Multidrug Resistance Protein Family Research Articles

Genomic Characteristics of an Extensive-Drug-Resistant Clinical Escherichia coli O99 H30 ST38 Recovered from Wound.

Antibiotic-resistant Escherichia coli is one of the major opportunistic pathogens that cause hospital-acquired infections worldwide. These infections include catheter-associated urinary tract infections (UTIs), ventilator-associated pneumonia, surgical wound infections, and bacteraemia. To understand the mechanisms of resistance and prevent its spread, we studied E. coli C91 (ST38), a clinical outbreak strain that was extensively drug-resistant. The strain was isolated from an intensive care unit (ICU) in one of Kuwait's largest hospitals from a patient with UTI. This study used whole-genome sequencing (Illumina, MiSeq) to identify the strain's multi-locus sequence type, resistance genes (ResFinder), and virulence factors. This study also measured the minimum inhibitory concentrations (MIC) of a panel of antibiotics against this isolate. The analysis showed that E. coli C-91 was identified as O99 H30 ST38 and was resistant to all antibiotics tested, including colistin (MIC > 32 mg/L). It also showed intermediate resistance to imipenem and meropenem (MIC = 8 mg/L). Genome analysis revealed various acquired resistance genes, including mcr-1, bla CTX-M-14, bla CTX-M-15, and bla OXA1. However, we did not detect bla NDM or bla VIM. There were also several point mutations resulting in amino acid changes in chromosomal genes: gyrA, parC, pmrB, and ampC promoter. Additionally, we detected several multidrug efflux pumps, including the multidrug efflux pump mdf(A). Eleven prophage regions were identified, and PHAGE_Entero_SfI_NC was detected to contain ISEc46 and ethidium multidrug resistance protein E (emrE), a small multidrug resistance (SMR) protein family. Finally, there was an abundance of virulence factors in this isolate, including fimbriae, biofilm, and capsule formation genes. This isolate has a diverse portfolio of antimicrobial resistance and virulence genes and belongs to ST38 O99 H30, posing a serious challenge to treating infected patients in clinical settings.

Correlating the transport cycle of small multidrug resistance transporters

Antibiotics have significantly improved human health around the globe. However, evolution has increased antibiotic resistance among bacteria, creating a critical public health concern. Thus, it is important to understand antibiotic resistance mechanisms bacteria. Members of the small multidrug resistance (SMR) protein family confer resistance to antibiotics by leveraging an established proton gradient. SMR proteins are small homodimers and are broadly classified into Qac and Gdx sub-families based on selective substrate transport across the membrane.

Single Cell RNA Sequencing Identifies Potential Molecular Indicators of Response to Melflufen in Multiple Myeloma

IntroductionMelphalan flufenamide (melflufen), is a novel peptide-drug conjugate that targets aminopeptidases and selectively delivers alkylating agents in tumors. Melflufen was recently FDA approved for the treatment of relapsed/refractory multiple myeloma (MM) patients. Considering the challenges in treating this group of patients, and the availability of several new drugs for MM, information that can support treatment selection is urgently needed. To identify potential indicators of response and mechanism of resistance to melflufen, we applied a multiparametric drug sensitivity assay to MM patient samples ex vivo and analyzed the samples by single cell RNA sequencing (scRNAseq). Ex vivo drug testing identified MM samples that were distinctly sensitive or resistant to melflufen, while differential gene expression analysis revealed pathways associated with response.MethodsBone marrow (BM) aspirates from 24 MM patients were obtained after written informed consent following approved protocols in compliance with the Declaration of Helsinki. BM mononuclear cells from 12 newly diagnosed (ND) and 12 relapsed/refractory (RR) patients were used for multi-parametric flow cytometry-based drug sensitivity and resistance testing (DSRT) evaluation to melflufen and melphalan, and for scRNAseq. Based on the results from the DSRT tests and drug sensitivity scores (DSS), we divided the samples into three groups - high sensitivity (HS, DSS > 40 (melflufen) or DSS > 16 (melphalan)), intermediate sensitivity (IS, 31 ≤ DSS ≤ 40 (melflufen) or 10 ≤ DSS ≤ 16 (melphalan)), and low sensitivity (LS, DSS < 31 (melflufen) or DSS < 10 (melphalan)). To identify genes, responsible for the general sensitivity to melphalan-based drugs we conducted differential gene expression (DGE) analyses separately for melphalan and melflufen focusing on the plasma cell populations, comparing gene expression between HS and LS samples for both drugs (“HS vs. LS melphalan” and “HS vs. LS for melflufen”, respectively). In addition, to explain the increased sensitivity of RR samples, we conducted the DGE analysis for ND vs. RR samples and searched for similarities between these three datasets.ResultsDSRT data indicated that samples from RRMM patients were significantly more sensitive to melflufen compared to samples from NDMM (Fig. 1A). In addition, we observed that samples with a gain of 1q (+1q) were more sensitive to melflufen while those with deletion of 13q (del13q) appeared to be less sensitive, although these results lacked significance (Fig. 1A). After separating the samples into different drug sensitivity groups (HS, IS, LS), DGE analysis showed significant downregulation of the drug efflux and multidrug resistance protein family member ABCB9 in the melflufen HS group opposed to the LS group (2.2-fold, p < 0.001). A similar pattern was detected for the melphalan HS vs. LS comparison suggesting that this alteration might be a common indicator of sensitivity to melphalan-based drugs. Furthermore, in the melflufen HS group we observed downregulation of the matrix metallopeptidase inhibitors TIMP1 and TIMP2 (3-fold and 1.6-fold, p < 0.001, respectively), and cathepsin inhibitors CST3 and CSTB (3.2-fold and 1.3-fold, p < 0.001, respectively) (Fig. 1B). This effect was observed in both “ND vs. RR” and “HS vs. LS for melflufen” comparisons, but not for melphalan, suggesting that these changes are associated with disease progression and specific indicators of sensitivity to melflufen. Moreover, gene set enrichment analysis (GSEA) showed activation of pathways related to protein synthesis, as well as amino acid starvation for malignant and normal cell populations in the HS group.ConclusionIn summary, our results indicate that melflufen is more active in RRMM compared to NDMM. In addition, samples from MM patients with +1q, which is considered an indicator of high-risk disease, tended to be more sensitive to melflufen. Based on differential GSEA and pathway enrichment, several synergizing mechanisms could potentially explain the higher sensitivity to melflufen, such as decreased drug efflux and increased drug uptake. Although these results indicate potential indicators of response and mechanisms of drug efficacy, further validation of these findings is required using data from melflufen treated patients. [Display omitted] DisclosuresSlipicevic: Oncopeptides AB: Current Employment. Nupponen: Oncopeptides AB: Consultancy. Lehmann: Oncopeptides AB: Current Employment. Heckman: Orion Pharma: Research Funding; Oncopeptides: Consultancy, Research Funding; Novartis: Research Funding; Celgene/BMS: Research Funding; Kronos Bio, Inc.: Research Funding.

Regulation of monocyte migration by amphoterin (HMGB1)

AbstractAmphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-γ (IFN-γ)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5′-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium. (Blood. 2004;104:1174-1182)

Complementary effects of Orthosiphon stamineus standardized ethanolic extract and rosmarinic acid in combination with gemcitabine on pancreatic cancer

BackgroundPancreatic cancer is one of the most notorious cancers and is known for its highly invasive characteristics, drug resistance, and metastatic progression. Unfortunately, many patients with advanced pancreatic cancer become insensitive towards gemcitabine treatment. Orthosiphon stamineus (O.s) is used widely as a traditional medicine for the treatment of multiple ailments, including cancer in South East Asia. The present in vitro study was designed to investigate the complementary effects of an ethanolic extract of O.s (Et. O.s) or rosmarinic acid in combination with gemcitabine on Panc-1 pancreatic cancer cells. MethodCell viability and colony formation assays were used to determine the 50% inhibitory concentration (IC50) of Et. O.s, rosmarinic acid, and gemcitabine. Different doses of gemcitabine in combination with Et. O.s or rosmarinic acid were tested against Panc-1 to select the best concentrations which possessed synergistic effects. Elucidation of molecular mechanisms responsible for mediating chemo-sensitivity in Panc-1 was performed using Quantitative Real-time PCR (QPCR), flow cytometry and immunohistochemistry. ResultsEt. O.s was found to significantly sensitise Panc-1 towards gemcitabine by reducing the gene expression of multidrug-resistant protein family (MDR) (MDR-1, MRP-4, and MRP-5) and molecules related to epithelial-mesenchymal transition (ZEB-1 and Snail-1). An induction of the human equilibrate nucleoside transporter-1 (hENT-1) gene was also found in cells treated with Et. O.s-gemcitabine. The Et. O.s-gemcitabine combination induced cellular senescence, cell death and cell cycle arrest in Panc-1. In addition, the inhibition of Notch signalling was demonstrated through the downregulation of Notch 1 intracellular domain in this treatment group. In contrast, rosmarinic acid-gemcitabine combination showed no additional effects on cellular senescence, apoptosis, epithelial mesenchymal transition (EMT) markers, the MRP-4 and MRP-5 multi-drug resistance protein family, hENT-1, and the Notch pathway through Notch 1 intracellular domain. ConclusionThis study provides valuable insights on the use of Et. O.s to complement gemcitabine in targeting pancreatic cancer in vitro, suggesting its potential use as a novel complementary treatment in pancreatic cancer patients.

Bacterial Ion Homeostasis through Biochemical Assays of Ion Transporters

Ion transport is essential to bacterial physiology, including processes like pathogenesis and biofilm formation. This study will take a biochemical and physiological perspective to understand the physiology of ion transport proteins in uropathogenic E. coli, including transporters from the small multi-drug resistance (SMR) protein family.

Interstitial Flow Recapitulates Gemcitabine Chemoresistance in A 3D Microfluidic Pancreatic Ductal Adenocarcinoma Model by Induction of Multidrug Resistance Proteins.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most lethal cancers due to a high chemoresistance and poor vascularization, which results in an ineffective systemic therapy. PDAC is characterized by a high intratumoral pressure, which is not captured by current 2D and 3D in vitro models. Here, we demonstrated a 3D microfluidic interstitial flow model to mimic the intratumoral pressure in PDAC. We found that subjecting the S2-028 PDAC cell line to interstitial flow inhibits the proliferation, while maintaining a high viability. We observed increased gemcitabine chemoresistance, with an almost nine-fold higher EC50 as compared to a monolayer culture (31 nM versus 277 nM), and an alleviated expression and function of the multidrug resistance protein (MRP) family. In conclusion, we developed a 3D cell culture modality for studying intratissue pressure and flow that exhibits more predictive capabilities than conventional 2D cell culture and is less time-consuming, and more scalable and accessible than animal models. This increase in microphysiological relevance might support improved efficiency in the drug development pipeline.

Influence of quaternary cation compound on the size of the Escherichia coli small multidrug resistance protein, EmrE

EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. It confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by the transmembrane proton motive force. We have expressed hexahistidinyl (His6) – myc epitope tagged EmrE, extracted it from membrane preparations using the detergent n-dodecyl-β-D-maltopyranoside (DDM), and purified it using nickel-affinity chromatography. The size of the EmrE protein, in DDM environment, was then examined in the presence and absence of a range of structurally different QCC ligands that varied in their chemical structure, charge and shape. We used dynamic light scattering and showed that the size and oligomeric state distributions are dependent on the type of QCC. We also followed changes in the Trp fluorescence and determined apparent dissociation constants (Kd). Overall, our in vitro analyses of epitope tagged EmrE demonstrated subtle but significant differences in the size distributions with different QCC ligands bound.

SugE belongs to the small multidrug resistance (SMR) protein family involved in tributyltin (TBT) biodegradation and bioremediation by alkaliphilic Stenotrophomonas chelatiphaga HS2

Tributyltin (TBT) used in a variety of industrial processes, subsequent discharge into the environment, its fate, toxicity and human exposure are topics of current concern. TBT degradation by alkaliphilic bacteria may be a key factor in the remediation of TBT in high pH contaminated sites. In this study, Stenotrophomonas chelatiphaga HS2 were isolated and identified from TBT contaminated site in Mediterranean Sea. S. chelatiphaga HS2 has vigor capability to transform TBT into dibutyltin and monobutyltin (DBT and MBT) at pH 9 and 7% NaCl (w/v). A gene was amplified and characterized from strain HS2 as SugE protein belongs to SMR protein family, a reverse transcription polymerase chain reaction analysis confirmed that SugE protein involved in the TBT degradation by HS2 strain. TBT bioremediation was investigated in stimulated TBT contaminated sediment samples (pH 9) using S chelatiphaga HS2 in association with E. coli BL21 (DE3)-pET28a(+)-sugE instead of S chelatiphaga HS2 alone reduced significantly the TBT half-life from 12d to 5d, although no TBT degradation appeared using E. coli BL21 (DE3)-pET28a(+)-sugE alone. This finding indicated that SugE gene increased the rate and degraded amount of TBT and is necessary in enhancing TBT bioremediation.

Investigating the Structure of the Drug Transporter EmrE

Multidrug resistance in bacteria is a critical challenge in public health and drug discovery. One of the primary mechanisms of resistance is efflux pumps, which couple an energetically favorable process with the export of a drug molecule against its concentration gradient.1 Efflux pumps from the small multidrug resistance protein family are ubiquitous among bacteria. These secondary active transporters couple the efflux of a wide variety of toxic compounds with the proton gradient of the inner membrane.2 To gain insight into this transport process we carried out a series of biophysical experiments, including solution and solid-state NMR spectroscopy using the multidrug transporter EmrE. EmrE is an asymmetric and antiparallel homodimer, which couples the efflux of cationic drugs with the import of protons. Previously, we revealed a large change in structure and dynamics due to the acid/base chemistry at a conserved glutamic acid residue.3 In this work we focus on describing the site-specific structural changes within the substrate binding pocket that drive the large conformational change in EmrE. Specifically, our oriented solid-state NMR and solution NMR experiments reveal backbone and side chain structural perturbations upon binding protons or drugs that reveal a specific mechanism involving aromatic residues in the binding pocket. Finally, we built on our previous findings 3 to show how conformationally biased mutants allow for the unambiguous determination of monomer-specific restraints for structural elucidation of EmrE in lipid bilayers.

Reserpine requires the D2-type receptor, dop-3, and the exoribonuclease, eri-1, to extend the lifespan in C. elegans.

Lifespan extension is an all systems encompassing event. Involvement of reduced insulin/IGF1 signalling is well worked out, first in the model organism Caenorhbaditis elegans followed by other systems including humans. But the role of neuronal component in lifespan extension is not well understood due to the refractory nature of neurons to small RNA interference (sRNAi) in C. elegans. Earlier, we have demonstrated that an antihypertensive drug, reserpine, extends lifespan through modulation of neurotransmitter release, especially, acetylcholine, in C. elegans. Intriguingly, the reserpine mediated lifespan extension (RMLE) does not happen through the known longevity pathways. Here, we report that the D2-type dopamine receptor (DOP-3), which acts through the inhibitory Gprotein coupled (G alpha i) pathway mediated signalling is partly required for RMLE. In the dop-3 loss of function mutant RMLE is shortened. DOP-3 acts through Gαo (goa-1). One of the downstream targets of G protein signalling is the transcription factor, jun-1. MRP-1, an ATP binding cassette transporter, belonging to the multidrug resistance protein family is one of the genes turned on by JUN-1. RMLE is shortened in dop-3-->goa-1-->jun1-->mrp-1 loss of function mutants, elucidating the contribution of dop-3 signalling. The dop-3 receptor system is known to inhibit acetylcholine release. This suggests dopamine receptor, dop-3 could be contributing to the modulation of acetylcholine release by reserpine. ERI-1 is a 3'-5' exoribonuclease, one of the negative regulators of sRNAi, whose loss of function makes neurons amenable to siRNA. In the absence of eri-1, RMLE is shortened. In the dop-3 loss-of-function background, lack of eri-1 completely abolishes RMLE. This suggests that dop-3 and eri-1 act in independent parallel pathways for RMLE and these two pathways are essential and sufficient for the longevity enhancement by reserpine in C. elegans.

Functional diversification of sea urchin ABCC1 (MRP1) by alternative splicing.

The multidrug resistance protein (MRP) family encodes a diverse repertoire of ATP-binding cassette (ABC) transporters with multiple roles in development, disease, and homeostasis. Understanding MRP evolution is central to unraveling their roles in these diverse processes. Sea urchins occupy an important phylogenetic position for understanding the evolution of vertebrate proteins and have been an important invertebrate model system for study of ABC transporters. We used phylogenetic analyses to examine the evolution of MRP transporters and functional approaches to identify functional forms of sea urchin MRP1 (also known as SpABCC1). SpABCC1, the only MRP homolog in sea urchins, is co-orthologous to human MRP1, MRP3, and MRP6 (ABCC1, ABCC3, and ABCC6) transporters. However, efflux assays revealed that alternative splicing of exon 22, a region critical for substrate interactions, could diversify functions of sea urchin MRP1. Phylogenetic comparisons also indicate that while MRP1, MRP3, and MRP6 transporters potentially arose from a single transporter in basal deuterostomes, alternative splicing appears to have been the major mode of functional diversification in invertebrates, while duplication may have served a more important role in vertebrates. These results provide a deeper understanding of the evolutionary origins of MRP transporters and the potential mechanisms used to diversify their functions in different groups of animals.

Membrane Transporters and Pharmacological Implications.

Membrane transporters serve to move chemicals in and out of the cells according to metabolic needs or the presence of toxic compounds. These processes are mediated by facilitated diffusion or active transport through the lipid bilayer that is the cell membrane. There exist two main categories of membrane transporters, the more passive solute carrier transporters (SLCs) and the ATP binding cassette transporters (ABCs). SLCs allow for passage of ions, sugars, lipids, amino acids and other compounds down a gradient, contributing to a cell’s passive permeability for such compounds. ABCs feature a highly conserved nucleotide binding domain (NBD) which contain peptide sequences responsible for ATP hydrolysis such as the Walker A and B motifs. ABCs utilize the energy stored in ATP to transport chemicals against their concentration and/or electrical gradient and consequently alter expected biological conditions. Transporters are now recognized as crucial barriers (e.g., efflux transporters) as well as possible delivery pathways to consider when designing new pharmaceutical agents as many traditional therapeutics are being recognized as transporter substrates [1–3]. Drug resistant tumors and the blood-brain barrier (BBB) for example have been shown to actively express efflux transporters preventing therapeutic agents from reaching clinically relevant intracellular concentrations and/or physiological targets in the brain [4–7]. Consequently, in 2010 the International Transporter Consortium put forth the ‘white paper’ detailing the structure, location, and known substrates of various pharmacologically relevant transporters. This prompted action from American and European regulatory agencies to release guidelines on transporter-drug interaction studies [3]. P-Glycoprotein (MDR1), Breast Cancer Resistance Protein (BCRP), Organic Anion Transporter peptide (OATP), Organic Anion Transporter (OAT), Organic Cation Transporter (OCT), and Multi Drug Resistance Protein (MRP) are mentioned directly in the white paper and have been a focus of the ITC to be included in the in vitro to in vivo extrapolation (IVIVE) which refers to the qualitative or quantitative transposition of in vitro experimental results to predicts a physiological and/or pathological phenomena in vivo. P-glycoproteins (Pgp) and the Cytochrome P450 enzyme CYP3A analogs share a significant number of substrates [8] and are both found in the intestines and liver [9–11]. Together they compose a first pass metabolism barrier for therapeutic agents taken orally [12,13]. Because Pgp effluxes compounds from the intestinal wall back into the lumen of the intestines, researchers may mistake increased mean residence time (MRT) for increased absorption when performing PK/PD studies [14]. Some of these CYP analogs such as CYP3A4, CYP2C9, CYP2C19, CYP2A6 and CYP2E1 are also expressed at the BBB endothelial level of the BBB under pathological conditions (e.g., drug resistant epilepsy). In some cases, the expression of a CYP enzyme (e.g., CYP3A CYP2C19 and CYP2C9) is regulated by the activation of the xenobiotic receptor pregnane X receptor (PXR) which also controls the expression of Pgp and other drug efflux systems [15,16]. With regard to the drug efflux transporters at the BBB level, multidrug resistance (MDR) is a major obstacle to treating patients with cancer and is often the result of overexpression of a 170- to 180-kDa plasma membrane glycoprotein known as P-glycoprotein (Pgp) [17,18] and multidrug resistance-related proteins (MRP1, 190 kDa) [19,20]. Human Pgp is encoded by MDR1 and rodent Pgp by Mdr1a and Mdr1b [21–24]. Pgp and MRPs belong to the superfamily of ATP-binding cassette transporters. Unlike other selective (classical) transport proteins, MDR proteins recognize a wide range of substrates. This wide substrate specificity explains the cross-resistance to several chemically unrelated compounds, the characteristic feature found in the multi-drug resistance phenotype. In addition to their overlapping substrates specificity, each transporter can handle unique compounds. Pgp-MDR1 is a transporter for large amphipathic compounds either uncharged or slightly charged while the MRP family is mostly transporting hydrophobic anionic conjugates with glucuronide, sulfate or glutathione and also extrudes hydrophobic uncharged drugs [19]. Experiments designed to define the structure of Pgp suggest that there is no simple single drug-binding site or pore in Pgp. Amino acid substitutions in, or near, most of the transmembrane segments affect substrate specificity efficiency or transport. In drug refractory patients a synergistic effect between MDRs and CYP enzymes has been recently observed which antagonizes the passage of drugs targeting the brain (e.g., antiepileptics, tumor suppressants, etc.) through a concerted set of mechanisms [15]. The efflux transporters extrude the drug from the brain across the BBB back into the blood circulation while the CYP enzyme metabolize the drug substrate into (at large) inactive derivate(s), thus rendering the drug bioavailability ineffective from a therapeutic standpoint. Other than MDR, altered activity of efflux transporters have been linked to a number of neurological disorders including Alzheimer’s disease [25], Parkinson’s diseases [26] and Creutzfeldt-Jakob disease [27].

Investigation of the role of multidrug resistance proteins (MRPs) in vascular homeostasis

Cellular levels of cyclic GMP (cGMP) are tightly controlled by synthetic and degradative mechanisms. Pharmacological manipulation of these processes (e.g. soluble guanylate cyclase stimulators and phosphodiesterase 5 inhibitors) augments cGMP-dependent signalling and is beneficial in treating cardiovascular disease (eg. pulmonary hypertension). An additional mechanism potentially important in the inactivation of cGMP is cellular extrusion, driven by a family of multidrug resistance proteins (MRPs). Herein, we investigated if MRPs modulate vascular reactivity, smooth muscle cell proliferation, and systemic hemodynamics. The functional reactivity of murine aortic rings and proliferation of human pulmonary artery smooth muscle cells (PASMC) were determined in the absence and presence of the MRP inhibitor MK571. Hemodynamic changes in vivo in response to MK571 were analysed acutely by bolus dosing and chronically by radiotelemetry. MK571 (1nM-50µM) caused a concentration-dependent relaxation of mouse aortic r...

Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE

EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His6) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-β-d-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants (Kd) and maximum specific one-site binding (Bmax) values for particular QCCs. In vitro analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.

Investigation of the Role of Multidrug Resistance Proteins (MRPs) in Vascular Homeostasis

Cellular levels of cyclic GMP (cGMP) are tightly controlled by synthetic and degradative mechanisms. Pharmacological manipulation of these processes (e.g. soluble guanylate cyclase stimulators and phosphodiesterase 5 inhibitors) augments cGMP-dependent signalling and is beneficial in treating cardiovascular disease (eg. pulmonary hypertension). An additional mechanism potentially important in the inactivation of cGMP is cellular extrusion, driven by a family of multidrug resistance proteins (MRPs). Herein, we investigated if MRPs modulate vascular reactivity, smooth muscle cell proliferation, and systemic hemodynamics. The functional reactivity of murine aortic rings and proliferation of human pulmonary artery smooth muscle cells (PASMC) were determined in the absence and presence of the MRP inhibitor MK571. Hemodynamic changes in vivo in response to MK571 were analysed acutely by bolus dosing and chronically by radiotelemetry. MK571 (1nM-50µM) caused a concentration-dependent relaxation of mouse aortic rings. In the presence of a threshold concentration of MK571 (3µM) vasorelaxant responses to NO and atrial natriuretic peptide were significantly augmented. MK571 (1nM-3µM) also caused a significant inhibition of PASMC growth. In vivo, MK571 (0.001-10mg/kg; iv.) elicited an acute, dose-dependent hypotensive activity and when delivered via the drinking water, caused a more sustained drop in mean arterial pressure (~5mmHg). These data suggest that extrusion by MRPs contributes to the dynamic equilibrium regulating intracellular levels of cGMP, and may represent a target amenable to drug intervention for the treatment of cardiovascular disease. Funded by the MRC

Molecular mechanisms mediating tolerance to ionic liquids in Listeria monocytogenes

Increased application of ionic liquids (ILs) in various chemical and biochemical processes as well as for pharmaceuticals or antimicrobial agents has prompted researchers to assess their biological functions, especially their toxicity. However, to date the molecular mechanisms underlying tolerance of certain bacterial species to ILs has not been studied in detail. The recently found transposon Tn6188 in Listeria monocytogenes has been shown to mediate tolerance to quaternary ammonium compounds (QACs) such as benzalkonium chloride, which are widely used as disinfectants in food processing plants, via QacH, a small multidrug resistance protein family (SMR) transporter. In this study the possible Tn6188 mediated tolerance of L. monocytogenes to ILs was investigated. It was found that Tn6188 confers tolerance of L. monocytogenes to ILs based on imidazolium and ammonium cations.

Glutamate-mediated upregulation of the multidrug resistance protein 2 in porcine and human brain capillaries.

As a member of the multidrug-resistance associated protein (MRP) family, MRP2 affects the brain entry of different endogenous and exogenous compounds. Considering the role of this transporter at the blood-brain barrier, the regulation is of particular interest. However, there is limited knowledge regarding the factors that regulate MRP2 in neurologic disease states. Thus, we addressed the hypothesis that MRP2 might be affected by a glutamate-induced signaling pathway that we previously identified as one key mechanism in the regulation of P-glycoprotein. Studies in isolated porcine brain capillaries confirmed that glutamate and N-methyl-d-aspartic acid (NMDA) exposure upregulates expression and function of MPR2. The involvement of the NMDA receptor was further suggested by the fact that the NMDA receptor antagonist MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], as well as the NMDA receptor glycine binding site antagonist L-701,324 [7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone], prevented the impact of glutamate. A role of cyclooxygenase-2 was indicated by coincubation with the cyclooxygenase-2 inhibitor celecoxib and the cyclooxygenase-1/-2 inhibitor indomethacin, which both efficaciously abolished a glutamate-induced upregulation of MRP2. Translational studies in human capillaries from surgical specimen demonstrated a relevant MRP2 efflux function and indicated an effect of glutamate exposure as well as its prevention by cyclooxygenase-2 inhibition. Taken together the findings provide first evidence for a role of a glutamate-induced NMDA receptor/cyclooxygenase-2 signaling pathway in the regulation of MRP2 expression and function. The response to excessive glutamate concentrations might contribute to overexpression of MRP2, which has been reported in neurologic diseases including epilepsy. The overexpression might have implications for brain access of various compounds including therapeutic drugs.

Abstract 1482: MicroRNA expression profiles classify renal cell carcinoma subtypes

Abstract Introduction Renal cell carcinoma (RCC) accounts for nearly 4% of all malignancies worldwide. According to the WHO classification of tumors of the urinary system, RCC is a collection of different subtypes rather than one entity. Besides the conventional clear cell RCC (ccRCC), the papillary RCC (pRCC), with an incidence of 10%-15%, is the second most common entity among renal malignancies. By histological characteristics, pRCCs can further be sub-divided into two distinct subtypes. Hereby, pRCCs type 2 are correlated with a worse clinical outcome. We sought to define characteristic miRNA expression profiles that could be useful for a molecular classification of RCC subtypes. Material and Methods Using miRNA microarrays (GeneChip miRNA arrays, Affymetrix), we established miRNA expression profiles of a discovery set of ccRCC and pRCC subtypes 1 and 2 tissue samples. The expression of a selected subset of miRNAs was validated by quantitative PCR. The best discriminating miRNAs of this initial set were used to establish a classification model for tissue samples. This core set of miRNAs was analyzed in a second, independent patient cohort of 69 tissue samples. Using the pre-defined classification rules, the samples of the independent patient cohort were classified in a double-blind fashion solely by their miRNA expression. Results Every RCC entity and subtype displayed a characteristic pattern of miRNA expression. Ten miRNAs were selected based on their ability to discriminate between tumor and normal tissue or between different subtypes of RCC. Binary logistic regression identified a core set of only five miRNAs that was able to fully classify any given sample with an overall accuracy of 88%. This classification scheme was applied to an independent patient cohort. Here five miRNAs (miRNAs miR-145, -200c, -210, -502-3p, and let-7c) were able to classify the samples with an overall accuracy of 64%. MiRNAs that are deregulated in every RCC entity target members of the family of multidrug-resistance proteins (MRPs) and miRNAs that distinguish between pRCC subtypes target components of the Jak-STAT signaling pathway. Conclusions Every entity or subtype of RCC displays a characteristic and unique pattern of miRNA expression. A core set of only five miRNAs can be used to distinguish not only between tumor and normal tissue samples but also between different tumor entities with high accuracy. Deregulated miRNAs might contribute to the high chemotherapy resistance of RCC. Furthermore, our results indicate that pRCC type 2 tumors could be dependent on oncogenic MYC signaling. Citation Format: Sven Wach, Elke Nolte, Theil Anne, Christine Stoehr, Tilman T. Rau, Arndt Hartmann, Arif B. Ekici, Bastian Keck, Helge Taubert, Bernd Wullich. MicroRNA expression profiles classify renal cell carcinoma subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1482. doi:10.1158/1538-7445.AM2014-1482

Tn6188 - A Novel Transposon in Listeria monocytogenes Responsible for Tolerance to Benzalkonium Chloride

Controlling the food-borne pathogen Listeria (L.) monocytogenes is of great importance from a food safety perspective, and thus for human health. The consequences of failures in this regard have been exemplified by recent large listeriosis outbreaks in the USA and Europe. It is thus particularly notable that tolerance to quaternary ammonium compounds such as benzalkonium chloride (BC) has been observed in many L. monocytogenes strains. However, the molecular determinants and mechanisms of BC tolerance of L. monocytogenes are still largely unknown. Here we describe Tn6188, a novel transposon in L. monocytogenes conferring tolerance to BC. Tn6188 is related to Tn554 from Staphylococcus (S.) aureus and other Tn554-like transposons such as Tn558, Tn559 and Tn5406 found in various Firmicutes. Tn6188 comprises 5117 bp, is integrated chromosomally within the radC gene and consists of three transposase genes (tnpABC) as well as genes encoding a putative transcriptional regulator and QacH, a small multidrug resistance protein family (SMR) transporter putatively associated with export of BC that shows high amino acid identity to Smr/QacC from S. aureus and to EmrE from Escherichia coli. We screened 91 L. monocytogenes strains for the presence of Tn6188 by PCR and found Tn6188 in 10 of the analyzed strains. These isolates were from food and food processing environments and predominantly from serovar 1/2a. L. monocytogenes strains harboring Tn6188 had significantly higher BC minimum inhibitory concentrations (MICs) (28.5 ± 4.7 mg/l) than strains without Tn6188 (14 ± 3.2 mg/l). Using quantitative reverse transcriptase PCR we could show a significant increase in qacH expression in the presence of BC. QacH deletion mutants were generated in two L. monocytogenes strains and growth analysis revealed that ΔqacH strains had lower BC MICs than wildtype strains. In conclusion, our results provide evidence that Tn6188 is responsible for BC tolerance in various L. monocytogenes strains.