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Abstract

The epithelial sodium channel (ENaC) and the proteolytic processing of its alpha- and γ-subunits by channel-activating proteases (CAPs) play a crucial role in sodium transport across different epithelial membranes, including the airways, and in airway surface liquid (ASL) homeostasis. The loss of ion transport homeostasis results in various diseases, including one of the most frequent lethal genetic disorders: Cystic fibrosis. The fundamental cause of the disease is a mutation of a chloride channel, resulting in loss or reduction of chloride secretion. Not only the chloride transport is affected, but also the sodium transport via ENaC which is increased in CF. It has been shown that CAP expression is elevated in primary bronchial epithelial cultures of Cystic Fibrosis (CF) patients compared to tissue from healthy subjects. However, the impact of CAPs on ENaC activity and ASL regulation in the CF lung disease is unknown. In order to study the role of CAPs in CF lung disease and their regulation in the disease context, substrate-based Förster Resonance Energy Transfer (FRET) probes were developed for measuring protease activity. In contrast to most commercial substrates, the probes presented in this work, are equipped with two fluorophores. Proteolytic cleavage of the probe resulted in a loss of FRET, giving a ratiometric readout reflecting enzyme activity. The attachment of a lipid anchor enabled the detection of membrane-associated enzyme activity in a spatially resolved manner. Different substrate sequences and fluorophore combinations were synthesized and tested towards their physical and biochemical properties. Not only extension or contraction in the amino acid sequence between both fluorophores affects the efficacy of FRET and maximal fold change of the ratio upon full cleavage of the probe, but also the identity of the flanking amino acids significantly contributes. For the lipidated probes, the combination of Coumarin 343 and TAMRA turned out to be the best working combination. Probes based on different substrate sequences have been evaluated with respect to selectivity for CAPs over other proteases. In vitro characterization using recombinant enzymes as well as cell-based characterization using broad-spectrum inhibitors identified the probe based on the CAP cleavage site in the human ENaC γ-subunit as the most selective one towards CAP activity. Probes based on the autoactivation cleavage site of CAP3, however, also have sufficient specificity and perform better with respect to localization and dynamic range compared lipidated versions. Furthermore, the fluorophore combination and potentially the insertion of the lipid anchor significantly affect the specificity. As proof of concept, the probes were successfully tested in primary human nasal epithelial cells and primary murine tracheal epithelial cells. Application of the probes to a small cohort of CF patients and a healthy control showed that the novel FRET probes are able to detect increased CAP activity in nasal cells from CF patients compared to healthy control samples. Murine tracheal epithelial cells of βENaC-transgenic mice, a mouse model for CF lung disease, however, have significantly reduced CAP activity compared to cells isolated from wild type mice. Both experimental sets suggested that our FRET probes are a useful tool to measure CAP1 and CAP3 as potential biomarkers in human specimens for different diseases as well as research tools for further investigation of CAP activity in chronic lung diseases, including CF, idiopathic pulmonary fibrosis and lung cancer.