RESUMO
CFTR possesses a large cluster of strict dibasic consensus sites for phosphorylation by protein kinase A (PKA) in the R-domain and an obligatory dependence on phosphorylation is a hallmark of CFTR Cl(-) channel function. Removal of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been reported, leaving open the possibility that the residual response may be mediated by associating ancillary phosphoproteins. We traced the residual PKA-catalyzed (32)P-labelling of the variant with 11 sites mutagenized (11SA) to distinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additional monobasic sites in these segments produced a 15SA variant in which Cl(-) channel response to PKA was abolished. Therefore, it can be concluded that ancillary phosphoproteins do not contribute to CFTR activation by PKA. Notably, however, the 15SA protein did exhibit a low level of constitutive channel activity not dependent on PKA, which might have reflected a down-regulating effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case.Since immature CFTR has been claimed to be active in the endoplasmic reticulum (ER), we also examined whether it can be phosphorylated in cells and what influence if any this might have on its susceptibility to degradation. Teleologically, activation by phosphorylation of CFTR Cl(-) channels in the ER might be undesirable to the cell. Using various phosphorylation site mutants and kinase and phosphatase inhibitors in pulse-chase experiments, we have found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore, we found that microsomes from cells expressing CFTR processing mutants such as DeltaF508 do not generate Cl(-) active channels when fused with planar bilayers unless maturation is promoted, e.g. by growth of cells at reduced temperature or other means. We conclude that the ER-retained mutant nascent chains which are incapable of maturation may be phosphorylated but do not form active channels. Stimulation by PKA of the insertion of CFTR containing vesicles into the plasma membrane as part of the mechanism of stimulation of chloride secretion has been reported, as has an influence of CFTR on the balance between endocytosis and exocytosis but these findings have not been universally confirmed.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Regulador de Condutância Transmembrana em Fibrose Cística/química , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Humanos , Ativação do Canal Iônico , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Conformação ProteicaRESUMO
BACKGROUND: Pulse wave velocity (PWV) is a marker of arterial stiffness and predicts cardiovascular events in the nontransplantation population. Cardiovascular events (CVE) are the leading cause of death and one of the leading causes of graft failure in renal transplant recipients. The present prospective study investigates whether there is a correlation between PWV and CVE in renal transplant recipients. METHODS: A prospective study assessing the incidence of a composite cardiovascular endpoint within ≥ 3 years after pulse wave analysis was performed in 64 stable renal transplant recipients. Measurement of PWV, augmentation index (AI75), and aortic systolic pressure was conducted using the SphygmoCor (AtCor) device. The composite endpoint of the study was the incidence of either death, myocardial infarction, stroke, or admission for symptomatic intermittent claudication or decompensated congestive heart failure. RESULTS: Fifteen patients (23%) reached the composite endpoint during a follow-up of 4.4 years. Binary logistic regression using PWV, AI75, central aortic systolic pressure, peripheral systolic pressure, and pulse pressure as covariates revealed that PWV was significantly associated with cardiovascular events (10.1 ± 3.6 m/s in subjects reaching the endpoint vs 8.5 ± 1.5 m/s in subjects not reaching the endpoint; P = .048). CONCLUSION: Increased arterial stiffness as assessed by PWV predicts CVE in renal transplant recipients and may be regarded as a footprint of accelerated arteriosclerosis for those patients.
Assuntos
Doenças Cardiovasculares/epidemiologia , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Complicações Pós-Operatórias , Medição de Risco/métodos , Rigidez Vascular , Adulto , Idoso , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Causas de Morte/tendências , Feminino , Seguimentos , Alemanha/epidemiologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
BACKGROUND: Urinary calprotectin has recently been identified as a promising biomarker for the differentiation of pre-renal and intrinsic acute kidney injury (AKI). This study compares the diagnostic performance of calprotectin and neutrophil gelatinase-associated lipocalin (NGAL) in this differential diagnosis. METHODS: Urinary calprotectin and NGAL concentrations were assessed in a study population of 87 subjects including 38 cases of intrinsic AKI, 24 cases of pre-renal AKI and 25 healthy controls. Urinary tract obstruction, renal transplantation and metastatic cancer were defined as exclusion criteria. RESULTS: Mean calprotectin concentrations were significantly lower in pre-renal (190.2 ± 205.7 ng mL(-1) ) than in intrinsic AKI (6250.1 ± 7167.2 ng mL(-1) , P < 0.001). Receiver-operating characteristic (ROC) analysis provided an AUC of 0.99. Mean NGAL concentrations were significantly higher in intrinsic than in pre-renal AKI as well (458.1 ± 695.3 vs. 64.8 ± 62.1 ng mL(-1) , P = 0.001) providing an AUC of 0.82. A combination of the present study population with the cohort of the proof of concept study led to a population of 188 subjects (58 pre-renal AKI, 90 intrinsic AKI, 40 healthy controls). ROC analyses provided an AUC of 0.97 for calprotectin and 0.76 for NGAL yielding sensitivity and specificity values of 93.3 and 94.8% (calprotectin) vs. 75.3 and 72.4% (NGAL). Optimal cut-off values were 440 ng mL(-1) (calprotectin) and 52 ng mL(-1) (NGAL). Pyuria increased calprotectin concentrations independent of renal failure. CONCLUSION: This study shows that both calprotectin and NGAL are able to differentiate between pre-renal and intrinsic AKI after exclusion of pyuria. In the present population, calprotectin presents a higher sensitivity and specificity than NGAL.
Assuntos
Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/urina , Proteínas de Fase Aguda/urina , Rim/metabolismo , Complexo Antígeno L1 Leucocitário/urina , Lipocalinas/urina , Proteínas Proto-Oncogênicas/urina , Injúria Renal Aguda/patologia , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores/urina , Biópsia , Estudos de Casos e Controles , Estudos Transversais , Diagnóstico Diferencial , Feminino , Humanos , Rim/patologia , Lipocalina-2 , Masculino , Pessoa de Meia-Idade , Curva ROC , Sensibilidade e EspecificidadeAssuntos
Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Animais , Baculoviridae , Células CHO , Células COS , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Canais de Cloreto/biossíntese , Canais de Cloreto/fisiologia , Clonagem Molecular/métodos , Cricetinae , Feminino , Humanos , Ativação do Canal Iônico , Potenciais da Membrana/fisiologia , Metotrexato/toxicidade , Oócitos/fisiologia , Plasmídeos , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Saccharomyces cerevisiae , Spodoptera , Transfecção/métodos , Xenopus laevisRESUMO
Translocation intermediates of the chimeric protein precursor Oee1-Dhfr were generated and used to identify envelope components in close proximity to the arrested precursor. The translocation of Oee1-Dhfr across the chloroplast envelope can be arrested at low ATP levels or by prebinding the fusion precursor with anti-Dhfr IgGs. The arrested Oee1-Dhfr precursor appears to span both the outer and inner envelope membranes. Translocational arrest of Oee1-Dhfr by low ATP levels was reversible, and import was restored upon resupplementation with higher ATP levels. Chemical cross-linking and co-immunoprecipitation with monospecific antibodies indicate that two outer envelope membrane proteins (Com44 and Com70) and at least one inner envelope protein (Cim44 and Cim97) were found to be in close proximity to Oee1-Dhfr during translocation. The Com70 protein was further studied and additional evidence for its role in chloroplast protein import is presented.
Assuntos
Proteínas de Algas , Cloroplastos/metabolismo , Proteínas de Plantas/metabolismo , Precursores de Proteínas/metabolismo , Transporte Biológico , Membranas Intracelulares/metabolismo , Pisum sativum/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Multidrug resistant cells may become acutely sensitive to the calcium channel blocker verapamil, in spite of the fact that its accumulation by these cells is negligible. We selected verapamil-resistant mutants from multidrug resistant Chinese hamster ovary cells. Levels of P-glycoprotein expression and cross-resistance profiles remained unaltered in the verapamil-resistant multidrug resistant cells. As well, a photoactive verapamil analog specifically bound to P-glycoprotein in these cells. We had previously used a photoactive anthracycline to show that calcium antagonists and several anticancer drugs bind to P-glycoprotein at overlapping or interacting sites. Verapamil and its analogues no longer inhibit the binding of either anticancer drugs or calcium channel blockers to P-glycoprotein. Sequencing of P-glycoprotein revealed that no change had occurred in the coding sequence as a result of the selection procedure.
Assuntos
Resistência a Múltiplos Medicamentos , Verapamil/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Daunorrubicina/análogos & derivados , Daunorrubicina/farmacologia , Técnicas In Vitro , Iodobenzenos/farmacologia , Fotoquímica , Ligação Proteica , Vimblastina/farmacologiaRESUMO
The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large superfamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bidirectional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.
Assuntos
Canais de Cloreto/química , Regulador de Condutância Transmembrana em Fibrose Cística/química , Fibrose Cística/metabolismo , Dobramento de Proteína , Catálise , Canais de Cloreto/genética , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/genética , HumanosRESUMO
To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wild-type CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 degrees C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P0) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P0, whereas the H949Y mutation doubled the P0 relative to wild-type. The changes in P0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range +/-80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.
Assuntos
Canais de Cloreto/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Mutação Puntual , Estrutura Secundária de Proteína , Animais , Células CHO , Células COS , Linhagem Celular , Membrana Celular/fisiologia , Canais de Cloreto/química , Cricetinae , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Citosol/metabolismo , Humanos , Iodetos/metabolismo , Rim , Cinética , Potenciais da Membrana , Modelos Estruturais , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , TransfecçãoRESUMO
A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Éxons , Mutação Puntual , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Células CHO , Membrana Celular/fisiologia , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Variação Genética , Humanos , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.
Assuntos
Polaridade Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Animais , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , AMP Cíclico/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Cães , Iodetos/farmacologia , Canais Iônicos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
Since little is known about the contribution to function of the N-terminal cytoplasmic loops (CL1, residues 139-194; CL2, residues 242-307) of cystic fibrosis transmembrane conductance regulator (CFTR), all nine point mutations identified in CLs 1 and 2 from patients with cystic fibrosis were reconstructed in the expression vector pcDNA3-CFTR and expressed transiently in COS-1 and HEK-293 cells and stably in Chinese hamster ovary (CHO) cells. Four amino acid substitutions retarded production of mature, fully glycosylated CFTR, suggesting that misprocessing of the channel causes the disease symptoms in the affected patients. Protein maturation could not be promoted by cell culture conditions of reduced temperature (26 degrees C). When properly processed mutants were evaluated for functional defects by the iodide efflux method, the G178R- and E193K-CFTR-expressing cell lines showed impaired anion translocation activities. Patch-clamp studies of single channels revealed that E193K variants had a significantly decreased open probability, which resulted from an increase in the mean closed time of the channels. This contrasted with a previous study of disease-associated point mutations in CL3 that mainly affected the mean open time. None of the maturation-competent CL 1 and 2 mutants had altered conductance. Thus, the N-terminal CLs appear not to contribute to the anion translocation pathway of CFTR; rather, mutations in CL1 can impede transition to the open state. Interestingly, the ability of the non-hydrolyzable ATP analogue adenylyl imidodiphosphate (AMP-PNP) to lock the channel into open bursts was abolished by the I148T and G178R amino acid substitutions.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Mutação , Adenilil Imidodifosfato/farmacologia , Animais , Células CHO , Cricetinae , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citoplasma/química , Humanos , Iodetos/metabolismo , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Técnicas de Patch-Clamp , Dobramento de ProteínaRESUMO
Hormonal regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is largely mediated via cAMP-dependent protein kinase (PKA). CFTR contains 10 dibasic consensus sites for potential PKA phosphorylation ((R/K) (R/K)X(S*/T*)). Previous studies (Chang, X.-B., Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon, N., Hanrahan, J. W., and Riordan, J.R (1993) J. Biol. Chem. 268, 11304-11311) showed that approximately 25% of the CFTR wild-type response to PKA activation remained upon inhibition of most detectable phosphorylation by in vitro mutagenesis of all 10 dibasic consensus sites (10SA CFTR). To identify potential additional sites responsible for the residual activity, large amounts of this mutant CFTR were phosphorylated with PKA using high specific activity [gamma-32P]ATP. Cyanogen bromide cleavage indicated that a large portion of the observed PKA phosphorylation occurred within a 5.8-kDa fragment of the R domain between residues 722-773. Removal of serines at potential PKA sites in this fragment showed that Ser-753 accounted for all of the gamma-32P labeling of the 5.8-kDa peptide. Replacement of Ser-753 with alanine reduced the level of residual CFTR activity by a further 40%, indicating that phosphorylation at this previously unidentified site contributes to the activation of 10SA CFTR.