The common ABCA3E292V variant disrupts AT2 cell quality control and increases susceptibility to lung injury and aberrant remodeling.

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.

Lutein and zeaxanthin reduce A2E and iso-A2E levels and improve visual performance in Abca4-/-/Bco2-/- double knockout mice.

Accumulation of bisretinoids such as A2E and its isomer iso-A2E is thought to mediate blue light-induced oxidative damage associated with age-related macular degeneration (AMD) and autosomal recessive Stargardt disease (STGD1). We hypothesize that increasing dietary intake of the macular carotenoids lutein and zeaxanthin in individuals at risk of AMD and STGD1 can inhibit the formation of bisretinoids A2E and iso-A2E, which can potentially ameliorate macular degenerative diseases. To study the beneficial effect of macular carotenoids in a retinal degenerative diseases model, we used ATP-binding cassette, sub-family A member 4 (Abca4-/-)/ß,ß-carotene-9',10'-oxygenase 2 (Bco2-/-) double knockout (KO) mice that accumulate elevated levels of A2E and iso-A2E in the retinal pigment epithelium (RPE) and macular carotenoids in the retina. Abca4-/-/Bco2-/- and Abca4-/- mice were fed a lutein-supplemented chow, zeaxanthin-supplemented chow or placebo chow (~2.6 mg of carotenoid/mouse/day) for three months. Visual function and electroretinography (ERG) were measured after one month and three months of carotenoid supplementation. The lutein and zeaxanthin supplemented Abca4-/-/Bco2-/- mice had significantly lower levels of RPE/choroid A2E and iso-A2E compared to control mice fed with placebo chow and improved visual performance. Carotenoid supplementation in Abca4-/- mice minimally raised retinal carotenoid levels and did not show much difference in bisretinoid levels or visual function compared to the control diet group. There was a statistically significant inverse correlation between carotenoid levels in the retina and A2E and iso-A2E levels in the RPE/choroid. Supplementation with retinal carotenoids, especially zeaxanthin, effectively inhibits bisretinoid formation in a mouse model of STGD1 genetically enhanced to accumulate carotenoids in the retina. These results provide further impetus to pursue oral carotenoids as therapeutic interventions for STGD1 and AMD.

Upregulation of ATP Binding Cassette Subfamily C Member 5 facilitates Prostate Cancer progression and Enzalutamide resistance via the CDK1-mediated AR Ser81 Phosphorylation Pathway.

The treatment of advanced prostate cancer, castration-resistant prostate cancer, remains challenging. The mechanisms of action of ATP binding cassette subfamily C member 5 (ABCC5) in prostate cancer and its relationship with drug resistance are still unclear. Expression and prognostic analyses of ABCC5 were performed through bioinformatic methods and immunohistochemistry analyses in multiple public databases as well as in our own prostate cancer cohort. The biological function of ABCC5 in prostate cancer cells was evaluated by in vitro and in vivo cell proliferation and migration and invasion assays. The regulation of CDK1 by ABCC5 was determined via RT-qPCR, western blots, and immunofluorescence. ABCC5 was significantly overexpressed in prostate cancer and positively associated with unfavorable clinicopathological features and prognosis. Upregulation of ABCC5 could enhance the cell proliferation, migration, and invasion of prostate cancer in vitro and in vivo. Mechanistically, ABCC5 exerts a protumor effect by binding to and inhibiting the protein degradation of CDK1, which promotes the phosphorylation of AR at Ser81 by CDK1 and activates the transcriptional activity of AR on target genes. Moreover, the addition of a CDK1 inhibitor or knockdown of CDK1 significantly improved the efficacy of enzalutamide on prostate cancer cells. The ABCC5-CDK1-AR regulatory pathway could be a potential therapeutic target for advanced prostate cancer, especially castration-resistant prostate cancer (CRPC), to enhance the therapeutic effect of enzalutamide.

The benzoate plant metabolite ethyl gallate prevents cellular- and vascular-lipid accumulation in experimental models of atherosclerosis.

Ethyl gallate (EG) is a well-known constituent of medicinal plants, but its effects on atherosclerosis development are not clear. In the present study, the anti-atherosclerosis effects of EG and the underlying mechanisms were explored using macrophage cultures, zebrafish and apolipoprotein (apo) E deficient mice. Treatment of macrophages with EG (20 µM) enhanced cellular cholesterol efflux to HDL, and reduced net lipid accumulation in response to oxidized LDL. Secretion of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) from activated macrophages was also blunted by EG. Fluorescence imaging techniques revealed EG feeding of zebrafish reduced vascular lipid accumulation and inflammatory responses in vivo. Similar results were obtained in apoE-/- mice 6.5 months of age, where plaque lesions and monocyte infiltration into the artery wall were reduced by 70% and 42%, respectively, after just 6 weeks of injections with EG (20 mg/kg). HDL-cholesterol increased 2-fold, serum cholesterol efflux capacity increased by ∼30%, and the levels of MCP-1 and IL-6 were reduced with EG treatment of mice. These results suggest EG impedes early atherosclerosis development by reducing the lipid and macrophage-content of plaque. Underlying mechanisms appeared to involve HDL cholesterol efflux mechanisms and suppression of pro-inflammatory cytokine secretion.

Expression, purification and microscopic characterization of human ATP-binding cassette sub-family B member 7 protein.

The ATP-binding cassette sub-family B member 7 (ABCB7) is a membrane transport protein located on the inner membrane of mitochondria, which could be involved in the transport of heme from the mitochondria to the cytosol. ABCB7 also plays a central role in the maturation of cytosolic iron-sulfur (Fe/S) cluster-containing proteins, and mutations can cause a series of mitochondrial defects. X-linked sideroblastic anemia and ataxia (XLSA-A) is a rare cause of early onset ataxia, which may be overlooked due to the usually mild asymptomatic anemia. The genetic defect has been identified as a mutation in the ABCB7 gene at Xq12-q13. Here, we report the expression, purification and the 2D projections derived from negatively stained electron micrographs of recombinant H. sapiens ABCB7 (hABCB7), paving the way from an atomic structure determination of ABCB7.

A LysR-type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens.

Small RNAs (sRNAs) are universal posttranscriptional regulators of gene expression and hundreds of sRNAs are frequently found in each and every bacterium. In order to coordinate cellular processes in response to ambient conditions, many sRNAs are differentially expressed. Here, we asked how these small regulators are regulated using Agrobacterium tumefaciens as a model system. Among the best-studied sRNAs in this plant pathogen are AbcR1 regulating numerous ABC transporters and PmaR, a regulator of peptidoglycan biosynthesis, motility, and ampicillin resistance. We report that the LysR-type regulator VtlR (also known as LsrB) controls expression of AbcR1 and PmaR. A vtlR/lsrB deletion strain showed growth defects, was sensitive to antibiotics and severely compromised in plant tumor formation. Transcriptome profiling by RNA-sequencing revealed more than 1,200 genes with altered expression in the mutant. Consistent with the function of VtlR/LsrB as regulator of AbcR1, many ABC transporter genes were affected. Interestingly, the transcription factor did not only control the expression of AbcR1 and PmaR. In the mutant, 102 sRNA genes were significantly up- or downregulated. Thus, our study uncovered VtlR/LsrB as the master regulator of numerous sRNAs. Thereby, the transcriptional regulator harnesses the regulatory power of sRNAs to orchestrate the expression of distinct sub-regulons.

Production of a human mitochondrial ABC transporter in E. coli.

Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

Low oxygen tension differentially regulates the expression of placental solute carriers and ABC transporters.

Low oxygen concentration, or hypoxia, is an important physiological regulator of placental function including chemical disposition. Here, we compared the ability of low oxygen tension to alter the expression of solute carriers (SLC) and ABC transporters in two human placental models, namely BeWo cells and term placental explants. We found that exposure to low oxygen concentration differentially regulates transporter expression in BeWo cells, including downregulation of ENT1, OATP4A1, OCTN2, BCRP, and MRP2/3/5, and upregulation of CNT1, OAT4, OATP2B1, SERT, SOAT, and MRP1. Similar upregulation of MRP1 and downregulation of MRP5 and BCRP were observed in explants, whereas uptake transporters were decreased or unchanged. Furthermore, a screening of transcriptional regulators of transporters revealed that hypoxia leads to a decrease in the mRNA levels of aryl hydrocarbon receptor, nuclear factor erythroid 2-related factor 2, and retinoid x receptor alpha in both human placental models. These data suggest that transporter expression is differentially regulated by oxygen concentration across experimental human placental models.

A twist in the ABC: regulation of ABC transporter trafficking and transport by FK506-binding proteins.

Post-transcriptional regulation of ATP-binding cassette (ABC) proteins has been so far shown to encompass protein phosphorylation, maturation, and ubiquitination. Yet, recent accumulating evidence implicates FK506-binding proteins (FKBPs), a type of peptidylprolyl cis-trans isomerase (PPIase) proteins, in ABC transporter regulation. In this perspective article, we summarize current knowledge on ABC transporter regulation by FKBPs, which seems to be conserved over kingdoms and ABC subfamilies. We uncover striking functional similarities but also differences between regulatory FKBP-ABC modules in plants and mammals. We dissect a PPIase- and HSP90-dependent and independent impact of FKBPs on ABC biogenesis and transport activity. We propose and discuss a putative new mode of transient ABC transporter regulation by cis-trans isomerization of X-prolyl bonds.

ABCA8 is regulated by miR-374b-5p and inhibits proliferation and metastasis of hepatocellular carcinoma through the ERK/ZEB1 pathway.

BACKGROUND: ATP binding cassette subfamily A member 8 (ABCA8) belongs to the ATP binding cassette (ABC) transporter superfamily. ABCA8 is a transmembrane transporter responsible for the transport of organics, such as cholesterol, and drug efflux. Some members of the ABC subfamily, such as ABCA1, may inhibit cancer development. However, the mechanism of ABCA8 in the process of cancer activation is still ambiguous. METHODS: The expression of ABCA8 in human hepatocellular carcinoma (HCC) tissues and cell lines was examined using qPCR, immunoblotting, and immunohistochemical staining. The effects of ABCA8 on the proliferation and metastasis of HCC were examined using in vitro and in vivo functional tests. A luciferase reporter assay was performed to explore the binding between microRNA-374b-5p (miR-374b-5p) and the ABCA8 3'-untranslated region (UTR). RESULTS: ABCA8 was frequently down-regulated in HCC and this down-regulation was negatively correlated with prognosis. The overexpression of ABCA8 inhibited growth and metastasis in HCC, whereas the knockdown of ABCA8 exerted the antithetical effects both in vivo and in vitro. ABCA8 was down-regulated by miR-374b-5p; this down-regulation can induce epithelial transformation to mesenchyme via the ERK/ZEB1 signaling pathway and promote HCC progression. CONCLUSION: We exposed the prognostic value of ABCA8 in HCC, and illuminated a novel pathway in ABCA8-regulated inhibition of HCC tumorigenesis and metastasis. These findings may lead to a new targeted therapy for HCC through the regulation of ABCA8, and miR-374b-5p.

Developmental patterns in human blood-brain barrier and blood-cerebrospinal fluid barrier ABC drug transporter expression.

When drugs exert their effects in the brain, linear extrapolation of doses from adults could be harmful for children as the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) function is still immature. More specifically, age-related variation in membrane transporters may impact brain disposition. As human data on brain transporter expression is scarce, age dependent [gestational age (GA), postnatal age (PNA), and postmenstrual age (PMA)] variation in immunohistochemical localization and staining intensity of the ABC transporters P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins 1, 2, 4, and 5 (MRP1/2/4/5) was investigated. Post mortem brain cortical and ventricular tissue was derived from 23 fetuses (GA range 12.9-39 weeks), 17 neonates (GA range 24.6-41.3 weeks, PNA range 0.004-3.5 weeks), 8 children (PNA range 0.1-3 years), and 4 adults who died from a wide variety of underlying conditions. In brain cortical BBB, immunostaining increased with age for Pgp and BCRP, while in contrast, MRP1 and MRP2 staining intensity appeared higher in fetuses, neonates, and children, as compared to adults. BCSFB was positively stained for Pgp, MRP1, and MRP2 and appeared stable across age, while BCRP was not detected. MRP4 and MRP5 were not detected in BBB or BCSFB. In conclusion, human BBB and BCSFB ABC membrane transporters show brain location and transporter-specific maturation.

Circ-ABCB10 accelerates the malignant progression of oral squamous cell carcinoma by absorbing miRNA-145-5p.

OBJECTIVE: Previous studies have confirmed the carcinogenic role of circ-ABCB10 in certain types of tumors. However, the role of circ-ABCB10 in oral squamous cell carcinoma (OSCC) has not been reported yet. This report investigated the biological function of circ-ABCB10 in aggravating the progression of OSCC by absorbing microRNA-145-5p (miRNA-145-5p) as a ceRNA. PATIENTS AND METHODS: Relative levels of circ-ABCB10 and miRNA-145-5p in OSCC tissues and cell lines were determined. The potential relation between circ-ABCB10 level and pathological indexes of OSCC patients was analyzed. Regulatory effects of circ-ABCB10 and miRNA-145-5p on proliferative and migratory capacities of CAL-27 and Tca8113 cells were assessed. The interaction between circ-ABCB10 and miRNA-145-5p was examined through dual-luciferase reporter gene assay and Chi-square test. At last, rescue experiments were carried out to uncover the role of the circ-ABCB10/miRNA-145-5p regulatory loop in regulating the progression of OSCC. RESULTS: Circ-ABCB10 was upregulated in OSCC tissues and cells. OSCC patients expressing a high level of circ-ABCB10 presented worse tumor staging and a higher rate of distant metastasis relative to those with low level. Knockdown of circ-ABCB10 attenuated proliferative and migratory capacities in CAL-27 and Tca8113 cells. Besides, miRNA-145-5p was downregulated in OSCC tissues and cells. The knockdown of miRNA-145-5p accelerated OSCC cells to proliferate and migrate. Dual-luciferase reporter gene assay proved the binding between circ-ABCB10 and miRNA-145-5p. Moreover, the miRNA-145-5p level was negatively correlated to circ-ABCB10 level in OSCC tissues. Rescue experiments indicated that miRNA-145-5p knockdown could reverse the regulatory effects of circ-ABCB10 on viability, colony formation, and migratory capacity in OSCC cells. CONCLUSIONS: Circ-ABCB10 is upregulated in OSCC, which is closely related to tumor staging and distant metastasis of OSCC patients. Circ-ABCB10 aggravates the progression of OSCC by absorbing miRNA-145-5p.

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection.

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.

Long noncoding RNA FAM201A mediates the metastasis of lung squamous cell cancer via regulating ABCE1 expression.

OBJECTIVE: Long noncoding RNA (lncRNA) family with sequence similarity 201-member A (FAM201A) is a novel lncRNA promoting the development of various cancers. However, the biological function of FAM201A on the metastasis of lung squamous cell carcinoma (LSCC) remains unknown. The aim of this study was to explore the molecular mechanism of FAM201A and its target protein in advanced LSCC. PATIENTS AND METHODS: Quantitative Polymerase Chain Reaction (qPCR) was applied to evaluate FAM201A expression in lung cancer tissues. The impact of high FAM201A expression on the overall survival in patients with lung cancer was tested using the log-rank test. The relevance between aberrant FAM201A and clinicopathological characteristics in patients with lung cancer was analyzed using the Chi-square tests. Cell proliferation was assayed using the Cell Counting Kit-8 (CCK-8) and a transwell assay, and the mice xenograft models were applied to determine the promoting effects of FAM201A on LSCC in vitro and in vivo. The underlying regulatory mechanism was explored through RNA transfection, qPCR, and Western blotting. The correlation between ATP-binding cassette transporter E1 (ABCE1) and FAM201A expression was verified using Spearman's correlation coefficient. RESULTS: FAM201A is aberrantly elevated in tissues from patients with non-small cell lung cancer. High levels of FAM201A expression were more likely to present in patients with squamous type, M1 stage, and inferior overall survival. Differential expression was found between non-metastatic and metastatic squamous carcinoma, but not in adenocarcinoma. FAM201A knockdown inhibits cell proliferation, migration, and invasion of LSCC cells in vitro, and represses tumor growth in vivo. Furthermore, ABCE1 in LSCC cells was downregulated by silencing FAM201A. The tissue level of ABCE1 was positively correlated with FAM201A expression in patients with LSCC. CONCLUSIONS: FAM201A may markedly induce migration and invasion of LSCC, resulting in the M1 stage and poor survival. These findings suggest the FAM201A-ABCE1 axis as a novel therapeutic target in LSCC.

Identification of candidate ATP-binding cassette transporter gene family members in Diaphorina citri (Hemiptera: Psyllidae) via adult tissues transcriptome analysis.

The ATP-binding cassette (ABC) transporters exist in all living organisms and play major roles in various biological functions by transporting a wide variety of substrates across membranes. The functions of ABC transporters in drug resistance have been extensively studied in vertebrates; however, they are rarely characterized in agricultural pests. The Asian citrus psyllid, Diaphorina citri, is one of the most damaging pests of the Citrus genus because of its transmission of Huanglongbing, also known as Yellow Dragon disease. In this study, the next-generation sequencing technique was applied to research the ABC transporters of D. citri. Fifty-three ABC transporter genes were found in the RNA-Seq data, and among these ABC transporters, 4, 4, 5, 2, 1, 4, 18 and 15 ABC proteins belonged to the ABCA-ABCH subfamilies, respectively. Different expression profiles of 52 genes between imidacloprid-resistant and imidacloprid-susceptible strains were studied by qRT-PCR; 5 ABCGs and 4 ABCHs were significantly upregulated in the imidacloprid-resistant strain. In addition, five of the nine upregulated genes were widely expressed in adult tissues in spatial expression analysis. The results suggest that these genes may play key roles in this phenotype. In general, this study contributed to our current understanding of D. citri resistance to insecticides.

Lidocine potentiates the cytotoxicity of 5-fluorouracil to choriocarcinoma cells by downregulating ABC transport proteins expression.

Choriocarcinoma is a gestational trophoblastic cancer, which often occurs in the first 3 months of pregnancy. 5-Fluorouracil (5-Fu) is the widely used chemotherapeutic drug for choriocarcinoma but limited by drug resistance. Lidocaine, an aminamide-type anesthetic, shows potential anticancer and chemosensitization effects in recent years. Herein, we tested the possible chemosensitization activity of lidocaine on the cytotoxicity of 5-Fu in choriocarcinoma cells. Viabilities and apoptosis of choriocarcinoma JEG-3 and JAR cells after lidocaine and/or 5-Fu treatment were detected using Cell Counting Kit-8 assay, annexin V-FITC/PI (fluorescein isothiocyanate/propidium iodide) staining and Western blot analysis, respectively. Quantitative reverse transcription polymerase chain reaction was done to measure breast cancer resistance protein (ABCG2) messenger RNA level. Western blot analysis was carried out to detect ABCG2, P-glycoprotein (P-gp), MRP1, and MRP2 protein levels. pEX-ABCG2 was transfected to elevate ABCG2 level. Then, the influence of ABCG2 on lidocaine + 5-Fu-caused cell viability loss, apoptosis, and inactivation of PI3K/AKT pathway were analyzed. We found that lidocaine in low concentration had no significant cytotoxicity to JEG-3 and JAR cells, but stimulated cell apoptosis in high concentration. Moreover, lidocaine potentiated the cytotoxicity of 5-Fu to JEG-3 and JAR cells through decreasing viability and increasing apoptosis. Lidocaine treatment reduced the ABCG2, P-gp, MRP1, and MRP2 protein levels in cells. Overexpression of ABCG2 reversed the synergistic effects of lidocaine + 5-Fu on JEG-3 and JAR cell viability and apoptosis, as well as PI3K/AKT pathway. Our research verified that lidocaine potentiated the cytotoxicity of 5-Fu to choriocarcinoma cells by downregulating ATP-binding cassette (ABC) transport proteins expression.

Proteomic evidence that ABCA4 is vital for traumatic proliferative vitreoretinopathy formation and development.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment failure. The mechanism of PVR development is complex and still not completely elucidated. There are no proven methods for early prevention or clinical treatment. Retinal proteins are abnormally expressed during the entire PVR disease process. Due to the limitations of research methods and techniques, we do not fully understand the retinal protein changes in PVR. This proteomics study systemically analyzed and identified differential protein expression between retinas of PVR and non-PVR (normal) eyes. Retinal samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) coupled with mass spectrometry. Raw data were processed and analyzed by Maxquant software and then searched against the human UniProKB (201510) protein database. Differentially expressed proteins were selected and further validated in a human retinal pigment epithelial (RPE) cell line. The effects of dysregulated proteins on cell proliferation, apoptosis, and migration were studied. Systemic proteomics analysis identified several PVR-enriched proteins. The differentially expressed proteins were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation to find abnormal pathways involved in PVR. Retinal-specific ATP-binding cassette transporter (ABCA4) expression was one of the most increased proteins in PVR tissue. ABCA4 knockdown significantly reduced proliferation and affected the cell cycle in the human RPE cell line. ABCA4 knockdown also induced apoptosis and inhibited retinal cell migration. In conclusion, systemic proteomics analysis identified differentially expressed proteins in traumatic PVR, with ABCA4 being highly expressed. Disruption of ABCA4 expression induced apoptosis and inhibited cell proliferation and migration in a human RPE cell line.

Cloning and expression of selected ABC transporters from the Arabidopsis thaliana ABCG family in Pichia pastoris.

Phytohormones play a major role in plant growth and development. They are in most cases not synthesized in their target location and hence need to be transported to the site of action, by for instance ATP-binding cassette transporters. Within the ATP-binding cassette transporter family, Pleiotropic Drug Resistance transporters are known to be involved in phytohormone transport. Interestingly, PDRs are only present in plants and fungi. In contrast to fungi, there are few biochemical studies of plant PDRs and one major reason is that suitable overexpression systems have not been identified. In this study, we evaluate the expression system Pichia pastoris for heterologous overexpression of PDR genes of the model plant Arabidopsis thaliana. We successfully cloned and expressed the potential phytohormone transporters PDR2 and PDR8 in P. pastoris. Sucrose gradient centrifugation confirmed that the overexpressed proteins were correctly targeted to the plasma membrane of P. pastoris and initial functional studies demonstrated ATPase activity for WBC1. However, difficulties in cloning and heterologous overexpression might be particular obstacles of the PDR family, since cloning and overexpression of White Brown Complex 1, a half-size transporter of the same ABCG subfamily with comparable domain organization, was more easily achieved. We present strategies and highlight critical factors to successfully clone plant PDR genes and heterologously expressed in P. pastoris.

Transcriptome analysis identifies activated signaling pathways and regulated ABC transporters and solute carriers after hyperosmotic stress in renal MDCK I cells.

Hyperosmolality is found under physiological conditions in the kidneys, whereas hyperosmolality in other tissues may be associated with pathological conditions. In such tissues an association between inflammation and hyperosmolality has been suggested. During hyperosmotic stress, an important phenomenon is upregulation of solute carriers (SLCs). We hypothesize that hyperosmolality affects the expression of many SLCs as well as ABC transporters. Through RNA-sequencing and topological pathway analysis, the cell cycle, the cytokine-cytokine receptor interaction pathway, and the chemokine-signaling pathway were significantly activated in MDCK I cells after hyperosmotic treatment (Δ200 mOsm) with raffinose or NaCl. 9065, 8052 and 5018 genes were significantly regulated by raffinose, NaCl or urea supplementation (500 mOsm), respectively, compared to control (300 mOsm). Cytokines, that have not previously been associated with hyperosmolality, were identified. We further provide an overview of transport proteins that could be of relevance in tissues exposed to hyperosmolality. Especially Slc5a8 was found highly up-regulated.

Curcumin potentiates the fungicidal effect of dodecanol by inhibiting drug efflux in wild-type budding yeast.

Drug resistance commonly occurs when treating immunocompromised patients who have fungal infections. Curcumin, is a compound isolated from Curcuma longa, has been reported to inhibit drug efflux in several human cell lines and nonpathogenic budding yeast Saccharomyces cerevisiae cells that overexpresses the ATP-binding cassette (ABC) transporters S. cerevisiae Pdr5p and pathogenic Candida albicans Cdr1p and Cdr2p. The aim of this study was to examine the effects of curcumin on multidrug resistance in a wild-type strain of the budding yeast with an intrinsic expression system of multidrug efflux-related genes. The antifungal activity of dodecanol alone was temporary against S. cerevisiae; however, restoration of cell viability was completely inhibited when the cells were co-treated with dodecanol and curcumin. Furthermore, restriction of rhodamine 6G (R6G) efflux from the cells and intracellular accumulation of R6G were observed with curcumin treatment. Reverse transcription-polymerase chain reaction analysis revealed that curcumin reduced the dodecanol-induced overexpression of the ABC transporter-related genes PDR1, PDR3 and PDR5 to their control levels in untreated cells. Curcumin can directly restrict the glucose-induced drug efflux and inhibits the expression of the ABC transporter gene PDR5, and can thereby inhibit the efflux of dodecanol from S. cerevisiae cells. Curcumin is effective in potentiating the efficacy of antifungal drugs via its effects on ABC transporters. SIGNIFICANCE AND IMPACT OF THE STUDY: Drug resistance is common in immunocompromised patients with fungal infections. Curcumin, isolated from Curcuma longa, inhibits drug efflux in nonpathogenic budding yeast Saccharomyces cerevisiae cells overexpressing ABC transporters S. cerevisiae Pdr5p and pathogenic Candida albicans Cdr1p and Cdr2p. We examined the effects of curcumin on multidrug resistance in a wild-type strain of the budding yeast with an intrinsic expression system of multidrug efflux-related genes. Curcumin directly inhibited drug efflux and also suppressed the PDR5 expression, thereby enhancing the antifungal effects. Thus, curcumin potentially promotes the efficacy of antifungals via its effects on ABC transporters in wild-type fungal strains.