Kinetic study of membrane protein interactions: from three to two dimensions.

Molecular interactions are contingent upon the system's dimensionality. Notably, comprehending the impact of dimensionality on protein-protein interactions holds paramount importance in foreseeing protein behaviour across diverse scenarios, encompassing both solution and membrane environments. Here, we unravel interactions among membrane proteins across various dimensionalities by quantifying their binding rates through fluorescence recovery experiments. Our findings are presented through the examination of two protein systems: streptavidin-biotin and a protein complex constituting a bacterial efflux pump. We present here an original approach for gauging a two-dimensional binding constant between membrane proteins embedded in two opposite membranes. The quotient of protein binding rates in solution and on the membrane represents a metric denoting the exploration distance of the interacting sites-a novel interpretation.

Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry.

The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol. Remarkably, whereas glycosylation occurs in the ER lumen, the initial steps of Glc3Man9GlcNAc2-PP-dolichol synthesis generate the lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic side of the ER. Glycolipid assembly is completed only after M5-DLO is translocated to the luminal side. The membrane protein (M5-DLO scramblase) that mediates M5-DLO translocation across the ER membrane has not been identified, despite its importance for N-glycosylation. Building on our ability to recapitulate scramblase activity in proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based 'activity correlation profiling' approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Data curation prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins is necessary for M5-DLO scramblase activity. Our results instead strongly suggest that M5-DLO scramblase activity is due to a protein, or protein complex, whose activity is regulated at the level of quaternary structure.

Transcriptomic insight into salinomycin mechanisms in breast cancer cell lines: synergistic effects with dasatinib and induction of estrogen receptor ß.

BACKGROUND: Tumors are heterogeneous in nature, composed of different cell populations with various mutations and/or phenotypes. Using a single drug to encounter cancer progression is generally ineffective. To improve the treatment outcome, multiple drugs of distinctive mechanisms but complementary anticancer activities (combination therapy) are often used to enhance antitumor efficacy and minimize the risk of acquiring drug resistance. We report here the synergistic effects of salinomycin (a polyether antibiotic) and dasatinib (a Src kinase inhibitor). METHODS: Functionally, both drugs induce cell cycle arrest, intracellular reactive oxygen species (iROS) production, and apoptosis. We rationalized that an overlapping of the drug activities should offer an enhanced anticancer effect, either through vertical inhibition of the Src-STAT3 axis or horizontal suppression of multiple pathways. We determined the toxicity induced by the drug combination and studied the kinetics of iROS production by fluorescence imaging and flow cytometry. Using genomic and proteomic techniques, including RNA-sequencing (RNA-seq), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western Blot, we subsequently identified the responsible pathways that contributed to the synergistic effects of the drug combination. RESULTS: Compared to either drug alone, the drug combination showed enhanced potency against MDA-MB-468, MDA-MB-231, and MCF-7 human breast cancer (BC) cell lines and tumor spheroids. The drug combination induces both iROS generation and apoptosis in a time-dependent manner, following a 2-step kinetic profile. RNA-seq data revealed that the drug combination exhibited synergism through horizontal suppression of multiple pathways, possibly through a promotion of cell cycle arrest at the G1/S phase via the estrogen-mediated S-phase entry pathway, and partially via the BRCA1 and DNA damage response pathway. CONCLUSION: Transcriptomic analyses revealed for the first time, that the estrogen-mediated S-phase entry pathway partially contributed to the synergistic effect of the drug combination. More importantly, our studies led to the discoveries of new potential therapeutic targets, such as E2F2, as well as a novel drug-induced targeting of estrogen receptor ß (ESR2) approach for triple-negative breast cancer treatment, currently lacking of targeted therapies.

Biochemical Reconstitution and Characterization of Multicomponent Drug Efflux Transporters.

Efflux pumps are the major determinants in bacterial multidrug resistance. In Gram-negative bacteria, efflux transporters are organized as macromolecular tripartite machineries that span the two-membrane cell envelope of the bacterium. Biochemical data on purified proteins are essential to draw a mechanistic picture of this highly dynamical, multicomponent, efflux system. We describe protocols for the reconstitution and the in vitro study of transporters belonging to RND and ABC superfamilies: the AcrAB-TolC and MacAB-TolC efflux systems from Escherichia coli and the MexAB-OprM efflux pump from Pseudomonas aeruginosa.

Light-independent phospholipid scramblase activity of bacteriorhodopsin from Halobacterium salinarum.

The retinylidene protein bacteriorhodopsin (BR) is a heptahelical light-dependent proton pump found in the purple membrane of the archaeon Halobacterium salinarum. We now show that when reconstituted into large unilamellar vesicles, purified BR trimers exhibit light-independent lipid scramblase activity, thereby facilitating transbilayer exchange of phospholipids between the leaflets of the vesicle membrane at a rate >10,000 per trimer per second. This activity is comparable to that of recently described scramblases including bovine rhodopsin and fungal TMEM16 proteins. Specificity tests reveal that BR scrambles fluorescent analogues of common phospholipids but does not transport a glycosylated diphosphate isoprenoid lipid. In silico analyses suggest that membrane-exposed polar residues in transmembrane helices 1 and 2 of BR may provide the molecular basis for lipid translocation by coordinating the polar head-groups of transiting phospholipids. Consistent with this possibility, extensive coarse-grained molecular dynamics simulations of a BR trimer in an explicit phospholipid membrane revealed water penetration along transmembrane helix 1 with the cooperation of a polar residue (Y147 in transmembrane helix 5) in the adjacent protomer. These results suggest that the lipid translocation pathway may lie at or near the interface of the protomers of a BR trimer.

Reconstitution of Membrane Proteins in Liposomes.

Membrane protein reconstitution in liposomes is an invaluable technique to study numerous properties of membrane proteins in vitro. Kinetics, substrate specificity, and protein-protein interaction of membrane proteins can be investigated once they are embedded in the liposome bilayer. Both protocols described here aim to investigate the efflux pump MexA-MexB-OprM from Pseudomonas aeruginosa, a proteins complex embedded in both membranes of this Gram-negative bacteria. This tri-partite system, which by-passes the periplams, is involved in antibiotic resistance. First, we describe a protocol to study MexB, the actual transporter, and second we propose an alternate protocol where the tripartite system is investigated as a whole.

Tripartite assembly of RND multidrug efflux pumps.

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa.

Efflux pumps are membrane transporters that actively extrude various substrates, leading to multidrug resistance (MDR). In this study, we have designed a new test that allows investigating the assembly of the MexA-MexB-OprM efflux pump from the Gram negative bacteria Pseudomonas aeruginosa. The method relies on the streptavidin-mediated pull-down of OprM proteoliposomes upon interaction with MexAB proteoliposomes containing a biotin function carried by lipids. We give clear evidence for the importance of MexA in promoting and stabilizing the assembly of the MexAB-OprM complex. In addition, we have investigated the effect of the role of the lipid anchor of MexA as well as the role of the proton motive force on the assembly and disassembly of the efflux pump. The assay presented here allows for an accurate investigation of the assembly with only tens of microgram of protein and could be adapted to 96 wells plates. Hence, this work provides a basis for the medium-high screening of efflux pump inhibitors (EPIs).

In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa.

Antibiotic resistance is a major public health issue and many bacteria responsible for human infections have now developed a variety of antibiotic resistance mechanisms. For instance, Pseudomonas aeruginosa, a disease-causing Gram-negative bacteria, is now resistant to almost every class of antibiotics. Much of this resistance is attributable to multidrug efflux pumps, which are tripartite membrane protein complexes that span both membranes and actively expel antibiotics. Here we report an in vitro procedure to monitor transport by the tripartite MexAB-OprM pump. By combining proteoliposomes containing the MexAB and OprM portions of the complex, we are able to assay energy-dependent substrate translocation in a system that mimics the dual-membrane architecture of Gram-negative bacteria. This assay facilitates the study of pump transport dynamics and could be used to screen pump inhibitors with potential clinical use in restoring therapeutic activity of old antibiotics.

In vitro investigation of the MexAB efflux pump from Pseudomonas aeruginosa.

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

Hoechst likes to play hide and seek...use it with caution!

We describe an unexpected feature of the dye Hoechst 33342 that may lead to misinterpretation of fluorescence assay results. When dissolved in aqueous solvent, Hoechst 33342 massively adsorbs on the polytetrafluoroethylene (PTFE)-coated stirrer and on the quartz cuvette. The interaction between the dye and the PTFE is stronger than that between the dye and the quartz. We show that Hoechst 33342 adsorption on quartz is due to its amphiphilic properties, as it depends on the buffer ionic strength and on pH. We suggest a procedure to detect and remove any residual Hoechst 33342.

Photo-induced proton gradients for the in vitro investigation of bacterial efflux pumps.

We describe an original activity assay for membrane transport that uses the proton motive force-dependent efflux pump MexAB from Pseudomonas aeruginosa. This pump is co-reconstituted into proteoliposomes together with bacteriorhodopsin (BR), a light-activated proton pump. In this system, upon illumination with visible light, the photo-induced proton gradient created by the BR is shown to be coupled to the active transport of substrates through the pump.