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1.
J Hartman Z Huang T A Rado S Peng T Jilling D D Muccio E J Sorscher 《The Journal of biological chemistry》1992,267(10):6455-6458
The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding. 相似文献
2.
Hartman J Daram P Frizzell RA Rado T Benos DJ Sorscher EJ 《Biotechnology and bioengineering》1992,39(8):828-832
Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase. 相似文献
3.
Linbo Liu Kengyeh K. Chu Grace H. Houser Bradford J. Diephuis Yao Li Eric J. Wilsterman Suresh Shastry Gregory Dierksen Susan E. Birket Marina Mazur Suzanne Byan-Parker William E. Grizzle Eric J. Sorscher Steven M. Rowe Guillermo J. Tearney 《PloS one》2013,8(1)
We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified. 相似文献
4.
Gudio Veit Radu G. Avramescu Annette N. Chiang Scott A. Houck Zhiwei Cai Kathryn W. Peters Jeong S. Hong Harvey B. Pollard William B. Guggino William E. Balch William R. Skach Garry R. Cutting Raymond A. Frizzell David N. Sheppard Douglas M. Cyr Eric J. Sorscher Jeffrey L. Brodsky Gergely L. Lukacs 《Molecular biology of the cell》2016,27(3):424-433
5.
Daniel Duran Xue Zeng Sheng Chih Jin Jungmin Choi Carol Nelson-Williams Bogdan Yatsula Jonathan Gaillard Charuta Gavankar Furey Qiongshi Lu Andrew T. Timberlake Weilai Dong Michelle A. Sorscher Erin Loring Jennifer Klein August Allocco Ava Hunt Sierra Conine Jason K. Karimy Kristopher T. Kahle 《Neuron》2019,101(3):429-443.e4
6.
Pyle LC Ehrhardt A Mitchell LH Fan L Ren A Naren AP Li Y Clancy JP Bolger GB Sorscher EJ Rowe SM 《American journal of physiology. Lung cellular and molecular physiology》2011,301(4):L587-L597
Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UC(CF)-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UC(CF)-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o(-) and Fischer rat thyroid cells, expressing various CFTR forms. UC(CF)-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UC(CF)-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR. 相似文献
7.
Jurkuvenaite A Varga K Nowotarski K Kirk KL Sorscher EJ Li Y Clancy JP Bebok Z Collawn JF 《The Journal of biological chemistry》2006,281(6):3329-3334
Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype. 相似文献
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9.
Rafael García-Mata Zsuzsa Beb?k Eric J. Sorscher Elizabeth S. Sztul 《The Journal of cell biology》1999,146(6):1239-1254
Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883-1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481-490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end-directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins. 相似文献
10.
The nucleotide binding domains (NBDs) within CFTR were initially predicted to lie in the cell cytoplasm, and to gate anion permeability through a pore that was present in membrane spanning helices of the overall polypeptide. Our studies designed to characterize CFTR suggest several important features of the isolated nucleotide binding domain. NBD-1 appears to bind nucleotides with similar affinity to the full-length CFTR protein. In solution, the domain contains a high sheet content and self-associates into ordered polymers with molecular mass greater than 300,000 Daltons. The domain is very lipophilic, disrupts liposomes, and readily enters the planar lipid bilayer. Clinically important mutations in the domain may disrupt the nucleotide binding capabilities of the protein, either through a direct effect on the nucleotide binding site, or through effects that influence the overall folding of the domain in vitro. Finally, after expression in human epithelial cells (including epithelial cells from a CF patient), the first nucleotide binding domain targets the plasma membrane even in the absence of other constituents of full-length CFTR and mediates anion permeability in these cells. 相似文献