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Background information. Cystic fibrosis results from mutations in the ABC transporter CFTR (cystic fibrosis transmembrane conductance regulator), which functions as a cAMP-regulated anion channel. The most prevalent mutation in CFTR, the Phe508 deletion, results in the generation of a trafficking and functionally deficient channel. The cellular machineries involved in modulating CFTR trafficking and function have not been fully characterized. In the present study, we identified a role for the COPI (coatomer protein I) cellular trafficking machinery in the development of the CFTR polypeptide into a functional chloride channel. To examine the role of COPI in CFTR biosynthesis, we employed the cell line ldlF, which harbours a temperature-sensitive mutation in ε-COP, a COPI subunit, to inhibit COPI function and then determined whether the CFTR polypeptide produced from the transfected gene developed into a cAMP-regulated chloride channel.Results. When COPI was inactivated in the ldlF cells by an elevated temperature pulse (39°C), the CFTR polypeptide was detected on the cell surface by immunofluorescence microscopy and cell-surface biotinylation. Therefore, CFTR proceeded upstream in the secretory pathway in the absence of COPI function, a result demonstrated previously by others. In contrast, electrophysiological measurements indicated an absence of cAMP-stimulated chloride efflux, suggesting that channel function was impaired. In comparison, expression of CFTR at the same elevated temperature (39°C) in an ε-COP-rescued cell line [ldlF(ldlF)], which has an introduced wild-type ε-COP gene in addition to the mutant ε-COP gene, showed restoration of cAMP-stimulated channel activity, confirming the requirement of COPI for channel function.Conclusions. These results therefore suggest that generation of the folded-functional conformation of CFTR requires COPI. [ABSTRACT FROM AUTHOR]

We have used site-directed mutagenesis and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase (AChE), ChE2 from amphioxus, by the sulfhydryl reagents 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM), creating various mutants, including C310A and C466A, and the double mutants C310A/C466A and C310A/F312I, to assess the relative roles of the two cysteines and a proposal that the increased rate of inactivation in the F312I mutant is due to increased access to Cys310. Our results suggest that both cysteines may be involved in inactivation by sulfhydryl reagents, but that the cysteine in the vicinity of the acyl pocket is more accessible. We speculate that the inactivation of aphid AChEs by sulfhydryl reagents is due to the presence of a cysteine homologous to Cys310. We also investigated the effects of various reversible cholinergic ligands, which bind to different subsites of the active site of the enzyme, on the rate of inactivation by DTNB of wild type ChE2 and ChE2 F312I. For the most part the inhibitors protect the enzymes from inactivation by DTNB. However, a notable exception is the peripheral site ligand propidium, which accelerates inactivation in the wild type ChE2, but retards inactivation in the F312I mutant. We propose that these opposing effects are the result of an altered allosteric signal transduction mechanism in the F312I mutant compared to the wild type ChE2. [ABSTRACT FROM AUTHOR]

We present an evaluation of our results for the first Critical Assessment of PRedicted Interaction (CAPRI). The methods used include the molecular docking program DOT, shape analysis tool FADE, cluster analysis and filtering based on biological data. Good results were obtained for most of the seven CAPRI targets, and for two systems, submissions having the highest number of correctly predicted contacts were produced. Proteins 2003;52:33–40. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]