Small-Angle Neutron Scattering Reveals the Nanostructure of Liposomes with Embedded OprF Porins of Pseudomonas aeruginosa.

The use of liposomes as drug delivery systems emerged in the last decades in view of their capacity and versatility to deliver a variety of therapeutic agents. By means of small-angle neutron scattering (SANS), we performed a detailed characterization of liposomes containing outer membrane protein F (OprF), the main porin of the Pseudomonas aeruginosa bacterium outer membrane. These OprF-liposomes are the basis of a novel vaccine against this antibiotic-resistant bacterium, which is one of the main hospital-acquired pathogens and causes each year a significant number of deaths. SANS data were analyzed by a specific model we created to quantify the crucial information about the structure of the liposome containing OprF, including the lipid bilayer structure, the amount of protein in the lipid bilayer, the average protein localization, and the effect of the protein incorporation on the lipid bilayer. Quantification of such structural information is important to enhance the design of liposomal delivery systems for therapeutic applications.

Cell-free expression of the outer membrane protein OprF of Pseudomonas aeruginosa for vaccine purposes.

Pseudomonas aeruginosa is the second-leading cause of nosocomial infections and pneumonia in hospitals. Because of its extraordinary capacity for developing resistance to antibiotics, treating infections by Pseudomonas is becoming a challenge, lengthening hospital stays, and increasing medical costs and mortality. The outer membrane protein OprF is a well-conserved and immunogenic porin playing an important role in quorum sensing and in biofilm formation. Here, we used a bacterial cell-free expression system to reconstitute OprF under its native forms in liposomes and we demonstrated that the resulting OprF proteoliposomes can be used as a fully functional recombinant vaccine against P. aeruginosa Remarkably, we showed that our system promotes the folding of OprF into its active open oligomerized state as well as the formation of mega-pores. Our approach thus represents an easy and efficient way for producing bacterial membrane antigens exposing native epitopes for vaccine purposes.

A PANI supported lipid bilayer that contains NhaA transporter proteins provides a basis for a biomimetic biocapacitor.

We designed a supported lipid bilayer (SLB) biomimetic membrane system that comprised polyaniline (PANI) to support a lipid bilayer membrane that incorporated Na+/H+ transporter proteins (NhaA) to give the system the capability of controllable electrogenic ion transport. The high turnover rate of NhaA (∼105 per min) provides the basis for this PANI-SLB-NhaA system to be a high-speed rechargeable biocapacitor that functions as a low-energy-consuming fast switch for biological engineering applications.

Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers.

OprF has a central role in Pseudomonas aeruginosa virulence and thus provides a putative target for either vaccines or antibiotic cofactors that could overcome the bacterium's natural resistance to antibiotics. Here we describe a procedure to optimize the production of highly pure and functional OprF porins that are then incorporated into a tethered lipid bilayer. This is a stable biomimetic system that provides the capability to investigate structural aspects and function of OprF using and neutron reflectometry and electrical impedance spectroscopy. The recombinant OprF produced using the optimized cell-free procedure yielded a quantity of between 0.5 to 1.0 mg/mL with a purity ranging from 85 to 91% in the proteoliposomes. The recombinant OprF is capable of binding IFN-γ and is correctly folded in the proteoliposomes. Because OprF proteins form pores the biomimetic system allowed the measurement of OprF conductance using impedance spectroscopy. The neutron reflectometry measurements showed that the OprF protein is incorporated into the lipid bilayer but with parts of the protein in both the regions above and below the lipid bilayer. Those structural aspects are coherent with the current assumed structure of a transmembrane N-terminal domain composed by eight stranded beta-barrels and a globular C-terminal domain located in the periplasm. Currently there are no crystal structures available for OprF. The experimental model system that we describe provides a basis for further fundamental studies of OprF and particularly for the ongoing biotechnological development of OprF as a target for antibacterial drugs.

[Biomimetic sensors in biomedical research]. / Les capteurs biomimétiques en recherche biomédicale.

The recent research on both the synthesis of membrane proteins by cell-free systems and the reconstruction of planar lipid membranes, has led to the development of a cross-technology to produce biosensors or filters. Numerous biomimetic membranes are currently being standardized and used by the industry, such as filters containing aquaporin for water desalination, or used in routine at the laboratory scale, for example the bacteriorhodopsin as a light sensor. In the medical area, several fields of application of these biomimetic membranes are under consideration today, particularly for the screening of therapeutic molecules and for the developing of new tools in diagnosis, patient monitoring and personalized medicine.

A common highly conserved cadmium detoxification mechanism from bacteria to humans: heavy metal tolerance conferred by the ATP-binding cassette (ABC) transporter SpHMT1 requires glutathione but not metal-chelating phytochelatin peptides.

Cadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers' yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the co-substrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO(3), As(III), As(V), CuSO(4), or HgCl(2). Abolishment of the catalytic activity by expression of SpHMT1(K623M) mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHMA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans.

Transport of antimony salts by Arabidopsis thaliana protoplasts over-expressing the human multidrug resistance-associated protein 1 (MRP1/ABCC1).

ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of "safe-food" strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.

Characterization of the typical multidrug resistance profile in human uveal melanoma cell lines and in mouse liver metastasis derivatives.

Uveal melanoma is the most common intraocular malignancy. To study its biology, stable cell lines provide a useful tool, but these are very difficult to obtain. A stable and rapidly growing human choroidal melanoma cell line composed of pure epithelioid cells was established and maintained for at least 4 years. In vivo transplantation into BALB/cByJ nude mice induced vascularized tumours at the injection sites. Interestingly, two of three cases produced a liver metastasis. Other uveal melanoma cell lines displaying different morphological aspects were also obtained. To avoid the bias due to uncertain immunologically based staining approaches, several methods were juxtaposed to establish the multidrug resistance (MDR) profile. All the uveal melanomas studied expressed significant levels of the MDR-related MDR1, MRP1 (MDR-related protein 1) and LRP/MVP (lung resistance protein/major vault protein) messenger RNAs (mRNAs), produced their corresponding proteins and were able to functionally extrude daunomycin. When compared with the established MEWO skin melanoma cell line, our data showed that both primary and metastatic uveal melanomas intrinsically expressed the typical MDR phenotype, which precludes the use of any anticancer drugs known to be substrates of MDR-related proteins to treat the disease. Moreover, it appears that the metastasizing process does not change the status of the MDR phenotype.

Major cytogenetic aberrations and typical multidrug resistance phenotype of uveal melanoma: current views and new therapeutic prospects.

Uveal melanoma is the most frequent intra-ocular cancer. The recent development of new chromosome-related technologies have permitted the elucidation of both the cytogenetics and the natural history of this disease. Fifty to 60% of uveal melanomas are linked to a monosomy 3, which appears as an early and determinant event in tumor progression. Tumors with this anomaly have a very poor prognosis. Recent work suggests that this category of uveal melanoma represents a distinct pathologic entity from that associated with normal disomy 3. Chromosome 6 aberrations probably constitute a second entry point into the process of cancerogenesis, while gains in 8q seem to appear later in the natural history of uveal melanomas due to their higher frequency in larger tumors. Other anomalies will be reviewed. In spite of significant improvements in the local treatment of uveal melanoma, many patients die due to tumor metastasis. This disease is characterized by a constitutive chemoresistance whose typical multidrug resistance phenotype (MDR) is particularly complex since different combinations of several resistance proteins are simultaneously produced. Regulation of the expression of these proteins is a research priority, increasingly so as gene therapy-dependent chemosensitization strategies expand. Therefore, the development and improvement of methods to determine the chemoresistance profile become a crucial objective today in the therapeutic strategies against uveal melanoma.

Control of P-glycoprotein activity by membrane cholesterol amounts and their relation to multidrug resistance in human CEM leukemia cells.

P-glycoprotein (P-gp) is the most well-known ATP-binding cassette (ABC) transporter involved in unidirectional substrate translocation across the membrane lipid bilayer, thereby causing the typical multidrug resistance (MDR) phenotype expressed in many cancers. We observed that in human CEM acute lymphoblastic leukemia cells expressing various degrees of chemoresistance and where P-gp was the sole MDR-related ABC transporter detected, the amount of esterified cholesterol increased linearly with the level of resistance to vinblastine while the amounts of total and free cholesterol increased in a nonlinear way. Membrane cholesterol controlled the ATPase activity of P-gp in a linear manner, whereas the P-gp-induced daunomycin efflux decreased nonlinearly with the depletion of membrane cholesterol. All these elements suggest that cholesterol controls both the ATPase and the drug efflux activities of P-gp. In addition, in CEM cell lines that expressed increasing levels of elevated chemoresistance, the amount of P-gp increases to a plateau value of 40% of the total membrane proteins and remained unvaried while the amount of membrane cholesterol increased with the elevation of the MDR level, strongly suggesting that cholesterol may be directly involved in the typical MDR phenotype. Finally, we showed that the decreased daunomycin efflux by P-gp due to the partial depletion of membrane cholesterol was responsible for the efficient chemosensitization of resistant CEM cells, which could be totally reversed after cholesterol repletion.

Multidrug-resistant cancer cells contain two populations of P-glycoprotein with differently stimulated P-gp ATPase activities: evidence from atomic force microscopy and biochemical analysis.

Considerable interest exists about the localization of P-gp (P-glycoprotein) in DRMs (detergent-resistant membranes) of multidrug resistant cancer cells, in particular concerning the potential modulating role of the closely related lipids and proteins on P-gp activity. Our observation of the opposite effect of verapamil on P-gp ATPase activity from DRM and solubilized-membrane fractions of CEM-resistant leukaemia cells, and results from Langmuir experiments on membrane monolayers from resistant CEM cells, strongly suggest that two functional populations of P-gp exist. The first is located in DRM regions: it displays its optimal P-gp ATPase activity, which is almost completely inhibited by orthovanadate and activated by verapamil. The second is located elsewhere in the membrane; it displays a lower P-gp ATPase activity that is less sensitive to orthovanadate and is inhibited by verapamil. A 40% cholesterol depletion of DRM caused the loss of 52% of the P-gp ATPase activity. Cholesterol repletion allowed recovery of the initial P-gp ATPase activity. In contrast, in the solubilized-membrane-containing fractions, cholesterol depletion and repletion had no effect on the P-gp ATPase activity whereas up to 100% saturation with cholesterol induced a 58% increased P-gp ATPase activity, while no significant modification was observed for the DRM-enriched fraction. DRMs were analysed by atomic force microscopy: 40-60% cholesterol depletion was necessary to remove P-gp from DRMs. In conclusion, P-gp in DRMs appears to contain closely surrounding cholesterol that can stimulate P-gp ATPase activity to its optimal value, whereas cholesterol in the second population seems deprived of this function.

New invMED1 element cis-activates human multidrug-related MDR1 and MVP genes, involving the LRP130 protein.

The MDR1 gene is a key component of the cytotoxic defense network and its overexpression results in the multidrug resistance (MDR) phenotype. However, the molecular mechanisms that regulate the MDR1 gene and coordinate multiple MDR-related genes expression are poorly understood. In a previous study, we identified a new 12 bp cis-activating region in the 5'-flanking region of the human MDR1 gene, which we called inverted MED1. In the present study, we characterized the precise binding element, which we named invMED1, and revealed the presence of the LRP130 protein as the nuclear factor. Its binding intensity increases with the endogenous MDR1 geneexpression and with the MDR level of CEM leukemia cells. Interestingly, the LRP130 level did not vary with the chemoresistance level. We observed the involvement of LRP130 in the transcriptional activity of the MDR1 gene promoter, and moreover, in that of the MDR-related, invMED1-containing, MVP gene promoter. We used siRNAs and transcriptional decoys in two unrelated human cancer cell lines to show the role of the invMED1/LRP130 couple in both MDR1 and MVP endogenous genes activities. We showed that invMED1 was localized in the -105/-100 and -148/-143 regions of the MDR1 and MVP gene promoters, respectively. In addition, since the invMED1 sequence is primarily located in the -160/-100 bp region of mammalian MDR-related genes, our results present the invMED1/LRP130 couple as a potential central regulator of the transcription of these genes.

Transcriptional regulation of the human MDR1 gene at the level of the inverted MED-1 promoter region.

The typical multidrug resistance phenotype (MDR), the major cause of failure of cancer chemotherapy, is the result of the overexpression of the human MDR1 gene, the regulation of which is still incompletely understood. Using several EMSA experiments, we have identified a new regulatory sequence located from -103 to -98 bp relative to the +1 start site in the MDR1 promoter region. This sequence, which we called inverted MED-1, acts as a cis-activator for this gene. In transient transfection experiments of highly resistant human lymphoblastic CEM/VLB5 cells, its deletion from the promoter region is responsible for 60% inhibition of the MDR1 transcriptional activity. This sequence specifically binds a nuclear protein of about 150-160 kDa. We showed that its binding capacity is related to the chemoresistance level of the studied cell lines and may reflect the increased transcriptional activity of the MDR1 gene in multidrug-resistant cells.

Transcriptional regulators of the human multidrug resistance 1 gene: recent views.

The multidrug resistance (MDR) phenotype is the major cause of failure of cancer chemotherapy. This phenotype is mainly due to the overexpression of the human MDR1 (hMDR1) gene. Several studies have shown that transcriptional regulation of this gene is unexpectedly complex and is far from being completely understood. Current work is aimed mainly at defining unclear and new control regions in the hMDR1 gene promoter as well as clarifying corresponding signaling pathways. Such studies provide new insights into the mechanisms by which xenobiotic molecules might modify the physiological hMDR1 expression as well as the possible role of oncogenes in the pathological dysregulation of the gene. Here we report recent findings on the regulation of hMDR1 which may help define specific targets aimed at modulating its transcription.