The reconstituted Escherichia coli MsbA protein displays lipid flippase activity

The MsbA protein is an essential ABC (ATP-binding-cassette) superfamily member in Gram-negative bacteria. This 65 kDa membrane protein is thought to function as a homodimeric ATP-dependent lipid translocase or flippase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. We have previously shown that purified MsbA from Escherichia coli displays high ATPase activity, and binds to lipids and lipid-like molecules, including lipid A, with affinity in the low micromolar range. Bacterial membrane vesicles isolated from E. coli overexpressing His6-tagged MsbA displayed ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Purified MsbA was reconstituted into proteoliposomes of E. coli lipid and its ability to translocate NBD-labelled lipid derivatives was characterized. In this system, the protein displayed maximal lipid flippase activity of 7.7 nmol of lipid translocated per mg of protein over a 20 min period for an acyl chain-labelled PE (phosphatidylethanolamine) derivative. The protein showed the highest rates of flippase activity when reconstituted into an E. coli lipid mixture. Substantial flippase activity was also observed for a variety of other NBD-labelled phospholipids and glycolipids, including molecules labelled on either the headgroup or the acyl chain. Lipid flippase activity required ATP hydrolysis, and was dependent on the concentration of ATP and NBD–lipid. Translocation of NBD–PE was inhibited by the presence of the putative physiological substrate lipid A. The present paper represents the first report of a direct measurement of the lipid flippase activity of purified MsbA in a reconstituted system

A new platform for high-throughput therapy testing on iPSC-derived lung progenitor cells from cystic fibrosis patients

For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies

Rescue of multiple class II CFTR mutations by elexacaftor+ tezacaftor+ivacaftor mediated in part by the dual activities of Elexacaftor as both corrector and potentiator

Positive results in preclinical studies of the triple combination of elexacaftor, tezacaftor and ivacaftor, performed in airway epithelial cell cultures obtained from patients harboring F508del-CFTR, translated to impressive clinical outcomes for subjects carrying this mutation in clinical trials and approval of TRIKAFTATM Encouraged by this correlation, we were prompted to evaluate the effect of the elexacaftor, tezacaftor and ivacaftor triple combination on primary nasal epithelial cultures obtained from individuals with rare Class II cystic fibrosis causing mutations; G85E, M1101K and N1303K for which TRIKAFTATM is not approved. Cultures from individuals homozygous for M1101K responded better than cultures harboring G85E and N1303K after treatment with the triple combination with respect to improvement in regulated channel function and protein processing. A similar genotype specific effect of the triple combination was observed when the different mutations were expressed in HEK-293 cells, supporting the hypothesis that these modulators may act directly on the mutant proteins. Detailed studies in nasal cultures and HEK-293 cells showed that the corrector: elexacaftor, exhibited dual activity as both corrector and potentiator and suggested that the potentiator activity contributes to its pharmacological activity. These preclinical studies using nasal epithelial cultures identified mutation genotypes for which elexacaftor, tezacaftor and ivacaftor may produce clinical responses that are comparable to, or inferior to those observed for F508del-CFTR

The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids

The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a ‘flippase’ for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD–GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD–GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD–GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the K(d) for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD–lactosylceramide drastically reduced the rate of flipping compared with NBD–PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids