BacA: a possible regulator that contributes to the biofilm formation of .

Previously, we pointed out in PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes ( to ) that we named for "Biofilm Associated Cluster." The present study focuses on the PA14_16140 protein, i.

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.

Starting with an Integral Membrane Protein Project for Structural Biology: Production, Purification, Detergent Quantification, and Buffer Optimization-Case Study of the Exporter CntI from Pseudomonas aeruginosa.

Production, extraction, purification, and stabilization of integral membrane proteins are key steps for successful structural biology studies, in particular for X-ray crystallography or single particle microscopy. Here, we present the purification protocol of CntI from Pseudomonas aeruginosa, a new metallophore exporter of the Drug Metabolite Transporter (DMT) family involved in pseudopaline secretion. Subsequent to CntI purification, we optimized the buffer pH, salts, and additives by differential scanning fluorimetry (DSF), also known as Thermofluor Assay (TFA) or fluorescent thermal stability assay (FTSA), with the use of dye 1-AnilinoNaphthalene-8-Sulfonic acid (ANS), a fluorescent molecule compatible with detergents.

The Structural and Functional Study of Efflux Pumps Belonging to the RND Transporters Family from Gram-Negative Bacteria.

Antimicrobial-resistant bacterial infections are a major and costly public health concern [...

Molecular Determinants for OMF Selectivity in Tripartite RND Multidrug Efflux Systems.

Tripartite multidrug RND efflux systems made of an inner membrane transporter, an outer membrane factor (OMF) and a periplasmic adaptor protein (PAP) form a canal to expel drugs across Gram-negative cell wall. Structures of MexA-MexB-OprM and AcrA-AcrB-TolC, from and , respectively, depict a reduced interfacial contact between OMF and PAP, making unclear the comprehension of how OMF is recruited. Here, we show that a Q93R mutation of MexA located in the α-hairpin domain increases antibiotic resistance in the MexA-MexB-OprM-expressed strain.

How to best estimate the viscosity of lipid bilayers.

The viscosity of lipid bilayers is a property relevant to biological function, as it affects the diffusion of membrane macromolecules. To determine its value, and hence portray the membrane, various literature-reported techniques lead to significantly different results. Herein we compare the results issuing from two widely used techniques to determine the viscosity of membranes: the Fluorescence Lifetime Imaging Microscopy (FLIM), and Fluorescence Recovery After Photobleaching (FRAP).

Phosphorylation of Extracellular Proteins in in Sessile Mode of Growth.

is a problematic nosocomial pathogen owing to its increasing resistance to antibiotics and its great ability to survive in the hospital environment, which is linked to its capacity to form biofilms. Structural and functional investigations of post-translational modifications, such as phosphorylations, may lead to identification of candidates for therapeutic targets against this pathogen. Here, we present the first S/T/Y phosphosecretome of two strains, the reference strain ATCC 17978 and the virulent multi-drug resistant strain AB0057, cultured in two modes of growth (planktonic and biofilm) using TiO chromatography followed by high resolution mass spectrometry.

MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of and .

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e.

Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex.

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form.

Studying dynamics without explicit dynamics: A structure-based study of the export mechanism by AcrB.

Resistance-nodulation-cell division family proteins are transmembrane proteins identified as large spectrum drug transporters involved in multidrug resistance. A prototypical case in this superfamily, responsible for antibiotic resistance in selected gram-negative bacteria, is AcrB. AcrB forms a trimer using the proton motive force to efflux drugs, implementing a functional rotation mechanism.

The Peptide ERα17p Is a GPER Inverse Agonist that Exerts Antiproliferative Effects in Breast Cancer Cells.

The inhibition of the G protein-coupled estrogen receptor (GPER) offers promising perspectives for the treatment of breast tumors. A peptide corresponding to part of the hinge region/AF2 domain of the human estrogen receptor α (ERα17p, residues 295-311) exerts anti-proliferative effects in various breast cancer cells including those used as triple negative breast cancer (TNBC) models. As preliminary investigations have evoked a role for the GPER in the mechanism of action of this peptide, we focused our studies on this protein using SkBr3 breast cancer cells, which are ideal for GPER evaluation.

VEGF (Vascular Endothelial Growth Factor) Functionalized Magnetic Beads in a Microfluidic Device to Improve the Angiogenic Balance in Preeclampsia.

Preeclampsia is a hypertensive pregnancy disease associated with a massive increase in sFlt-1 (soluble form of the vascular endothelial growth factor 1) in the maternal circulation, responsible for angiogenic imbalance and endothelial dysfunction. Pilot studies suggest that extracorporeal apheresis may reduce circulating sFlt-1 and prolong pregnancy. Nonspecific apheresis systems have potential adverse effects because of the capture of many other molecules.

Minimal nanodisc without exogenous lipids for stabilizing membrane proteins in detergent-free buffer.

Membrane protein stabilization after detergent solubilization presents drawbacks for structural and biophysical studies, in particular that of a reduced stability in detergent micelles. Therefore, alternative methods are required for efficient stabilization. Lipid nanodisc made with the membrane scaffold protein MSP is a valuable system but requires a fine optimization of the lipid to protein ratio.

From Vascular Smooth Muscle Cells to Folliculogenesis: What About Vasorin?

First described in 1988, vasorin (VASN) is a transmembrane glycoprotein expressed during early mouse development, and with a less extent, in various organs and tissues (e.g., kidney, aorta, and brain) postnatally.

LC-MS/MS-based quantification of efflux transporter proteins at the BBB.

Targeted protein quantification using tandem mass spectrometry coupled to high performance chromatography (LC-MS/MS) has been used to quantify proteins involved in the absorption, distribution, metabolism and excretion (ADME) of xenobiotics to better understand these processes. At the blood-brain barrier (BBB), these proteins are particularly important for the maintenance of brain homeostasis, but also regulate the distribution of therapeutic drugs. Absolute quantification (AQUA) is achieved by using stable isotope labeled surrogate peptides specific to the target protein and analyzing the digested proteins in a triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode to achieve a high specificity, sensitivity, accuracy and reproducibility.

Functional Mechanism of the Efflux Pumps Transcription Regulators From Based on 3D Structures.

Bacterial antibiotic resistance is a worldwide health problem that deserves important research attention in order to develop new therapeutic strategies. Recently, the World Health Organization (WHO) classified as one of the priority bacteria for which new antibiotics are urgently needed. In this opportunistic pathogen, antibiotics efflux is one of the most prevalent mechanisms where the drug is efficiently expulsed through the cell-wall.

Lysine Succinylation and Acetylation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a multi-drug-resistant human opportunistic pathogen largely involved in nosocomial infections. Unfortunately, effective antibacterial agents are lacking. Exploring its physiology at the post-translational modifications (PTMs) level may contribute to the renewal of combat tactics.

Constitutive Activation of MexT by Amino Acid Substitutions Results in MexEF-OprN Overproduction in Clinical Isolates of Pseudomonas aeruginosa.

When overproduced, the multidrug efflux system MexEF-OprN increases the resistance of to fluoroquinolones, chloramphenicol, and trimethoprim. In this work, we demonstrate that gain-of-function mutations in the regulatory gene result in oligomerization of the LysR regulator MexT, constitutive upregulation of the efflux pump, and increased resistance in clinical isolates.

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.

Xenon for tunnelling analysis of the efflux pump component OprN.

Tripartite efflux pumps are among the main actors responsible for antibiotics resistance in Gram-negative bacteria. In the last two decades, structural studies gave crucial information about the assembly interfaces and the mechanistic motions. Thus rigidifying the assembly seems to be an interesting way to hamper the drug efflux.

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.

Targeted unlabeled multiple reaction monitoring analysis of cell markers for the study of sample heterogeneity in isolated rat brain cortical microvessels.

Liquid chromatography coupled to tandem mass spectrometry-based targeted absolute protein quantification (in fmol of the analyte protein per μg of total protein) is employed for the molecular characterization of the blood-brain barrier using isolated brain microvessels. Nevertheless, the heterogeneity of the sample regarding the levels of different cells co-isolated within the microvessels and bovine serum albumin (BSA) contamination (from buffers) are not always evaluated. We developed an unlabeled targeted liquid chromatography coupled to tandem mass spectrometry method to survey the levels of endothelial cells (ECs), astrocytes, and pericytes, as well as BSA contaminant in rat cortical microvessels.

VEGFR1 domain 2 covalent labeling with horseradish peroxidase: Development of a displacement assay on VEGF.

The VEGFR1 has been shown to play a role in the regulation of angiogenesis, and has therefore been associated to several pathologies. In order to extend our toolbox of screening methods for the identification of compounds disrupting the VEGF receptor 1/VEGF interaction, we developed a fast and accurate displacement assay, in which VEGF receptor 1 domain 2 is directly labeled with an enzyme, bypassing the classical streptavidin-biotin interaction system. A description of this straightforward strategy is provided here, including its advantages and disadvantages.

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.