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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.
Mol Cell Proteomics 2003 Jul
PMID:The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: I. Statistically annotated datasets for peptide sequences and proteins identified via the application of ICAT and tandem mass spectrometry to proteins copurifying with T cell lipid rafts. 1293 Aug 72

Members of the odorant-binding protein (OBP) and chemosensory protein (CSP) families were identified and characterised in the sensory tissues of the social wasp Polistes dominulus (Hymenoptera: Vespidae). Unlike most insects so far investigated, OBPs were detected in antennae, legs and wings, while CSPs appeared to be preferentially expressed in the antennae. The OBP is very different from the homologous proteins of other Hymenopteran species, with around 20% of identical residues, while the CSP appears to be much better conserved. Both OBP and CSP, not showing other post-translational modifications apart from disulphide bridges, were expressed with high yields in a bacterial system. Cysteine pairing in the recombinant and native proteins follows the classical arrangements described for other members of these classes of proteins. OBPs isolated from the wings were found to be associated with a number of long-chain aliphatic amides and other small organic molecules. Binding of these ligands and other related compounds was measured for both recombinant OBP and CSP.
Cell Mol Life Sci 2003 Sep
PMID:Soluble proteins of chemical communication in the social wasp Polistes dominulus. 1452 53

alpha-Fetoprotein (AFP), known largely as a growth-promoting agent, also possesses a growth inhibitory motif recently identified as an occult epitopic segment of the molecule. This segment, a 34-amino acid stretch termed the growth inhibitory peptide (GIP), has been chemically synthesized, purified, and characterized. The purified 34-mer exhibits complex aggregation behaviors; initially, trimeric oligomers were formed that possess growth inhibitory activity in rodent uterine bioassays. These rodent growth assays have served as a prelude to the anticancer studies that followed. In solution, the trimers convert slowly to dimers containing intrapeptide disulfide bonds; such dimers are inactive in the antigrowth assays. Cysteine-to-alanine analogues of the GIP retain the antigrowth properties, while similar cysteine-to-glycine and cysteine-to-serine analogues demonstrate little, if any, growth regulatory activity. Chemical modifications of the cysteine residues also have little influence on the antigrowth activity of the GIP. Fragments of the 34-mer possess variable growth activities of their own, with an octamer from near the carboxyl terminus displaying estrogen-dependent antigrowth activity similar to that of the 34-mer. It was further observed that the GIP can bind both Zn(2+) and Co(2+); the Co(2+) peptide complex was shown to have a distorted tetrahedral symmetry, involving coordination of two cysteine and two histidine residues. The Zn(2+)-GIP complex had antigrowth activity and did not form the intrapeptide disulfide bond characteristic of the free GIP in aqueous solution. The GIP was tested in vitro for anticancer activity and was found to suppress the growth in 38 of 60 human cancer cell lines, representing nine different cancer types. In vivo studies of the GIP, certain analogues, and its fragments revealed anticancer activities in both isograft and xenograft animal tumor transplants. Furthermore, the 2C --> 2A replacement analogue was active against a breast tumor in vivo and in vitro and a prostate cancer in vitro. Thus, it is proposed that the GIP, its analogues, and its fragment peptides can potentially serve as lead compounds for cancer therapeutics.
Mol Cancer Ther 2003 Nov
PMID:Alpha-fetoprotein growth inhibitory peptides: potential leads for cancer therapeutics. 1461 98

Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrates.
Insect Biochem Mol Biol 2004 Apr
PMID:Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris. 1504 Oct 15

Cysteine proteases of the caspase family play key roles in the execution of apoptosis and in the maturation of proinflammatory cytokines. During apoptosis signaling, the latent forms of caspase precursors undergo rapid proteolytic processing and activation. Thus, the measurement of caspase activation provides a quick and convenient mean to assess apoptosis. This chapter outlines the various commonly used assays for determining caspase activity in cultured cells or tissue extracts.
Methods Mol Biol 2004
PMID:Measurement of caspase activity in cells undergoing apoptosis. 1510 54

Cysteine proteases are currently targets for drug development in a number of parasitic diseases, including malaria. In Plasmodium falciparum, the parasite responsible for the most virulent form of human malaria, there are four members of the cathepsin L-like family of cysteine proteases. Three of these (falcipains 2A, 2B and 3) are thought to be primarily involved in haemoglobin digestion, whereas falcipain 1 has recently been linked to erythrocyte invasion. Neither their expression nor their role in P. falciparum gametocytogenesis, which is required for malaria transmission, has been evaluated. In this study, RNA transcripts for the falcipain family members were identified as the parasite developed through all five stages of gametocytogenesis. Falcipain 1 transcript was upregulated in gametocytes, while levels of falcipain 2A/2B decreased in late-stage gametocytes and gametes. To evaluate the function of falcipain 1, the gene was disrupted, and clones from independent transformations were isolated. The asexual growth of the falcipain 1 minus clones was not overtly affected, and they produced morphologically normal gametocytes and gametes. However, when falcipain 1 minus parasites were fed to a mosquito, oocyst production was reduced by 70-90%, suggesting an important role for falcipain 1 during parasite development in the mosquito midgut.
Mol Microbiol 2004 Jul
PMID:Targeted disruption of Plasmodium falciparum cysteine protease, falcipain 1, reduces oocyst production, not erythrocytic stage growth. 1522 18

Cysteine-capped ZnS nanometer-sized fluorescent particles were produced by a colloidal aqueous synthesis. The functionalized nanoparticles are water-soluble and suitable for biological application. A synchronous fluorescence method has been developed for the rapid determination of DNA with functionalized nano-ZnS as a fluorescence probe, based on the synchronous fluorescence enhancement of cysteine-capped nano-ZnS in the presence of DNA. When Deltalambda =190 nm, maximum synchronous fluorescence is produced at 267 nm at pH 5.12. Under optimum conditions, the synchronous fluorescence intensity is proportional to the concentration of nucleic acids in the range 0.1-1.2 microg ml(-1) for calf thymus DNA, 0.1-0.6 microg ml(-1) for fish sperm DNA. The corresponding detection limit is 32.9 ng ml(-1) for calf thymus DNA and 24.6 ng ml(-1) for fish sperm DNA. This method is simple, inexpensive, rapid and sensitive. The recovery and relative standard deviation are satisfactory.
Spectrochim Acta A Mol Biomol Spectrosc 2004 Jul
PMID:Preparation and application of cysteine-capped ZnS nanoparticles as fluorescence probe in the determination of nucleic acids. 1524 43

Cysteine proteases are predominant in thrips guts (TGs) and, therefore, a suitable target for selecting effective protease inhibitors against western flower thrips (Frankliniella occidentalis). We report the isolation of four full-length cysteine protease cDNA clones from thrips in a two-step PCR approach with degenerate oligonucleotides designed on conserved cathepsin L domains. At the deduced amino acid level, the clones possessed all functional and structural characteristics of cathepsin L, and showed high mutual identity and strong similarity with cathepsin L-like cysteine proteases from other insects and arthropods. Southern analysis indicated that a family of four closely related and 10-12 less-related genes encode the cathepsin L-like cysteine proteases in the thrips genome. Partial sequencing of genomic DNA demonstrated the presence of three introns in the coding DNA.
Comp Biochem Physiol B Biochem Mol Biol 2004 Sep
PMID:Isolation and molecular characterization of cathepsin L-like cysteine protease cDNAs from western flower thrips (Frankliniella occidentalis). 1536 89

Cysteine proteinases are involved in a variety of important biological processes and have been implicated in molting and tissue remodeling in free living and parasitic nematodes. We show that in the lymphatic filarial nematode Brugia pahangi molting of third-stage larvae (L3) to fourth-stage larvae is dependent on the activity of a cathepsin L-like cysteine protease (CPL), which can be detected in the excretory/secretory (ES) products of molting L3. Directed cloning of a cysteine protease gene in B. pahangi and analysis of the expressed sequence tag (EST) and genomic sequences of the closely related human lymphatic filarial nematode Brugia malayi have identified a family of CPLs. One group of these enzymes, Bm-cpl-1, -4, -5 and Bp-cpl-4, is highly expressed in the B. malayi and B. pahangi infective L3 larvae. Immunolocalization indicates that the corresponding enzymes are synthesized and stored in granules of the glandular esophagus of L3 and released during the molting process. Functional analysis of these genes in Brugia and closely related CPL genes identified in the filarial nematode Onchocerca volvulus and the free living model nematode Caenorhabditis elegans indicate that these genes are also involved in cuticle and eggshell remodeling.
Mol Biochem Parasitol 2004 Aug
PMID:A gene family of cathepsin L-like proteases of filarial nematodes are associated with larval molting and cuticle and eggshell remodeling. 1547 1

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.
J Mol Biol 2004 Dec 03
PMID:Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo. 1554 2


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