from Bcl-2 transgenic mice. Omi release also occurs in apoptotic dying but not in necrotic dying fibrosarcoma L929 cells, treated with anti-Fas antibodies and TNF, respectively. The amino acid sequence reveals the presence of an XIAP interaction motif at the N-terminus of mature Omi. We demonstrate an interaction between endogeneous Omi and recombinant XIAP. Furthermore we show that endogenous Omi is involved in enhanced activation of caspases in cytosolic extracts.
Amino Acid Sequence↗
Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex.
BACKGROUND: The Rho GTPases Rho, Rac, and Cdc42 regulate the organization of the actin cytoskeleton by interacting with multiple, distinct downstream effector proteins. Cdc42 controls the formation of actin bundle-containing filopodia at the cellular periphery. The molecular mechanism for this remains as yet unclear. RESULTS: We report here that Cdc42 interacts with IRSp53/BAP2 alpha, an SH3 domain-containing scaffold protein, at a partial CRIB motif and that an N-terminal fragment of IRSp53 binds, via an intramolecular interaction, to the CRIB motif-containing central region. Overexpression of IRSp53 in fibroblasts leads to the formation of filopodia, and both this and Cdc42-induced filopodia are inhibited by expression of the N-terminal IRSp53 fragment. Using affinity chromatography, we have identified Mena, an Ena/VASP family member, as interacting with the SH3 domain of IRSp53. Mena and IRSp53 act synergistically to promote filopodia formation. CONCLUSION: We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia.
3T3 Cells↗
A novel receptor-mediated regulation mechanism of type I inositol polyphosphate 5-phosphatase by calcium/calmodulin-dependent protein kinase II phosphorylation.
D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)) are both substrates of the 43-kDa type I inositol polyphosphate 5-phosphatase. Transient and okadaic acid-sensitive inhibition by 70-85% of Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) 5-phosphatase activities was observed in homogenates from rat cortical astrocytes, human astrocytoma 1321N1 cells, and rat basophilic leukemia RBL-2H3 cells after incubation with carbachol. The effect was reproduced in response to UTP in rat astrocytic cells and Chinese hamster ovary cells overexpressing human type I 5-phosphatase. Immunodetection as well as mass spectrometric peptide mass fingerprinting and post-source decay (PSD) sequence data analysis after immunoprecipitation permitted unambiguous identification of the major native 5-phosphatase isoform hydrolyzing Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) as type I inositol polyphosphate 5-phosphatase. In ortho-(32)P-preincubated cells, the phosphorylated 43 kDa-enzyme could be identified after receptor activation by immunoprecipitation followed by electrophoretic separation. Phosphorylation of type I 5-phosphatase was blocked after cell preincubation in the presence of Ca(2+)/calmodulin kinase II inhibitors (i.e. KN-93 and KN-62). In vitro phosphorylation of recombinant type I enzyme by Ca(2+)/calmodulin kinase II resulted in an inhibition (i.e. 60-80%) of 5-phosphatase activity. In this study, we demonstrated for the first time a novel regulation mechanism of type I 5-phosphatase by phosphorylation in intact cells.
Animals↗
Proteome analysis of the Chlamydia pneumoniae elementary body.
Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniae cultivated in Hep-2 cells were 35S-labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two-dimensional gel electrophoresis. Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry. In the pH range from 3-11, 263 C. pneumoniae protein spots encoded from 167 genes were identified. These genes constitute 15% of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.
Amino Acids↗
Protein identification based on matrix assisted laser desorption/ionization-post source decay-mass spectrometry.
Due to its very short analysis time, its high sensitivity and ease of automation, matrix-assisted laser desorption/ionization (MALDI)-peptide mass fingerprinting has become the preferred method for identifying proteins of which the sequences are available in databases. However, many protein samples cannot be unambiguously identified by exclusively using their peptide mass fingerprints (e.g., protein mixtures, heavily posttranslationally modified proteins and small proteins). In these cases, additional sequence information is needed and one of the obvious choices when working with MALDI-mass spectrometry (MS) is to choose for post source decay (PSD) analysis on selected peptides. This can be performed on the same sample which is used for peptide mass fingerprinting. Although in this type of peptide analysis, fragmentation yields are very low and PSD spectra are often very difficult to interpret manually, we here report upon our five years of experience with the use of PSD spectra for protein identification in sequence (protein or expressed sequence tag (EST)) databases. The combination of peptide mass fingerprinting and PSD and analysis described here generally leads to unambiguous protein identification in the amount of material range generally encountered in most proteome studies.
Peptide Fragments↗
Time-dependent expression and processing of a hypothetical protein of possible importance for regulation of the Chlamydia pneumoniae developmental cycle.
Chlamydia pneumoniae is an obligate intracellular human pathogen infecting epithelial cells of the upper respiratory tract. It is a Gram-negative bacteria and has a unique biphasic developmental cycle. In this study, we use two-dimensional gel electrophoresis in combination with radioactive labeling to investigate time-dependent expression and processing of C. pneumoniae proteins. We report on (i) the identification of a hypothetical protein which is expressed late in the developmental cycle and subsequently processed; we speculate that this protein may be of importance for the developmental cycle of Chlamydia; (ii) the identification of the major outer membrane protein in three different variants, which may all be present in vivo.
Bacterial Proteins↗
Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation.
A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated.
Animals↗
Targeting of aminopeptidase I to the yeast vacuole is mediated by Ssa1p, a cytosolic member of the 70-kDa stress protein family.
The two cytosolic members of the highly conserved 70-kDa stress protein family, Ssa1p and Ssa2p, were specifically retained by the prepro-NH(2) extension of the vacuolar aminopeptidase I precursor (pAPI) conjugated to agarose (Sulfolink). A temperature-sensitive mutant strain a1(ts)a234 (ssa1(ts) ssa2 ssa3 ssa4), when incubated at the restrictive temperature, was able to assemble the API precursor into dodecamers, but failed to pack pAPI into vesicles and to convert it into mature API (mAPI), a process that occurs in the vacuole. Altogether these results indicate that Ssa1p mediates the targeting of pAPI to the vacuole.
Adenosine Triphosphatases↗
Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16.
We have identified a rapid protein phosphorylation event at residue serine 16 of stathmin using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry in combination with post-source decay analysis, which is induced by the epidermal growth factor receptor. Phosphorylation is specifically mediated by the small GTPases Rac and Cdc42 and their common downstream target, the serine/threonine kinase p65PAK. Both GTPases have previously been shown to regulate the dynamics of actin polymerization. Because stathmin destabilizes microtubules, and this process is inhibited by phosphorylation at residue 16, Rac and Cdc42 can potentially regulate both F-actin and microtubule dynamics.
Cell Line↗
A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin.
The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.
14-3-3 Proteins↗
A fast and convenient MALDI-MS based proteomic approach: identification of components scaffolded by the actin cytoskeleton of activated human thrombocytes.
A recently developed concentration and purification method (Gevaert, K., Demol, H., Puype, M., Broekaert, D., De Boeck, S., Houthaeve, T., Vandekerckhove, J., 1997. Electrophoresis 18, 2950-2960) for the analysis of diluted peptide samples by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is compared with conventional MALDI sample preparation methods. In the procedure developed, reverse-phase chromatographic beads are added to diluted peptide solutions and act as a peptide-trapping device. Peptides concentrated on the added beads are subsequently harvested, transferred to the MALDI-target disc and efficiently on target desorbed from the beads in a very small volume of an organic-aqueous mixture containing the aromatic MALDI-matrix components. Using this procedure, we show that it is possible to use the totality of in gel protein digests without negative interference of buffers and chaotropes that may be present in the digestion mixture. This method links MALDI-MS peptide analysis more efficiently to 2-D gel electrophoresis in the concept of proteome analysis. The procedure is illustrated by the identification of a class of proteins, which translocate to the actin cytoskeleton of human platelets upon thrombin stimulation.
Actins↗
Protein identification methods in proteomics.
A combination of high-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis, highly sensitive biological mass spectrometry, and the rapidly growing protein and DNA databases has paved the way for high-throughput proteomics. This review concentrates on protein identification. We first discuss the use of protein electroblotting and Edman sequencing as tools for de novo sequencing and protein identification. In the second part, we highlight matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as one of the main contemporary analytical methods for linking gel-separated proteins to entries in sequence databases. In this context we describe the two main MALDI-MS-based identification methods: (i) peptide mass fingerprinting, and (ii) post-source decay (PSD) analysis. In the last part, we briefly emphasize the importance of sample preparation for obtaining highly sensitive and high-quality MALDI-MS spectra.
Animals↗
Techniques for sample preparation including methods for concentrating peptide samples.
In the current era of proteomics two main analytical techniques are employed for protein identification. By far the fastest and most sensitive procedure for protein identification employs biological mass spectrometry, while de novo sequence analysis by classical Edman degradation is currently diminishing. In order to achieve the highest sensitivity for both techniques, great demands need to be put on sample preparation. In this paper we review three different aspects of protein sample preparation. Firstly, we discuss the use of polyacrylamide or agarose gel systems in which, during electrophoresis, proteins present in multiple primary gel pieces are eluted and simultaneously concentrated in a small secondary gel volume, whereby the overall sensitivity of Edman sequencing can be greatly increased. In a second chapter we review automation strategies occurring in the protein field which allow the automatic handling of multiple protein spots at the same time. In this context, we describe the use of auto-sampling techniques for further mass spectrometric studies and protein digestion robots allowing the simultaneous preparation of tens of gel-separated proteins. Finally we discuss various strategies for the preparation of biological peptide samples such as protein digests for both matrix-assisted laser desorption ionisation and electrospray ionisation mass spectrometry.
Automation↗
A peptide concentration and purification method for protein characterization in the subpicomole range using matrix assisted laser desorption/ionization-postsource decay (MALDI-PSD) sequencing.
We here describe the use of added reversed-phase chromatographic beads to concentrate peptides from highly diluted solutions. In the procedure developed, peptide-bead suspensions are dried under vacuum to complete dryness; peptides are subsequently eluted in a small volume of matrix-assisted laser desorption/ionization (MALDI)-matrix containing organic/aqueous solvent and transferred to a MALDI-target for mass analysis. We show that by using this bead-peptide concentration procedure, low femtomole amounts of peptides are efficiently concentrated, up to 1000 times, to volumes smaller than 0.7 microL. We have used this concentration procedure in combination with MALDI-post-source decay analysis to identify subpicomole amounts of proteins present in polyacrylamide gels. Furthermore, we show that the bead-peptide concentration method can be elegantly used to clean up samples contaminated with high concentrations of substances normally deleterious to MALDI-mass spectrometry (MS) experiments. We have found additionally that the bead-peptide concentration procedure can be successfully used to store low femtomole amounts of peptide for prolonged periods of time without severe losses of peptide material. This bead-peptide concentration procedure therefore seems to be a simple and convenient step in the MALDI-MS sample preparation process.
Chromatography, High Pressure Liquid↗
Sample preparation procedures for ultrasensitive protein identification by PSD-MALDI-TOF mass spectrometry.
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Proteins↗
Peptides adsorbed on reverse-phase chromatographic beads as targets for femtomole sequencing by post-source decay matrix assisted laser desorption ionization-reflectron time of flight mass spectrometry (MALDI-RETOF-MS).
We here describe a procedure for concentrating peptides from solutions by adsorbing them onto reverse-phase beads that were added to these solutions. The beads are then transferred to the target disc of the matrix assisted laser desorption ionization-reflectron time of flight (MALDI-RETOF) mass spectrometer. Because of their hydrophobic nature, these beads cluster in a very small area on the target disc assuring an important concentration step. After drying, peptides are desorbed from the beads by adding a small volume of 50% acetonitrile in 0.1% trifluroacetic acid in water containing the matrix components. Hereby we focus the original amount of peptide material on the target disc on a very small surface, producing highly concentrated peptide-matrix mixtures. This permits high yield identification and sequence tagging by post-source-decay analysis on peptides derived from proteins only available in the femtomole range from one-dimensional (1-D) or two-dimensional (2-D) gels. The procedure is illustrated by the identification of 38 proteins from human thrombocyte membrane skeletons.
Acetonitriles↗