A robust method to screen detergents for membrane protein stabilization, revisited.

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of ß-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents.

Cholesterol and Sphingomyelin-Containing Model Condensed Lipid Monolayers: Heterogeneities Involving Ordered Microdomains Assessed by Two Cholesterol Derivatives.

Lipid monolayers are often considered as model membranes, but they are also the physiologic lipid part of the peripheral envelope of lipoproteins and cytosolic lipid bodies. However, their structural organization is still rather elusive, in particular when both cholesterol and sphingomyelin are present. To investigate such structural organization of hemimembranes, we measured, using alternative current voltammetry, the differential capacitance of condensed phosphatidylcholine-based monolayers as a function of applied potential, which is sensitive to their lipid composition and molecular arrangement. Especially, monolayers containing both sphingomyelin and cholesterol, at 15% w/w, presented specific characteristics of the differential capacitance versus potential curves recorded, which was indicative of specific interactions between these two lipid components. We then compared the behavior of two cholesterol derivatives (at 15% w/w), 21-methylpyrenyl-cholesterol (Pyr-met-Chol) and 22-nitrobenzoxadiazole-cholesterol (NBD-Chol), with that of cholesterol when present in model monolayers. Indeed, these two probes were chosen because of previous findings reporting opposite behaviors within bilayer membranes regarding their interaction with ordered lipids, with only Pyr-met-Chol mimicking cholesterol well. Remarkably, in monolayers containing sphingomyelin or not, Pyr-met-Chol and NBD-Chol presented contrasting behaviors, and Pyr-met-Chol mimicked cholesterol only in the presence of sphingomyelin. These two observations (i.e., optimal amounts of sphingomyelin and cholesterol, and the ability to discriminate between Pyr-met-Chol and NBD-Chol) can be interpreted by the existence of heterogeneities including ordered patches in sphingomyelin- and cholesterol-containing monolayers. Since such monolayer lipid arrangement shares some properties with the raft-type lipid microdomains well-described in sphingomyelin- and cholesterol-containing bilayer membranes, our data thus strongly suggest the existence of compact and ordered microdomains in model lipid monolayers.

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: a new labelling tool.

Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described "lipid rafts" in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD-Chol non-specifically delivered to the cells.

Recognition of sulfonylurea receptor (ABCC8/9) ligands by the multidrug resistance transporter P-glycoprotein (ABCB1): functional similarities based on common structural features between two multispecific ABC proteins.

ATP-sensitive K(+) (K(ATP)) channels are the target of a number of pharmacological agents, blockers like hypoglycemic sulfonylureas and openers like the hypotensive cromakalim and diazoxide. These agents act on the channel regulatory subunit, the sulfonylurea receptor (SUR), which is an ABC protein with homologies to P-glycoprotein (P-gp). P-gp is a multidrug transporter expressed in tumor cells and in some healthy tissues. Because these two ABC proteins both exhibit multispecific recognition properties, we have tested whether SUR ligands could be substrates of P-gp. Interaction with P-gp was assayed by monitoring ATPase activity of P-gp-enriched vesicles. The blockers glibenclamide, tolbutamide, and meglitinide increased ATPase activity, with a rank order of potencies that correlated with their capacity to block K(ATP) channels. P-gp ATPase activity was also increased by the openers SR47063 (a cromakalim analog), P1075 (a pinacidil analog), and diazoxide. Thus, these molecules bind to P-gp (although with lower affinities than for SUR) and are possibly transported by P-gp. Competition experiments among these molecules as well as with typical P-gp substrates revealed a structural similarity between drug binding domains in the two proteins. To rationalize the observed data, we addressed the molecular features of these proteins and compared structural models, computerized by homology from the recently solved structures of murine P-gp and bacterial ABC transporters MsbA and Sav1866. Considering the various residues experimentally assigned to be involved in drug binding, we uncovered several hot spots, which organized spatially in two main binding domains, selective for SR47063 and for glibenclamide, in matching regions of both P-gp and SUR.

Overexpression of the ABC Transporter BmrA Within Intracellular Caveolae in Escherichia coli.

We describe here the overproduction and oriented membrane insertion of membrane protein inside intracellular vesicles named heterologous caveolae within E. coli. The method is described with BmrA, a multidrug efflux pump from Bacillus subtilis. BmrA is produced in these vesicles, thanks to the coexpression with the canine caveolin-1ß, one of the two isoforms of caveolin-1. Enriched by sucrose gradient, the caveolae-containing fraction allows to probe the ATPase and Hoechst 33342 transport activities, the latter displaying a higher specific activity than the same without caveolin-1ß.

Caveolin-1ß promotes the production of active human microsomal glutathione S-transferase in induced intracellular vesicles inSpodoptera frugiperda21 insect cells.

The heterologous expression in Spodoptera frugiperda 21 (Sf21) insect cells of the ß isoform of canine caveolin-1 (caveolin-1ß), using a baculovirus-based vector, resulted in intracellular vesicles enriched in caveolin-1ß. We investigated whether these vesicles could act as membrane reservoirs, and promote the production of an active membrane protein (MP) when co-expressed with caveolin-1ß. We chose hMGST1 (human microsomal glutathione S-transferase 1) as the co-expressed MP. It belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral MPs, and, as a phase II detoxification enzyme, it catalyzes glutathione conjugation of lipophilic drugs present in the lipid membranes. In addition to its pharmaceutical interest, its GST activity can be conveniently measured. The expression of both MPs were followed by Western blots and membrane fractionation on density gradient, and their cell localization by immunolabeling and transmission electron microscopy. We showed that caveolin-1ß kept its capacity to induce intracellular vesicles in the host when co-expressed with hMGST1, and that hMGST1 is in part addressed to these vesicles. Remarkably, a fourfold increase in the amount of active hMGST1 was found in the most enriched membrane fraction, along with an increase of its specific activity by 60% when it was co-expressed with caveolin-1ß. Thus, heterologously expressed caveolin-1ß was able to induce cytoplasmic vesicles in which a co-expressed exogenous MP is diverted and sequestered, providing a favorable environment for this cargo.

Coronaviruses, cholesterol and statins: Involvement and application for Covid-19.

The infectious power of coronaviruses is dependent on cholesterol present in the membranes of their target cells. Indeed, the virus enters the infected cell either by fusion or by endocytosis, in both cases involving cholesterol-enriched membrane microdomains. These membrane domains can be disorganized in-vitro by various cholesterol-altering agents, including statins that inhibit cell cholesterol biosynthesis. As a consequence, numerous cell physiology processes, such as signaling cascades, can be compromised. Also, some examples of anti-bacterial and anti-viral effects of statins have been observed for infectious agents known to be cholesterol dependent. In-vivo, besides their widely-reported hypocholesterolemic effect, statins display various pleiotropic effects mediated, at least partially, by perturbation of membrane microdomains as a consequence of the alteration of endogenous cholesterol synthesis. It should thus be worth considering a high, but clinically well-tolerated, dose of statin to treat Covid-19 patients, in the early phase of infection, to inhibit virus entry into the target cells, in order to control the viral charge and hence avoid severe clinical complications. Based on its efficacy and favorable biodisposition, an option would be considering Atorvastatin, but randomized controlled clinical trials are required to test this hypothesis. This new therapeutic proposal takes benefit from being a drug repurposing, applied to a widely-used drug presenting a high efficiency-to-toxicity ratio. Additionally, this therapeutic strategy avoids any risk of drug resistance by viral mutation since it is host-targeted. Noteworthy, the same pharmacological approach could also be proposed to address different animal coronavirus endemic infections that are responsible for heavy economic losses.

Ectopic Neo-Formed Intracellular Membranes in Escherichia coli: A Response to Membrane Protein-Induced Stress Involving Membrane Curvature and Domains.

Bacterial cytoplasmic membrane stress induced by the overexpression of membrane proteins at high levels can lead to formation of ectopic intracellular membranes. In this review, we report the various observations of such membranes in Escherichia coli, compare their morphological and biochemical characterizations, and we analyze the underlying molecular processes leading to their formation. Actually, these membranes display either vesicular or tubular structures, are separated or connected to the cytoplasmic membrane, present mono- or polydispersed sizes and shapes, and possess ordered or disordered arrangements. Moreover, their composition differs from that of the cytoplasmic membrane, with high amounts of the overexpressed membrane protein and altered lipid-to-protein ratio and cardiolipin content. These data reveal the importance of membrane domains, based on local specific lipid⁻protein and protein⁻protein interactions, with both being crucial for local membrane curvature generation, and they highlight the strong influence of protein structure. Indeed, whether the cylindrically or spherically curvature-active proteins are actively curvogenic or passively curvophilic, the underlying molecular scenarios are different and can be correlated with the morphological features of the neo-formed internal membranes. Delineating these molecular mechanisms is highly desirable for a better understanding of protein⁻lipid interactions within membrane domains, and for optimization of high-level membrane protein production in E. coli.

Structural model, functional modulation by ivermectin and tissue localization of Haemonchus contortus P-glycoprotein-13.

Haemonchus contortus, one of the most economically important parasites of small ruminants, has become resistant to the anthelmintic ivermectin. Deciphering the role of P-glycoproteins in ivermectin resistance is desirable for understanding and overcoming this resistance. In the model nematode, Caenorhabditis elegans, P-glycoprotein-13 is expressed in the amphids, important neuronal structures for ivermectin activity. We have focused on its ortholog in the parasite, Hco-Pgp-13. A 3D model of Hco-Pgp-13, presenting an open inward-facing conformation, has been constructed by homology with the Cel-Pgp-1 crystal structure. In silico docking calculations predicted high affinity binding of ivermectin and actinomycin D to the inner chamber of the protein. Following in vitro expression, we showed that ivermectin and actinomycin D modulated Hco-Pgp-13 ATPase activity with high affinity. Finally, we found in vivo Hco-Pgp-13 localization in epithelial, pharyngeal and neuronal tissues. Taken together, these data suggest a role for Hco-Pgp-13 in ivermectin transport, which could contribute to anthelmintic resistance.

Interaction of macrocyclic lactones with P-glycoprotein: structure-affinity relationship.

P-glycoprotein (P-gp) is involved in the ATP-dependant cellular efflux of a large number of drugs including ivermectin, a macrocyclic lactone (ML) endectocide, widely used in livestock and human antiparasitic therapy. The interactions of P-gp with ivermectin and other MLs were studied. In a first approach, the ability of ivermectin (IVM), eprinomectin (EPR), abamectin (ABA), doramectin (DOR), selamectin (SEL), or moxidectin (MOX) to inhibit the rhodamine123 efflux was measured in recombinant cells overexpressing P-gp. Then, the influence of these compounds on the P-gp ATPase activity was tested on membrane vesicles prepared from fibroblasts overexpressing P-gp. All the MLs tested increased the intracellular rhodamine123. However, the potency of MOX to inhibit P-gp function was 10 times lower than the other MLs. They all inhibited the basal and decreased the verapamil-stimulated P-gp ATPase activity. But SEL and MOX were less potent than the other MLs when competing with verapamil. According to the structural specificity of SEL and MOX, we conclude that the integrity of the sugar moiety is determinant to achieve the optimal interaction of macrocyclic lactones with P-gp. The structure-affinity relationship for interaction with P-gp is important information for improving ML bioavailability and reversal of multidrug resistance (MDR).

Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

In vitro increase of the fluid-phase endocytosis induced by pulsed radiofrequency electromagnetic fields: importance of the electric field component.

Nowadays, due to the wide use of mobile phones, the possible biological effects of electromagnetic fields (EMF) become a public health general concern. Despite intensive research, there are no widely accepted theories about the interactions between EMFs and living cells, and the experimental data are often controversial. We examined the effects of mobile phones EMF (envelope frequency of 217 Hz, carrier frequency of 900 MHz and pulse duration of 580 micros) or its pure, low-frequency pulsed electric field component on fluid-phase endocytosis. In both cases, with exposures exceeding 10 min, an increase of the fluid-phase endocytosis rate was observed ( approximately 1.5-fold), on three different cell types. This increase is an all-or-nothing type of response that is occurring for threshold values comprised between 1.3 and 2.6 W/kg for the delivered EMF powers and between 1.1 and 1.5 V/cm for the electric fields intensities depending upon the cell type. The electric component of these EMFs is shown to be responsible for the observed increase. Variations of frequency or pulse duration of the electric pulses are shown to be without effect. Thus, EMF, via their electrical component, can perturb one of the most fundamental physiological functions of the cells-endocytosis.

Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3).

Ivermectin is a potent antiparasitic drug from macrocyclic lactone (ML) family, which interacts with the ABC multidrug transporter P-glycoprotein (Pgp). We studied the interactions of ivermectin with the multidrug resistance proteins (MRPs) by combining cellular and subcellular approaches. The inhibition by ivermectin of substrate transport was measured in A549 cells (calcein or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, BCECF) and in HL60-MRP1 (calcein). Ivermectin induced calcein and BCECF retention in A549 cells (IC(50) at 1 and 2.5microM, respectively) and inhibited calcein efflux in HL60-MRP1 (IC(50)=3.8microM). The action of ivermectin on the transporters ATPase activity was followed on membranes from Sf9 cells overexpressing human Pgp, MRP1, 2 or 3. Ivermectin inhibited the Pgp, MRP1, 2 and 3 ATPase activities after stimulation by their respective activators. Ivermectin showed a rather good affinity for MRPs, mainly MRP1, in the micromolar range, although it was lower than that for Pgp. The transport of BODIPY-ivermectin was followed in cells overexpressing selectively Pgp or MRP1. In both cell lines, inhibition of the transporter activity induced intracellular retention of BODIPY-ivermectin. Our data revealed the specific interaction of ivermectin with MRP proteins, and its transport by MRP1. Although Pgp has been considered until now as the sole active transporter for this drug, the MRPs should be taken into account for the transport of ivermectin across cell membrane, modulating its disposition in addition to Pgp. This could be of importance for optimizing clinical efficacy of ML-based antiparasitic treatments. This offers fair perspectives for the use of ivermectin or non-toxic derivatives as multidrug resistance-reversing agents.

Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.

In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1.

Macrocyclic lactones (ML) are important anthelmintics used in animals and humans against parasite nematodes, but their therapeutic success is compromised by the spread of ML resistance. Some ABC transporters, such as p-glycoproteins (Pgps), are selected and overexpressed in ML-resistant nematodes, supporting a role for some drug efflux proteins in ML resistance. However, the role of such proteins in ML transport remains to be clarified at the molecular level. Recently, Caenorhabditis elegans Pgp-1 (Cel-Pgp-1) has been crystallized, and its drug-modulated ATPase function characterized in vitro revealed Cel-Pgp-1 as a multidrug transporter. Using this crystal structure, we have developed an in silico drug docking model in order to study the binding of ML and other anthelmintic drugs to Cel-Pgp-1. All tested ML bound with high affinity in a unique site, within the inner chamber of the protein, supporting that ML may be transported by Cel-Pgp-1. Interestingly, interacting residues delineate a ML specific fingerprint involving H-bonds, including T1028. In particular, benzofurane and spiroketal moieties bound to specific sub-sites. When compared with the aglycone ML, such as moxidectin and ivermectin aglycone, avermectin anthelmintics have significant higher affinity for Cel-Pgp-1, likely due to the sugar substituent(s) that bind to a specific area involving H-bonds at Y771. Triclabendazole, closantel and emodepside bound with good affinities to different sub-sites in the inner chamber, partially overlapping with the ML binding site, suggesting that they could compete for Cel-Pgp-1-mediated ML transport. In conclusion, this work provides novel information on the role of nematode Pgps in transporting anthelmintics, and a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of clinically-relevant nematode Pgps, to improve anthelmintic therapeutics.

Specific cellular incorporation of a pyrene-labelled cholesterol: lipoprotein-mediated delivery toward ordered intracellular membranes.

In the aim of testing tools for tracing cell trafficking of exogenous cholesterol, two fluorescent derivatives of cholesterol, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), with distinctive chemico-physical characteristics, have been compared for their cell incorporation properties, using two cell models differently handling cholesterol, with two incorporation routes. In the Caco-2 cell model, the cholesterol probes were delivered in bile salt micelles, as a model of intestinal absorption. The two probes displayed contrasting behaviors for cell uptake characteristics, cell staining, and efflux kinetics. In particular, Pyr-met-Chol cell incorporation involved SR-BI, while that of NBD-Chol appeared purely passive. In the PC-3 cell model, which overexpresses lipoprotein receptors, the cholesterol probes were delivered via the serum components, as a model of systemic delivery. We showed that Pyr-met-Chol-labelled purified LDL or HDL were able to specifically deliver Pyr-met-Chol to the PC-3 cells, while NBD-Chol incorporation was independent of lipoproteins. Observations by fluorescence microscopy evidenced that, while NBD-Chol readily stained the cytosolic lipid droplets, Pyr-met-Chol labelling led to the intense staining of intracellular structures of membranous nature, in agreement with the absence of detectable esterification of Pyr-met-Chol. A 48 h incubation of PC-3 cells with either Pyr-met-Chol-labelled LDL or HDL gave same staining patterns, mainly colocalizing with Lamp1, caveolin-1 and CD63. These data indicated convergent trafficking downwards their respective receptors, LDL-R and SR-BI, toward the cholesterol-rich internal membrane compartments, late endosomes and multivesicular bodies. Interestingly, Pyr-met-Chol staining of these structures exhibited a high excimer fluorescence emission, revealing their ordered membrane environment, and indicating that Pyr-met-Chol behaves as a fair cholesterol tracer regarding its preferential incorporation into cholesterol-rich domains. We conclude that, while NBD-Chol is a valuable marker of cholesterol esterification, Pyr-met-Chol is a reliable new lipoprotein fluorescent marker which allows to probe specific intracellular trafficking of cholesterol-rich membranes.

ATP binding cassette multidrug transporters limit the anti-HIV activity of zidovudine and indinavir in infected human macrophages.

OBJECTIVES: To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro. DESIGN: We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes. METHODS: P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR. MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs. We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor. Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS. P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp. RESULTS: Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment. In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir. AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors. Indinavir (10 nM) gave 14% inhibition alone and 81% in combination. The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration. However, unlike PIs, neither AZT nor its metabolites interacted with P-gp. CONCLUSION: AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity. The cellular efflux of AZT probably involves MRP4 or MRP5. In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.

A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation.

The growing array of in vitro models of the blood-brain barrier (BBB) which have been used makes it difficult to draw firm conclusions concerning the BBB penetration of HIV-1 protease inhibitors. What is needed is a combined in vivo and in vitro study on biological models that mimic as closely as possible the normal human BBB, to establish whether and how indinavir crosses the BBB. We developed a new human BBB model using primary endothelial cells and astrocytes. The biological relevance of this model was checked with respect on the one hand, to the close relationship between the log of drug permeability coefficient normalized to molecular weight and the log of the 1-octanol/water partition coefficient, and on the other hand to the functional P-glycoprotein (P-gp) expression. We employed this model to perform transport studies with indinavir and showed that the rate of in vitro indinavir transport from the basal to apical compartment was higher than the rate of apical to basal transport. Pretreatment of the BBB model with the P-gp inhibitor, quinidine, significantly increased apical to basal transport. Intracellular indinavir accumulation was increased in BBB as a result of inhibition of active transport. These data were correlated with the indinavir-mediated P-gp ATPase modulation showing that indinavir specifically interacted with a binding site on P-gp. Moreover, the activation of P-gp ATPase by indinavir was inhibited by quinidine. In addition, the in vivo brain to plasma concentration ratio of indinavir into mice showed that indinavir concentration was up to five times higher in the brain of mdr1a(-/-) mice than in the brain of mdr1a(+/+) mice. All these results confirm the role of P-gp in preventing the passage of indinavir across BBB and thus its entry into the central nervous system (CNS). Our human BBB model represents a useful tool for the evaluation of drug penetration into the CNS.

Lipid rafts: dream or reality for cholesterol transporters?

As a key constituent of the cell membranes, cholesterol is an endogenous component of mammalian cells of primary importance, and is thus subjected to highly regulated homeostasis at the cellular level as well as at the level of the whole body. This regulation requires adapted mechanisms favoring the handling of cholesterol in aqueous compartments, as well as its transfer into or out of membranes, involving membrane proteins. A membrane exhibits functional properties largely depending on its lipid composition and on its structural organization, which very often involves cholesterol-rich microdomains. Then there is the appealing possibility that cholesterol may regulate its own transmembrane transport at a purely functional level, independently of any transcriptional regulation based on cholesterol-sensitive nuclear factors controling the expression level of lipid transport proteins. Indeed, the main cholesterol "transporters" presently believed to mediate for instance the intestinal absorption of cholesterol, that are SR-BI, NPC1L1, ABCA1, ABCG1, ABCG5/G8 and even P-glycoprotein, all present privileged functional relationships with membrane cholesterol-containing microdomains. In particular, they all more or less clearly induce membrane disorganization, supposed to facilitate cholesterol exchanges with the close aqueous medium. The actual lipid substrates handled by these transporters are not yet unambiguously determined, but they likely concern the components of membrane microdomains. Conversely, raft alterations may provide specific modulations of the transporter activities, as well as they can induce indirect effects via local perturbations of the membrane. Finally, these cholesterol transporters undergo regulated intracellular trafficking, with presumably some relationships to rafts which remain to be clarified.

Molecular dynamics simulations of E. coli MsbA transmembrane domain: formation of a semipore structure.

The human P-glycoprotein (MDR1/P-gp) is an ATP-binding cassette (ABC) transporter involved in cellular response to chemical stress and failures of anticancer chemotherapy. In the absence of a high-resolution structure for P-gp, we were interested in the closest P-gp homolog for which a crystal structure is available: the bacterial ABC transporter MsbA. Here we present the molecular dynamics simulations performed on the transmembrane domain of the open-state MsbA in a bilayer composed of palmitoyl oleoyl phosphatidylethanolamine lipids. The system studied contained more than 90,000 atoms and was simulated for 50 ns. This simulation shows that the open-state structure of MsbA can be stable in a membrane environment and provides invaluable insights into the structural relationships between the protein and its surrounding lipids. This study reveals the formation of a semipore-like structure stabilized by two key phospholipids which interact with the hinge region of the protein during the entire simulation. Multiple sequence alignments of ABC transporters reveal that one of the residues involved in the interaction with these two phospholipids are under a strong selection pressure specifically applied on the bacterial homologs of MsbA. Hence, comparison of molecular dynamics simulation and phylogenetic data appears as a powerful approach to investigate the functional relevance of molecular events occurring during simulations.