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A photochemical approach to the lipid accessibility of engineered cysteinyl residues.

ABSTRACT: Ordinary electrophilic reagents react too slowly in a nonpolar environment to be useful for the determination of the accessibility to lipid of continuous stretches of residues mutated to cysteine. By contrast, photoactivated 5-iodonaphthyl-1-azide (INA) reacted readily with 2-mercaptoethanol and dodecanethiol in nonpolar solvents and in liposomes. Continuous stretches of residues in the amphipathic N-terminal helix and first transmembrane helix of the bacterial potassium channel KcsA were replaced with cysteine, and the mutants were expressed in Escherichia coli and isolated in inner membranes. These membranes were dissolved in detergent and reconstituted into asolectin liposomes incorporating INA. The extent of light-induced reaction of INA with each cysteine was assayed by subsequent reaction with the gel-shifting, SH-specific methoxy-polyethylene glycol-2-pyridine disulfide. The pattern of apparent second-order rate constants for the photoreactions of eight substituted cysteines in the N-terminal helix conformed to other measures of lipid exposure. The pattern of the rate constants for the photoreactions of 15 cysteines in the first transmembrane helix had peaks every third residue, which partly conformed to other measures of lipid exposure.

SUBMITTER: Li J

PROVIDER: S-EPMC298696 | biostudies-literature | 2003 Feb

REPOSITORIES: biostudies-literature

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A photochemical approach to the lipid accessibility of engineered cysteinyl residues.

Li Jing J Shi Lei L Karlin Arthur A

Proceedings of the National Academy of Sciences of the United States of America 20030117 3

Ordinary electrophilic reagents react too slowly in a nonpolar environment to be useful for the determination of the accessibility to lipid of continuous stretches of residues mutated to cysteine. By contrast, photoactivated 5-iodonaphthyl-1-azide (INA) reacted readily with 2-mercaptoethanol and dodecanethiol in nonpolar solvents and in liposomes. Continuous stretches of residues in the amphipathic N-terminal helix and first transmembrane helix of the bacterial potassium channel KcsA were replac ...[more]

PMID: 12533666

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Project description:BackgroundCysteinyl leukotrienes (CysLTs) are potent prophlogistic mediators in asthmatic patients; however, inhibition of CysLT receptor 1 is not a consistently effective treatment, suggesting additional regulatory mechanisms. Other cysteinyl-containing lipid mediators (LMs) derived from docosahexaenoic acid, namely maresin conjugates in tissue regeneration (MCTRs), were recently discovered. Therefore their production and actions in the lung are of considerable interest.ObjectiveWe sought to determine MCTR production, bioactions, and mechanisms in the human lung and in patients with experimental allergic airway inflammation.MethodsLM metabololipidomic profiling of the lung was performed by using liquid chromatography with tandem mass spectrometry. Donor-derived human precision-cut lung slices were exposed to leukotriene (LT) D4, MCTRs, or both before determination of airway contraction. The actions of exogenous MCTRs on murine allergic host responses were determined in the setting of ovalbumin- and house dust mite-induced lung inflammation.ResultsLipidomic profiling showed that the most abundant cysteinyl LMs in healthy human lungs were MCTRs, whereas CysLTs were most prevalent in patients with disease. MCTRs blocked LTD4-initiated airway contraction in human precision-cut lung slices. In mouse allergic lung inflammation MCTRs were present with temporally regulated production. With ovalbumin-induced inflammation, MCTR1 was most potent for promoting resolution of eosinophils, and MCTR3 potently decreased airway hyperreactivity to methacholine, bronchoalveolar lavage fluid albumin, and serum IgE levels. MCTR1 and MCTR3 inhibited lung eosinophilia after house dust mite-induced inflammation.ConclusionThese results identified lung MCTRs that blocked human LTD4-induced airway contraction and promoted resolution of murine allergic airway responses when added exogenously. Together, these findings uncover proresolving mechanisms for lung responses that can be disrupted in patients with disease.

Dataset Information

A photochemical approach to the lipid accessibility of engineered cysteinyl residues.

Publications

A photochemical approach to the lipid accessibility of engineered cysteinyl residues.

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