Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of the hamster fibrosarcoma cell lines (Met B, D and E) and BHK-21 hamster fibroblast cells with the glucocorticoid dexamethasone led to a powerful dose-dependent mRNA-synthesis-dependent increase in transglutaminase activity, which can be correlated with dexamethasone-responsive receptor numbers in each cell line. Increasing the number of dexamethasone-responsive receptors by transfection of cells with the HG1 glucocorticoid receptor protein caused an increase in transglutaminase activity that was proportional to the level of transfected receptor. In all experiments the levels of the tissue transglutaminase-mediated detergent-insoluble bodies was found to be comparable with increases in transglutaminase activity. Despite an increase in detergent-insoluble body formation, an increase in apoptosis as measured by DNA fragmentation was not found. Incubation of cells with the non-toxic competitive transglutaminase substrate fluorescein cadaverine led to the incorporation of this fluorescent amine into cellular proteins when cells were damaged after exposure to trypsin during cell passage. These cross-linked proteins containing fluorescein cadaverine were shown to be present in the detergent-insoluble bodies, indicating that the origin of these bodies is via activation of tissue transglutaminase after cell damage by trypsinization rather than apoptosis per se, since Met B cells expressing the bcl-2 cDNA were not protected from detergent-insoluble body formation. We describe a novel mechanism of cell death related to tissue transglutaminase expression and cell damage.
...
PMID:Induction of tissue transglutaminase by dexamethasone: its correlation to receptor number and transglutaminase-mediated cell death in a series of malignant hamster fibrosarcomas. 951 67

The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.
...
PMID:Limited proteolysis for assaying ligand binding affinities of nuclear receptors. 963 26

Steroid-induced changes in receptor protein conformation constitute a logical means of translating the variations in steroid structures into the observed array of whole cell biological activities. One conformational change in the rat glucocorticoid receptor (GR) can be readily discerned by following the ability of trypsin digestion to afford a 16-kDa fragment. This fragment is seen after proteolysis of steroid-free receptors but disappears in digests of either glucocorticoid- or antiglucocorticoid-bound receptors. The location of this cleavage site has now been located unambiguously as R651, in helix 6 of the ligand binding domain, by a combination of point mutagenesis, arginine specific protease digestion, and radiochemical sequencing. This 16-kDa species, corresponding to amino acids 652-795, was non-covalently associated with another, approximately 17-kDa species that was determined to be amino acids 518-651 after a comparison of co-immunoprecipitated fragments from wild type and two chimeric receptors. These assignments revise our earlier report of amino acids 537-673 being the 16-kDa fragment and suggest that sequences of the entire ligand binding domain are required for high affinity and specificity binding. This was supported by the observation that trypsin digestion of the steroid-free R651A mutant GR gave rise to the 30-kDa meroreceptor (amino acids 518-795), which displayed wild type affinity. This 30-kDa species is thus the smallest non-associated fragment of GR possessing wild type steroid binding affinity. This suggests that other GR regions do not influence steroid binding affinity. The above results are reminiscent of those observed for the estrogen receptor. However, unlike the estrogen receptor or the more closely related progesterone receptor, the precise proteolytic cleavage points of both the steroid-free and -bound GR fall within regions that are predicted, on the basis of X-ray crystal structures of related receptors, to be alpha-helical and resistant to proteolysis. Thus, the tertiary structure of the GR ligand binding domain may be distinctly different from that of estrogen and progesterone receptors.
...
PMID:Steroid-induced conformational changes of rat glucocorticoid receptor cause altered trypsin cleavage of the putative helix 6 in the ligand binding domain. 1058 Aug 42

Trypsin digestion of steroid-free, but not steroid-bound, rat glucocorticoid receptor (GR) has recently been reported to occur at arginine-651 (R651). This residue is close to the affinity labeled Cys-656 and thus could be a sensitive probe of steroid binding. This hypothesis is supported by the current model of the GR ligand binding domain (LBD), which is based on the X-ray structures of several related receptor LBDs and places R651 in the middle of the putative alpha-helix 6 (649-EQRMS-653 of rat GR), close to the bound steroid. To test this model, R651, which could be involved in hydrophilic and/or hydrogen bonding, was mutated to alanine (A), which favors alpha-helices, the helix breakers proline (P) and glycine (G), or tryptophan (W). All receptors were expressed at about the same level, as determined by Western blots, but the cell-free binding activity of R651P was reduced twofold. The cell-free binding affinities were all within a factor of 10 of wild type receptors. Whole cell biological activity with transiently transfected receptors was determined with a variety of GR agonists (dexamethasone and deacylcortivazol) or antagonists (dexamethasone mesylate, RU486, and progesterone). Reporters containing both simple (GRE) and complex (MMTV) enhancers were used to test for alterations in GR interactions with enhancer/promoter complexes. Surprisingly, no correlation was observed between biological activity and ability to preserve alpha-helical structures for each point mutation. Finally, similar trypsin digestion patterns indicated no major differences in the tertiary structure of the mutant receptors. Collectively, these results argue that the polar/ionizable residue R651 is not required for GR activity and is not part of an alpha-helix in the steroid-free or bound GR. The effect of these mutations on GR structure and activity may result from a cascade of initially smaller perturbations. These LBD alterations were the most varied for interactions with deacylcortivazol and RU 486, which have recently been predicted to be sub-optimal binders due to their large size. However, further analyses of ligand size versus affinity suggest that there is no narrowly defined optimal size for ligand binding, although larger ligands may be more sensitive to modifications of LBD structure. Finally, the changes in GR activity with the various mutations seem to result from altered LBD interactions with common, as opposed to enhancer specific, transcription factors.
...
PMID:Functional analysis of R651 mutations in the putative helix 6 of rat glucocorticoid receptors. 1063 Apr 12

The amnion, a single layer of epithelial cells (EC) overlying layers of mesenchymal cells (MC) has been identified as a source of intrauterine prostaglandins (PG). The objectives of the present study were: (1) to establish a technique for the isolation and culture of pure amnion EC and MC preparations, (2) to characterize the cellular expression of PGHS-II and PGHS activity within these separated amnion cells and (3) to characterize the pattern of glucocorticoid stimulation of these separated amnion cells. Term gestation human amnion was collected after elective caesarean section or vaginal delivery. A trypsin digestion was used to isolate EC and a mechanical digestion and collagenase dispersion was used to isolate MC. Following 48 or 96 h in culture, cells were incubated for 24 h in the presence or absence of 1 microm arachidonic acid and treated with cortisol (F: 10-1000 nm) or 1 microm dexamethasone (DEX). Cell types were identified by immunohistochemistry (IHC). Immunoreactive PGHS-II (ir-PGHS-II) and glucocorticoid receptor (ir-GR) were localized by IHC. PGHS activity was measured as PGE(2)output determined by radioimmunoassay. Mean PGE(2)production by MC at 72 h was 22-fold greater (P<0.05) and at 120 h was 32-fold greater (P<0.03) than PGE(2)output by EC. Administration of arachidonic acid stimulated a 5.0-fold increase in PGE(2)output (P<0.0002) by EC after 72 h and a 3.6-fold increase (P<0.05) after 120 h but did not alter MC PGE(2)output. Despite exogenous substrate, EC PGE(2)output remained significantly less than PGE(2)output by MC. There was no difference in PG production by EC and MC with the onset of labour. Ir-GR expression was found in both EC and MC. F and/or DEX with and without arachidonic acid (AA) stimulated PGE(2)output by EC. Only DEX and not F increased PGE(2)output by MC. These data suggest that relatively pure EC and MC preparations can be established from amnion. PG output and its regulation appears to differ within these two amnion cell types, dependent upon (1) substrate availability and (2) the regulation of PGHS activity.
...
PMID:The characterization of human amnion epithelial and mesenchymal cells: the cellular expression, activity and glucocorticoid regulation of prostaglandin output. 1083 75

The activation domains of many transcription factors appear to exist naturally in an unfolded or only partially folded state. This seems to be the case for AF1/tau1, the major transactivation domain of the human glucocorticoid receptor. We show here that in buffers containing the natural osmolyte trimethylamine N-oxide (TMAO), recombinant AF1 folds into more a compact structure, as evidenced by altered fluorescence emission, circular dichroism spectra, and ultracentrifugal analysis. This conformational transition is cooperative, a characteristic of proteins folding to natural structures. The structure resulting from incubation in TMAO causes the peptide to resist proteolysis by trypsin, chymotrypsin, endoproteinase Arg-C and endoproteinase Gluc-C. Ultracentrifugation studies indicate that AF1/tau1 exists as a monomer in aqueous solution and that the presence of TMAO does not lead to oligomerization or aggregation. It has been suggested that recombinant AF1 binds both the ubiquitous coactivator CBP and the TATA box-binding protein, TBP. Interactions with both of these are greatly enhanced in the presence of TMAO. Co-immunoadsorption experiments indicate that in TMAO each of these and the coactivator SRC-1 are found complexed with AF1. These data indicate that TMAO induces a conformation in AF1/tau1 that is important for its interaction with certain co-regulatory proteins.
...
PMID:The conformation of the glucocorticoid receptor af1/tau1 domain induced by osmolyte binds co-regulatory proteins. 1127 38

The fragments of the androgen receptor (amino acids: 359-732) and of the glucocorticoid receptor (amino acids: 396-548) were expressed in E. coli as fusion proteins with GST. Both fusion proteins, denoted GST-AR and GST-GR, contained the DNA-binding domain and some flanking amino acids. In gel retardation assay both fusion proteins could bind the androgen/glucocorticoid response element (ARE/GRE). We found that both cytosol and nuclear extracts from rat ventral prostate (v.p), but not from other source tested could abolish the interaction of GST-AR and GST-GR with ARE/GRE (from C3 (1) gene and MMTV LTR). The inhibition was androgen-dependent and sensitive to temperature and trypsin treatment. It implies that a protein inhibitor was present in the rat ventral prostate.
...
PMID:A prostate-specific Protein Factor Inhibits the Interaction of Androgen Receptors with Hormone Response Elements. 1216 99


<< Previous 1 2 3 4 5 6

---