The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites

MsbA is an essential ABC (ATP-binding cassette) transporter involved in lipid A transport across the cytoplasmic membrane of Gram-negative bacteria. The protein has also been linked to efflux of amphipathic drugs. Purified wild-type MsbA was labelled stoichiometrically with the fluorescent probe MIANS [2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid] on C315, which is located within the intracellular domain connecting transmembrane helix 6 and the nucleotide-binding domain. MsbA–MIANS displayed high ATPase activity, and its folding and stability were unchanged. The initial rate of MsbA labelling by MIANS was reduced in the presence of amphipathic drugs, suggesting that binding of these compounds alters the protein conformation. The fluorescence of MsbA–MIANS was saturably quenched by nucleotides, lipid A and various drugs, and estimates of the Kd values for binding fell in the range of 0.35–10 μM. Lipid A and daunorubicin were able to bind to MsbA–MIANS simultaneously, implying that they occupy different binding sites. The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered. The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity. These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other. One is a high affinity binding site for the physiological substrate, lipid A, and the other site interacts with drugs with comparable affinity. Thus MsbA may function as both a lipid flippase and a multidrug transporter

Liposomes as a model for the biological membrane : studies on daunorubicin bilayer interaction

In this study the interaction of the antitumoral drug daunorubicin with egg phosphatidylcholine (EPC) liposomes, used as a cell membrane model, was quantified by determination of the partition coefficient (Kp). The liposome/aqueous-phase Kp of daunorubicin was determined by derivative spectrophotometry and measurement of the zeta-potential. Mathematical models were used to fit the experimental data, enabling determination of Kp. In the partition of daunorubicin within the membrane both superficial electrostatic and inner hydrophobic interactions seem to be involved. The results are affected by the two types of interaction since spectrophotometry measures mainly hydrophobic interactions, while zeta-potential is affected by both interpenetration of amphiphilic charged molecules in the bilayer and superficial electrostatic interaction. Moreover, the degree of the partition of daunorubicin with the membrane changes with the drug concentration, due mainly to saturation factors. Derivative spectrophotometry and zeta-potential variation results, together with the broad range of concentrations studied, revealed the different types of interactions involved. The mathematical formalism applied also allowed quantification of the number of lipid molecules associated with one drug molecule

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

Discovery of the inhibitory effect of a phosphatidylinositol derivative on P-glycoprotein by virtual screening followed by <i>in vitro</i> cellular studies

P-glycoprotein is capable of effluxing a broad range of cytosolic and membrane penetrating xenobiotic substrates, thus leading to multi-drug resistance and posing a threat for the therapeutic treatment of several diseases, including cancer and central nervous disorders. Herein, a virtual screening campaign followed by experimental validation in Caco-2, MDKCII, and MDKCII mdr1 transfected cell lines has been conducted for the identification of novel phospholipids with P-gp transportation inhibitory activity. Phosphatidylinositol-(1,2-dioctanoyl)-sodium salt (8∶0 PI) was found to significantly inhibit transmembrane P-gp transportation in vitro in a reproducible-, cell line-, and substrate-independent manner. Further tests are needed to determine whether this and other phosphatidylinositols could be co-administered with oral drugs to successfully increase their bioavailability. Moreover, as phosphatidylinositols and phosphoinositides are present in the human diet and are known to play an important role in signal transduction and cell motility, our finding could be of substantial interest for nutrition science as well

ER-Bound Protein Tyrosine Phosphatase PTP1B Interacts with Src at the Plasma Membrane/Substrate Interface

PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research

Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B

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