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

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

Of 12 cell lines derived from human lung cancers, only Calu-3 cells showed high transepithelial resistance (Rte) and increases in short-circuit current (Isc) in response to mediators. Calu-3 cells formed polarized monolayers with tight junctions and Rte of approximately 100 omega.cm2. Baseline Isc was approximately 35 microA/cm2 and was increased by approximately 75 microA/cm2 on elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by isoproterenol. Flux studies showed that the increase in Isc was due to Cl- secretion. Forskolin and permeant analogues of cAMP also increased Isc. Consistent with the presence of cAMP-dependent Cl- secretion, immunoprecipitation demonstrated the presence of the cystic fibrosis transmembrane conductance regulator (CFTR). Bradykinin, methacholine, trypsin, and histamine all transiently (15-30 s) elevated Isc, probably by increasing intracellular Ca concentration. Experiments in which the basolateral membrane was permeabilized with nystatin indicated that CFTR was substantially activated under baseline conditions and that Ca-activated Cl- channels were absent from the apical membrane. We anticipate that Calu-3 cells will prove useful in the study of Cl- secretion and other functions of human airway epithelial cells.
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PMID:Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl- secretion. 751 78

Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20-40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.
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PMID:CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance. 751 79

Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Ad12-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 microgram/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca+2 and Mg(+2)-free Hanks', balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the delta F508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differentiation of immortalized epithelial cells derived from cystic fibrosis airway submucosal glands. 752 86

This paper describes a method for measuring the increase in halide permeability of isolated airway epithelial cells induced by adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of isolated cells, known to contain the cystic fibrosis transmembrane conductance regulator (CFTR), were placed in the upper part of a Swinnex filter holder containing a filter with pores of 0.65 micron diameter. Medium was perfused over the cells at room temperature and collected at minute intervals following its passage through the filter. Experiments were performed on Calu-3 and T84 cells (human lung and colonic epithelial cell lines), primary cultures of dog and human tracheal epithelium, and Swiss 3T3 fibroblasts stably transfected with CFTR. In all cell types, addition of agents that elevate cAMP led to increases in the rates of loss of 36Cl and 125I. However, in human tracheal epithelial cells, warming the medium from room temperature to 37 degrees C was a more effective way of stimulating tracer efflux. Increases in efflux in response to either temperature or cAMP-elevating agents were inhibited by diphenylamine-2-carboxylate, a blocker of CFTR. Reproducible increases in tracer efflux were seen with as few as 10(6) cells. Cells that had been trypsinized off their culture dishes responded better than cells that had been scraped off, although treatment of scraped cells with trypsin enhanced their responsiveness to cAMP-elevating agents. Cystic fibrosis is characterized by the lack of a cAMP-activated Cl conductance in the apical membrane of airway epithlia.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A method for measuring Cl efflux from dispersed cells of airway epithelium. 753 84

Phosphorylation of P-glycoprotein (Pgp) by protein kinase C occurs on apparently the same sites in vitro and in intact cells (in situ) and is implicated in modulation of Pgp function. The region of the molecule which contains the in vitro phosphorylation sites and two specific sites within this region are now determined by peptide sequencing. Membrane vesicles from multidrug-resistant human KB-V1 cells were incubated with purified protein kinase C and [gamma-32P]ATP, and Pgp (containing 1 mol of phosphate/mol of protein) was purified to apparent homogeneity. Phosphorylation occurred exclusively on serine residues. Phosphopeptides were generated by digestion with Lys-C endoproteinase or trypsin, partially purified by high performance liquid chromatography, and further purified with strategies developed for individual phosphopeptides. Sequence analysis by Edman degradation and comparison with the deduced amino acid sequence of human (mdr 1) Pgp identified serines 661 and 671, and one or more of serines 667, 675, and 683, as sites of phosphorylation. These sites are clustered in the linker region located between the two homologous halves of Pgp. Our results identify a previously undefined, phosphorylatable domain of Pgp, smaller in size but analogous in location to the R-domain of the cystic fibrosis transmembrane conductance regulator. These data provide a basis for a better understanding of the role of phosphorylation in the mechanism of action and regulation of this important multidrug pump protein.
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PMID:Identification of specific sites in human P-glycoprotein phosphorylated by protein kinase C. 809 61

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.
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PMID:Membrane topology and glycosylation of the human multidrug resistance-associated protein. 864 33

A series of recombinant peptides, each including the sequence proposed to be the first nucleotide-binding fold of cystic fibrosis transmembrane conductance regulator (CFTR), has been produced in an attempt to find a model peptide that would autologously fold into a soluble structure with native-like properties. The peptide NBDIF, which contains the 267-amino acid sequence of CFTR from 384 to 650, meets these requirements. The peptide was produced with a high expression bacterial plasmid pRSET, purified from inclusion bodies following solubilization with 6 M guanidine-HCl and refolded from 8 M urea. Competitive displacement of trinitrophenol-ATP by nucleotides reveals binding of ATP and related nucleotides with KDs in the low micromolar range; the KD for ATP gamma S is 1.0 +/- 0.4 microM and for ADP 8.8 +/- 3.1 microM. The native-like character of the model peptide's structure is further supported by the findings that the KD for the ATP analog, 5'-adenylimidodiphosphate, is fourfold lower than the KD for the methylene analog, 5'-adenylmethylenediphosphonate, and that ATP binding slows the trypsin proteolysis of NBDIF. The CD spectra of NBDIF and the parallel peptide containing the most common cystic fibrosis mutation, deletion of Phe 508, are essentially indistinguishable, both spectra indicating 28% alpha-helix and 23% beta-sheet, with insignificant differences in the amounts of beta-turns and random structure. Extensive investigation using multiple conditions with highly purified preparations of the model peptides demonstrates that they do not support ATP hydrolysis. These large recombinant peptides offer practical models for the investigation of the first nucleotide-binding domain of CFTR.
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PMID:A recombinant peptide model of the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator: comparison of wild-type and delta F508 mutant forms. 877 Dec

The cystic fibrosis transmembrane conductance regulator (CFTR) is known to function as a regulated chloride channel and, when genetically impaired, to cause the disease cystic fibrosis. The novel studies reported here were undertaken to gain greater molecular insight into possible interactions among CFTR's soluble domains, which include two nucleotide binding domains (NBF1 and NBF2) and a regulatory domain (R). The NBF1+R and NBF2 regions of CFTR were highly expressed in Escherichia coli, purified to near homogeneity under denaturing conditions, and refolded. Both refolded proteins bound TNP-ATP and TNP-ADP, which could be readily replaced with ATP. Four different approaches were then used to determine whether the NBF1+R and NBF2 proteins interact. First, the purified NBF2 protein was labeled near its C-terminus with a fluorescent probe, 7-diethyl amino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Addition of the unlabeled NBF1+R to the CPM-labeled NBF2 caused a red-shift in lambda(max) of the CPM fluorescence, consistent with a direct interaction between the two proteins. Second, when the NBF1+R protein, the NBF2 protein, and a mixture of the two proteins were folded separately and analyzed by molecular sieve chomatography, the mixture was found to elute prior to either NBF1+R or NBF2. Third, na-tive-PAGE gel studies revealed that the mixture of the NBF1+R and NBF2 domains migrated as a single band with an R(F) value between that of NBF1+R and NBF2. Fourth, trypsin digestion of a mixture of the NBF1+R and NBF2 proteins occurred at a slower rate than that for the individual proteins. Finally, studies were carried out to determine whether an NBF1+R/NBF2 interaction could be demonstrated after expressing one of the two proteins in soluble, native form, thus avoiding the inclusion body, denaturation, and renaturation approach. Specifically, the NBF1+R protein was overexpressed in E. coli in fusion with glutathione-S-transferase near a thrombin cleavage site. Following binding of the GST-(NBF1+R) fusion protein to a GST Sepharose affinity column, added NBF2 was shown to bind and then to coelute with NBF1+R upon addition of glutathione or thrombin. Collectively, these experiments demonstrate that CFTR's NBF1+R region and its NBF2 domain, after folding separately as distinct units, have a strong propensity to interact and that this interaction is stable in the absence of added nucleotides or exogenously induced phosphorylation. These findings, together with the additional observation that the NBF1+R/NBF2 interaction induces a change in the C-terminus of NBF2, which resides within the C-terminal region of CFTR, may have important implications not only for the function of CFTR per se, but its interaction with other proteins.
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PMID:Cystic fibrosis transmembrane conductance regulator: the purified NBF1+R protein interacts with the purified NBF2 domain to form a stable NBF1+R/NBF2 complex while inducing a conformational change transmitted to the C-terminal region. 1068 44

Inefficient maturation and trafficking to the cell surface of the cystic fibrosis transmembrane conductance regulator (CFTR) is the primary cause of cystic fibrosis. CFTR protein that fails to mature accumulates as an immature core-glycosylated protein and is rapidly degraded. To determine how the structures of mature and immature CFTR are different, we compared the properties of CFTR that had been expressed in the presence or absence of the proteasome inhibitor, MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal). Transient expression of wild-type CFTR in the presence of submicromolar concentrations of MG-132 blocks maturation of the protein. We found that expression of CFTR in the presence of MG-132 trapped the protein in a trypsin-sensitive conformation. In addition, the structure of the carboxyl-terminus of immature and mature CFTR differed as histidine-tagged mature CFTR was preferentially recovered by metal-chelate chromatography. No chloride channel activity was detected when membranes containing immature CFTR were fused with planar lipid bilayers. These results show that expression of CFTR in the presence of MG-132 traps the protein in an altered conformation that may be inactive.
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PMID:Cystic fibrosis transmembrane conductance regulator has an altered structure when its maturation is inhibited. 1073 80

After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.
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PMID:Differential interactions of nucleotides at the two nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator. 1127 83


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