Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Transporters Revealed.

The subfamily C ATP-binding cassette (ABCC) transporters mediate multidrug resistance and ion conductance regulation. Recent atomic or near-atomic resolution structures of three physiologically significant ABCC transporters (MRP1, SUR1, and CFTR), determined by using single-particle cryo-electron microscopy (cryo-EM), reveal structural details that help explain the wide functional diversity of this ABC transporter subfamily.

Structural Basis of Substrate Recognition by the Multidrug Resistance Protein MRP1.

The multidrug resistance protein MRP1 is an ATP-binding cassette (ABC) transporter that confers resistance to many anticancer drugs and plays a role in the disposition and efficacy of several opiates, antidepressants, statins, and antibiotics. In addition, MRP1 regulates redox homeostasis, inflammation, and hormone secretion. Using electron cryomicroscopy, we determined the molecular structures of bovine MRP1 in two conformations: an apo form at 3.5 Å without any added substrate and a complex form at 3.3 Å with one of its physiological substrates, leukotriene C4. These structures show that by forming a single bipartite binding site, MRP1 can recognize a spectrum of substrates with different chemical structures. We also observed large conformational changes induced by leukotriene C4, explaining how substrate binding primes the transporter for ATP hydrolysis. Structural comparison of MRP1 and P-glycoprotein advances our understanding of the common and unique properties of these two important molecules in multidrug resistance to chemotherapy.

ABCC1 transporter exports the immunostimulatory cyclic dinucleotide cGAMP.

The DNA sensor cyclic GMP-AMP synthase (cGAS) is important for antiviral and anti-tumor immunity. cGAS generates cyclic GMP-AMP (cGAMP), a diffusible cyclic dinucleotide that activates the antiviral response through the adaptor protein stimulator of interferon genes (STING). cGAMP cannot passively cross cell membranes, but recent advances have established a role for extracellular cGAMP as an "immunotransmitter" that can be imported into cells. However, the mechanism by which cGAMP exits cells remains unknown. Here, we identifed ABCC1 as a direct, ATP-dependent cGAMP exporter in mouse and human cells. We show that ABCC1 overexpression enhanced cGAMP export and limited STING signaling and that loss of ABCC1 reduced cGAMP export and potentiated STING signaling. We demonstrate that ABCC1 deficiency exacerbated cGAS-dependent autoimmunity in the Trex1-/- mouse model of Aicardi-Goutières syndrome. Thus, ABCC1-mediated cGAMP export is a key regulatory mechanism that limits cell-intrinsic activation of STING and ameliorates STING-dependent autoimmune disease.

Lipid-derived electrophiles inhibit the function of membrane channels during ferroptosis.

The therapeutic targeting of ferroptosis requires full understanding of the molecular mechanism of this regulated cell death pathway. While lipid-derived electrophiles (LDEs), including 4-hydroxy-2-nonenal (4-HNE), are important biomarkers of ferroptosis, a functional role for these highly reactive species in ferroptotic cell death execution has not been established. Here, through mechanistic characterization of LDE-detoxification impairment, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that export of glutathione-LDE-adducts through multidrug resistance-associated protein (MRP) channels is inhibited following exposure to a panel of ferroptosis inducers (FINs) with different modes of action (type I-IV FINs erastin, RSL3, FIN56, and FINO2). This channel inhibition was recreated by both initiation of lipid peroxidation and treatment with 4-HNE. Importantly, treatment with radical-trapping antioxidants prevented impaired LDE-adduct export when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of this inhibited MRP activity observed during ferroptosis. Our findings, when combined with reports of widespread LDE alkylation of key proteins following ferroptosis induction, including MRP1, set a precedent for LDEs as critical mediators of ferroptotic cell damage. Lipid hydroperoxide breakdown to form truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function downstream of lipid peroxidation, offering a unified explanation of the molecular cell death mechanism of ferroptosis.

Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

The structure of Plasmodium falciparum multidrug resistance protein 1 reveals an N-terminal regulatory domain.

Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), an adenosine triphosphate (ATP)-binding cassette (ABC) transporter on the digestive vacuole (DV) membrane of the parasite, is associated with the resistance to antimalarial drugs. To understand the mechanisms of PfMDR1, we determined the cryo-electron microscopy structures of this transporter in different states. The transporter in the apo state shows an inward-facing conformation with a large cavity opening to the cytoplasm. Upon ATP binding and dimerization of the nucleotide-binding domains (NBDs), PfMDR1 displays an outward-facing conformation with a cavity toward the DV lumen. Drug resistance-associated mutations were investigated in both structures for their effects, and Y184F was identified as an allosteric activity-enhancing mutation. The amphiphilic substrate-binding site of PfMDR1 was revealed by the complex structure with the antimalarial drug mefloquine and confirmed by mutagenesis studies. Remarkably, a helical structure was found to hinder NBD dimerization and inhibit PfMDR1 activity. The location of this regulatory domain in the N terminus is different from the well-studied R domain in the internal linker region of other ABC transporter family members. The lack of the phosphorylation site of this domain also suggests a different regulation mechanism.

A macrocyclic peptide inhibitor traps MRP1 in a catalytically incompetent conformation.

Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT.

Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite's susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite's susceptibility to lumefantrine and mefloquine-a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite's digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine's access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.

Role of cAMP in Cardiomyocyte Viability: Beneficial or Detrimental?

BACKGROUND: 3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. METHODS: We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. RESULTS: For prodeath GsPCRs, we explored ß1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in ß1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. ß1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. CONCLUSIONS: Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].

ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity.

The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

The role of ABCC10/MRP7 in anti-cancer drug resistance and beyond.

Multidrug resistance protein 7 (MRP7), also known as ATP-binding cassette (ABC) transporter subfamily C10 (ABCC10), is an ABC transporter that was first identified in 2001. ABCC10/MRP7 is a 171 kDa protein located on the basolateral membrane of cells. ABCC10/MRP7 consists of three transmembrane domains and two nucleotide binding domains. It mediates multidrug resistance of tumor cells to a variety of anticancer drugs by increasing drug efflux and results in reducing intracellular drug accumulation. The transport substrates of ABCC10/MRP7 include antineoplastic drugs such as taxanes, vinca alkaloids, and epothilone B, as well as endobiotics such as leukotriene C4 (LTC4) and estradiol 17 ß-D-glucuronide. A variety of ABCC10/MRP7 inhibitors, including cepharanthine, imatinib, erlotinib, tariquidar, and sildenafil, can reverse ABCC10/MRP7-mediated MDR. Additionally, the presence or absence of ABCC10/MRP7 is also closely related to renal tubular dysfunction, obesity, and other diseases. In this review, we discuss: 1) Structure and functions of ABCC10/MRP7; 2) Known substrates and inhibitors of ABCC10/MRP7 and their potential therapeutic applications in cancer; and 3) Role of ABCC10/MRP7 in non-cancerous diseases.

ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification.

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are clinically distinct genetic entities of ectopic calcification associated with differentially reduced circulating levels of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification. Variants in ENPP1, the gene mutated in GACI, have not been associated with classic PXE. Here we report the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. All patients were found to carry biallelic variants in ENPP1. Among ten ENPP1 variants, one homozygous variant demonstrated uniparental disomy inheritance. Functional assessment of five previously unreported ENPP1 variants suggested pathogenicity. The two PXE patients, currently 57 and 27 years of age, had diagnostic features of PXE and had not manifested the GACI phenotype. The similarly reduced PPi plasma concentrations in the PXE and GACI patients in our study correlate poorly with their disease severity. This study demonstrates that in addition to GACI, ENPP1 variants can cause classic PXE, expanding the clinical and genetic heterogeneity of heritable ectopic calcification disorders. Furthermore, the results challenge the current prevailing concept that plasma PPi is the only factor governing the severity of ectopic calcification.

MRP5 and MRP9 play a concerted role in male reproduction and mitochondrial function.

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter because mrp-5 mutants are unviable due to their inability to export heme from the intestine to extraintestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans, raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO (DKO) mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes, dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes, and RNA sequencing shows significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.

Nonadditive functional interactions between ligand-binding sites of the multidrug efflux pump AdeB from Acinetobacter baumannii.

Multidrug efflux is one of the major mechanisms of antibiotic resistance identified in clinical isolates of the human pathogen Acinetobacter baumannii. The multiple antibiotic resistance in this species is often enabled by the overproduction of the tripartite efflux pump AdeABC. In this pump, AdeB is the inner membrane transporter from the resistance-nodulation-division (RND) superfamily of proteins, which is responsible for the recognition and efflux of multiple structurally unrelated compounds. Like other RND transporters, AdeB is a trimeric protein with ligand-binding sites located in the large periplasmic domains. Previous structural studies, however, highlighted the uniqueness of AdeB interactions with ligands. Up to three ligand molecules were bound to one protomer of AdeB, mapping its substrate translocation path. In this study, we introduced single and double substitutions in the identified ligand-binding sites of AdeB. Our results show that the mechanism of substrate translocation by AdeB is different from that of other characterized RND transporters and that the functional interactions between the sites are nonadditive. We identified AdeB mutants with both the loss and the gain of antibiotic susceptibility phenotypes, as well as AdeB mutations making A. baumannii cells overproducing such pump variants even more susceptible to multiple antibiotics than efflux-deficient cells. IMPORTANCE Multidrug efflux pumps of the resistance-nodulation-division superfamily of proteins are important contributors to various aspects of bacterial physiology and antibiotic resistance. Studies of the best-characterized model transporter AcrB from Escherichia coli suggested that these transporters operate by a functional rotation mechanism in which various substrates bind to at least two different binding sites. This study suggests that the mechanism of AdeB is distinct and that the binding sites in this transporter are functionally linked.

Hydrophobic moment drives penetration of bacterial membranes by transmembrane peptides.

With antimicrobial resistance (AMR) remaining a persistent and growing threat to human health worldwide, membrane-active peptides are gaining traction as an alternative strategy to overcome the issue. Membrane-embedded multi-drug resistant (MDR) efflux pumps are a prime target for membrane-active peptides, as they are a well-established contributor to clinically relevant AMR infections. Here, we describe a series of transmembrane peptides (TMs) to target the oligomerization motif of the AcrB component of the AcrAB-TolC MDR efflux pump from Escherichia coli. These peptides contain an N-terminal acetyl-A-(Sar)3 (sarcosine; N-methylglycine) tag and a C-terminal lysine tag-a design strategy our lab has utilized to improve the solubility and specificity of targeting for TMs previously. While these peptides have proven useful in preventing AcrB-mediated substrate efflux, the mechanisms by which these peptides associate with and penetrate the bacterial membrane remained unknown. In this study, we have shown peptide hydrophobic moment (µH)-the measure of concentrated hydrophobicity on one face of a lipopathic α-helix-drives bacterial membrane permeabilization and depolarization, likely through lateral-phase separation of negatively-charged POPG lipids and the disruption of lipid packing. Our results show peptide µH is an important consideration when designing membrane-active peptides and may be the determining factor in whether a TM will function in a permeabilizing or non-permeabilizing manner when embedded in the bacterial membrane.

Distribution patterns of molecular markers of antimalarial drug resistance in Plasmodium falciparum isolates on the Thai-Myanmar border during the periods of 1993-1998 and 2002-2008.

BACKGROUND: Polymorphisms of Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multi-drug resistance 1 (pfmdr1) and Plasmodium falciparum kelch 13-propeller (pfk13) genes are accepted as valid molecular markers of quinoline antimalarials and artemisinins. This study investigated the distribution patterns of these genes in P. falciparum isolates from the areas along the Thai-Myanmar border during the two different periods of antimalarial usage in Thailand. RESULTS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect pfcrt mutations at codons 76, 220, 271, 326, 356, and 371 as well as pfmdr1 mutation at codon 86. The prevalence of pfcrt mutations was markedly high (96.4-99.7%) in samples collected during both periods. The proportions of mutant genotypes (number of mutant/total isolate) at codons 76, 220, 271, 326, 356 and 371 in the isolates collected during 1993-1998 (period 1) compared with 2002-2008 (period 2) were 97.9% (137/140) vs. 97.1% (401/413), 97.9% (140/143) vs. 98.8% (171/173), 97.2% (139/143) vs. 97.1% (333/343), 98.6% (140/142) vs. 99.7% (385/386), 96.4% (134/139) vs. 98.2% (378/385) and 97.8% (136/139) vs. 98.9% (375/379), respectively. Most isolates carried pfmdr1 wild-type at codon 86, with a significant difference in proportions genotypes (number of wild type/total sample) in samples collected during period 1 [92.9% (130/140)] compared with period 2 [96.9% (379/391)]. Investigation of pfmdr1 copy number was performed by real-time PCR. The proportions of isolates carried 1, 2, 3 and 4 or more than 4 copies of pfmdr1 (number of isolates carried correspondent copy number/total isolate) were significantly different between the two sample collecting periods (65.7% (90/137) vs. 87.8% (390/444), 18.2% (25/137) vs. 6.3%(28/444), 5.1% (7/137) vs. 1.4% (6/444) and 11.0% (15/137) vs. 4.5% (20/444), for period 1 vs. period 2, respectively). No pfk13 mutation was detected by nested PCR and nucleotide sequencing in all samples with successful analysis (n = 68). CONCLUSIONS: The persistence of pfcrt mutations and pfmdr1 wild-types at codon 86, along with gene amplification in P. falciparum, contributes to the continued resistance of chloroquine and mefloquine in P. falciparum isolates in the study area. Regular surveillance of antimalarial drug resistance in P. falciparum, incorporating relevant molecular markers and treatment efficacy assessments, should be conducted.

Downregulation of ABCC3 activates MAPK signaling through accumulation of deoxycholic acid in colorectal cancer cells.

ABCC3 (also known as MRP3) is an ATP binding cassette transporter for bile acids, whose expression is downregulated in colorectal cancer through the Wnt/ß-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. Gene expression analysis of colorectal adenoma isolated from familial adenomatous polyposis patients revealed that genes related to bile acid secretion including ABCC3 were downregulated as early as at the stage of adenoma formation. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid, a secondary bile acid, respectively, in colorectal cancer cells. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that nonsteroidal anti-inflammatory drugs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of familial adenomatous polyposis. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling.

ABCC4 suppresses glioblastoma progression and recurrence by restraining cGMP-PKG signalling.

BACKGROUND: Cyclic nucleotides are critical mediators of cellular signalling in glioblastoma. However, the clinical relevance and mechanisms of regulating cyclic nucleotides in glioblastoma progression and recurrence have yet to be thoroughly explored. METHODS: In silico, mRNA, and protein level analyses identified the primary regulator of cyclic nucleotides in recurrent human glioblastoma. Lentiviral and pharmacological manipulations examined the functional impact of cyclic nucleotide signalling in human glioma cell lines and primary glioblastoma cells. An orthotopic xenograft mice model coupled with aspirin hydrogels verified the in vivo outcome of targeting cyclic nucleotide signalling. RESULTS: Elevated intracellular levels of cGMP, instead of cAMP, due to a lower substrate efflux from ATP-binding cassette sub-family C member 4 (ABCC4) is engaged in the recurrence of glioblastoma. ABCC4 gene expression is negatively associated with recurrence and overall survival outcomes in glioblastoma specimens. ABCC4 loss-of-function activates cGMP-PKG signalling, promoting malignancy in glioblastoma cells and xenografts. Hydrogels loaded with aspirin, inhibiting glioblastoma progression partly by upregulating ABCC4 expressions, augment the efficacy of standard-of-care therapies in orthotopic glioblastoma xenografts. CONCLUSION: ABCC4, repressing the cGMP-PKG signalling pathway, is a tumour suppressor in glioblastoma progression and recurrence. Aspirin hydrogels impede glioblastoma progression through ABCC4 restoration and constitute a viable translational approach.

NorA efflux pump mediates Staphylococcus aureus response to Pseudomonas aeruginosa pyocyanin toxicity.

Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.