Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species.

Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.

The non-Newtonian behavior of detergents during concentration is increased by macromolecules, in trans, and results in their over-concentration.

Concentration of pure membrane proteins in detergent solution results in detergent concentration, albeit in unknown amounts. This phenomenon is observed in every lab working on membrane proteins, but has seldom been investigated. In this study, we explored the behavior of detergents mixed with membrane proteins during the step of sample concentration using centrifugal devices. We show that detergent over-concentrate with the presence of polymers, typically membrane or soluble proteins but also polysaccharides. The over-concentration of detergents depends on centrifugal force applied to the device. With the use of a specific dye, we observed the formation of a mesh on the concentrator device. Importantly, reducing the centrifugal speed allows to reduce the concentration of detergents when mixed to macromolecules, as tested with 3 different membrane proteins. All together, these results highlight the non-Newtonian behavior of detergents and provides a solid framework to investigators to improve drastically biochemical and structural studies of membrane proteins.

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.

Production and Purification of a GFP-Tagged ABC Transporter CaCdr1p.

The production and purification are the first steps required in any functional or structural study of a protein of interest. In the case of membrane proteins, these tasks can be difficult due to low expression levels and the necessity to extract them from their membrane environment. This chapter describes a convenient method based on GFP tagged to the membrane protein to facilitates these steps. Production is carried out in the yeast S. cerevisiae and purification steps are carried out and monitored taking advantage of an anti-GFP nanobody. We show how GFP can be a very helpful tool for controlling the correct addressing of the protein and for probing and optimizing purification. These methods are described here for producing and purifying CaCdr1p, an ABC exporter conferring multiantifungal resistance to C. albicans. This purification method can be amenable to any other GFP-tagged protein.

Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter.

Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg2+-bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in B. subtilis. Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5'-6' of the other. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.

Glycosyl-Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins.

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl-substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane-cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b, 8 c, and 9 b preserved the ATPase function of BmrA, an ATP-binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a, 8 b, 8 f, 9 a, and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G-protein-coupled adenosine receptor A2A R. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.

Quantification of Detergents Complexed with Membrane Proteins.

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.

Collateral sensitivity of resistant MRP1-overexpressing cells to flavonoids and derivatives through GSH efflux.

The multidrug resistance protein 1 (MRP1) is involved in multidrug resistance of cancer cells by mediating drug efflux out of cells, often in co-transport with glutathione (GSH). GSH efflux mediated by MRP1 can be stimulated by verapamil. In cells overexpressing MRP1, we have previously shown that verapamil induced a huge intracellular GSH depletion which triggered apoptosis of the cells. That phenomenon takes place in the more global anticancer strategy called "collateral sensitivity" and could be exploited to eradicate some chemoresistant cancer cells. Seeking alternative compounds to verapamil, we screened a library of natural flavonoids and synthetic derivatives. A large number of these compounds stimulate MRP1-mediated GSH efflux and the most active ones have been evaluated for their cytotoxic effect on MRP1-overexpressing cells versus parental cells. Interestingly, some are highly and selectively cytotoxic for MRP1-cells, leading them to apoptosis. However, some others do not exhibit any cytotoxicity while promoting a strong GSH efflux, indicating that GSH efflux is necessary but not sufficient for MRP1-cells apoptosis. In support to this hypothesis, structure activity relationships show that the absence of a hydroxyl group at position 3 of the flavonoid C ring is an absolute requirement for induction of MRP1-cells death, but is not for GSH efflux stimulation. Chrysin (compound 8) and its derivatives, compounds 11 and 22, exhibit a high selectivity toward MRP1-cells with a IC50 value of 4.1 µM for compound 11 and 4.9 µM for chrysin and compound 22, making them among the best described selective killer compounds of multidrug ABC transporter-overexpressing cells.

Identification of xanthones as selective killers of cancer cells overexpressing the ABC transporter MRP1.

Multidrug-resistance protein 1 (MRP1) belongs to the ATP-binding cassette (ABC) transporter family. MRP1 mediates MDR (multidrug resistance) by causing drug efflux either by conjugation to glutathione (GSH) or by co-transport with free GSH (without covalent bonding between the drug and GSH). We recently reported that the calcium channel blocker verapamil can activate massive GSH efflux in MRP1-overexpressing cells, leading to cell death through apoptosis. However, clinical use of verapamil is hampered by its cardiotoxicity. Then, in the search for compounds that act similarly to verapamil, but without major side effects, we investigated xanthones. Herein we show that xanthones induce apoptosis among resistant cells overexpressing MRP1 similarly to the verapamil effect. Among the xanthones studied, 1,3-dihydroxy-6-methoxyxanthone was identified as the most active derivative, able to specifically kill cells transfected with human MRP1 with even greater potency than verapamil. Under the same conditions, the active xanthones have no toxic effect on control (sensitive) cells. Xanthones could therefore be considered as new potential anticancer agents for the selective treatment of MRP1-positive tumors.

Leucine-rich protein 130 contributes to apoptosis resistance of human hepatocarcinoma cells.

LRP130 is a ubiquitous protein involved in cellular homeostasis, microtubule alteration, and transactivation of a few multidrug resistance genes. Its role in resistance to apoptosis in HepG2 and HUH7 hepatocarcinoma cells was investigated. Using shRNA-producing lentiviruses to down-regulate the LRP130 gene, we showed that i) LRP130 did not affect the capacity of hepatocarcinoma cells to extrude drugs since LRP130 down-regulation was insufficient to significantly reduce P-glycoprotein production in these cells, and ii) the expression of 11 apoptosis-related genes measured by PCR-array was significantly reduced. Interestingly, six of these genes encode extrinsic pathway proapoptotic proteins whose expression was higher in LRP130-non producing than in LRP130-producing HepG2 cells. Fluorescence microscopy confirmed this new anti-apoptotic role of LRP130, which is strengthened by a significantly reduced cytochrome c oxidase activity in LRP130-down-regulated hepatocarcinoma cells.