Functional and structural comparison of the ABC exporter MsbA studied in detergent and reconstituted in nanodiscs.

Purified membrane proteins are most frequently studied solubilized in detergent, but the properties of detergent micelles are very different from those of lipid bilayers. Therefore, there is an increasing interest in studying membrane proteins under conditions that resemble the membrane protein native environment more closely. Although there are indications of differences between membrane proteins in detergent and in lipid bilayers, direct functional and structural comparisons are very hard to find. Nanodiscs have been established as a new platform that consists of two molecules of a membrane scaffold protein that surround a small lipid-bilayer patch. Here, we undertook the task of comparing the function and conformational states of the transport protein MsbA in detergent and nanodiscs using ATPase activity and luminescence resonance energy transfer (LRET) measurements to assess differences in activity and conformational states, respectively. MsbA is a prototypical member of the ATP binding cassette protein superfamily. MsbA activity was higher in nanodiscs vs detergent, which had clear structural correlates: an increase in the fraction of molecules displaying closed nucleotide-binding domain dimers in the apo state, and a decrease in the distance of the "dissociated" nucleotide-binding domains. Our LRET studies support the notion that the widely separated nucleotide binding domains observed in the MsbA x-ray structures in detergent do not correspond to physiological conformations. Although our studies focus on a particular ABC exporter, the possibility of similar environment effects on other membrane proteins should be carefully considered.

Physiological and pathophysiological factors affecting the expression and activity of the drug transporter MRP2 in intestine. Impact on its function as membrane barrier.

The gastrointestinal epithelium functions as a selective barrier to absorb nutrients, electrolytes and water, but at the same time restricts the passage into the systemic circulation of intraluminal potentially toxic compounds. This epithelium maintains its selective barrier function through the presence of very selective and complex intercellular junctions and the ability of the absorptive cells to reject those compounds. Accordingly, the enterocytes metabolize orally incorporated xenobiotics and secrete the hydrophilic metabolites back into the intestinal lumen through specific transporters localized apically. In the recent decades, there has been increasing recognition of the existence of the intestinal cellular barrier. In the present review we focus on the role of the multidrug resistance-associated protein 2 (MRP2, ABCC2) in the apical membrane of the enterocytes, as an important component of this intestinal barrier, as well as on its regulation. We provide a detailed compilation of significant contributions demonstrating that MRP2 expression and function vary under relevant physiological and pathophysiological conditions. Because MRP2 activity modulates the availability and pharmacokinetics of many therapeutic drugs administered orally, their therapeutic efficacy and safety may vary as well.

Deletion of pilA, a Minor Pilin-Like Gene, from Xanthomonas citri subsp. citri Influences Bacterial Physiology and Pathogenesis.

Type IV pili (Tfp) are widely distributed adhesins of bacterial surfaces. In plant pathogenic bacteria, Tfp are involved in host colonization and pathogenesis. Xanthomonas citri subsp. citri (Xcc) is the phytopathogen responsible for citrus canker disease. In this work, three Tfp structural genes, fimA, fimA1, and pilA from Xcc were studied. A pilA mutant strain from Xcc (XccΔpilA) was constructed and differences in physiological features, such as motilities, adhesion, and biofilm formation, were observed. A structural study of the purified Tfp fractions from Xcc wild-type and Xcc∆pilA showed that pilins are glycosylated in both strains and that FimA and FimA1 are the main structural components of the pili. Furthermore, smaller lesion symptoms and reduced bacterial growth were produced by Xcc∆pilA in orange plants compared to the wild-type strain. These results indicate that the minor pilin-like gene, pilA, is involved in Tfp performance during the infection process.

Intraluminal nutrients acutely strengthen rat intestinal MRP2 barrier function by a glucagon-like peptide-2-mediated mechanism.

AIM: MRP2 is an intestinal ABC transporter that prevents the absorption of dietary xenobiotics. The aims of this work were: (1) to evaluate whether a short-term regulation of intestinal MRP2 barrier function takes place in vivo after luminal incorporation of nutrients and (2) to explore the underlying mechanism. METHODS: MRP2 activity and localization were assessed in an in vivo rat model with preserved irrigation and innervation. Nutrients were administered into distal jejunum. After 30-minutes treatments, MRP2 activity was assessed in proximal jejunum by quantifying the transport of the model substrate 2,4-dinitrophenyl-S-glutathione. MRP2 localization was determined by quantitative confocal microscopy. Participation of extracellular mediators was evaluated using selective inhibitors and by immunoneutralization. Intracellular pathways were explored in differentiated Caco-2 cells. RESULTS: Oleic acid, administered intraluminally at dietary levels, acutely stimulated MRP2 insertion into brush border membrane. This was associated with increased efflux activity and, consequently, enhanced barrier function. Immunoneutralization of the gut hormone glucagon-like peptide-2 (GLP-2) prevented oleic acid effect on MRP2, demonstrating the participation of this trophic factor as a main mediator. Further experiments using selective inhibitors demonstrated that extracellular adenosine synthesis and its subsequent binding to enterocytic A2B adenosine receptor (A2BAR) take place downstream GLP-2. Finally, studies in intestinal Caco-2 cells revealed the participation of A2BAR/cAMP/PKA intracellular pathway, ultimately leading to increased MRP2 localization in apical domains. CONCLUSION: These findings reveal an on-demand, acute regulation of MRP2-associated barrier function, constituting a novel physiological mechanism of protection against the absorption of dietary xenobiotics in response to food intake.