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1.
Proc Biol Sci ; 287(1934): 20201311, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32873204

RESUMO

Phytophagous insects can tolerate and detoxify toxic compounds present in their host plants and have evolved intricate adaptations to this end. Some insects even sequester the toxins for their defence. This necessitates specific mechanisms, especially carrier proteins that regulate uptake and transport to specific storage sites or protect sensitive tissues from noxious compounds. We identified three ATP-binding cassette subfamily B (ABCB) transporters from the transcriptome of the cardenolide-sequestering leaf beetle Chrysochus auratus and analysed their functional role in the sequestration process. These were heterologously expressed and tested for their ability to interact with various potential substrates: verapamil (standard ABCB substrate), the cardenolides digoxin (commonly used), cymarin (present in the species's host plant) and calotropin (present in the ancestral host plants). Verapamil stimulated all three ABCBs and each was activated by at least one cardenolide, however, they differed as to which they were activated by. While the expression of the most versatile transporter fits with a protective role in the blood-brain barrier, the one specific for cymarin shows an extreme abundance in the elytra, coinciding with the location of the defensive glands. Our data thus suggest a key role of ABCBs in the transport network needed for cardenolide sequestration.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Besouros/fisiologia , Proteínas de Insetos/metabolismo , Proteínas de Plantas/toxicidade , Animais
3.
PLoS Pathog ; 16(8): e1008763, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32834002

RESUMO

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Colite/patologia , Variação Genética , Macrófagos/imunologia , Salmonelose Animal/patologia , Salmonella typhimurium/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem da Célula , Colite/induzido quimicamente , Colite/imunologia , Colite/microbiologia , Análise Mutacional de DNA , Modelos Animais de Doenças , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Replicação Viral
4.
Ecotoxicol Environ Saf ; 204: 111069, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32758696

RESUMO

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glutationa/análogos & derivados , Mucosa Intestinal/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Oncorhynchus mykiss/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Saxitoxina/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Catalase/metabolismo , Dinoflagelados/metabolismo , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Mucosa Intestinal/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lisossomos/metabolismo , Frutos do Mar
5.
Life Sci ; 259: 118352, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32860804

RESUMO

AIMS: Lipopolysaccharide (LPS) induces inflammatory cholestasis by impairing expression, localization, and function of carriers involved in bile formation, e.g. bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). A specific therapy against this disease is still lacking. Therefore, we evaluated the anticholestatic effects of spironolactone (SL), a PXR ligand that regulates bile salt homeostasis, up-regulates Mrp2, and bears anti-inflammatory properties. MAIN METHODS: Male Wistar rats were divided into four groups: Control, SL (83.3 mg/kg/day of SL, i.p., for 3 days), LPS (2.5 mg/kg/day, i.p., at 8 am of the last 2 days, and 1.5 mg/kg/day at 8 pm of the last day), and SL + LPS. Biliary and plasma parameters and the expression, function, and localization of Mrp2 and Bsep were evaluated. KEY FINDINGS: SL partially prevented LPS-induced drop of basal bile flow by normalizing the bile salt-independent fraction of bile flow (BSIBF), via improvement of glutathione output. This was due to a recovery in Mrp2 transport function, the major canalicular glutathione transporter, estimated by monitoring the output of its exogenously administered substrate dibromosulfophthalein. SL counteracted the LPS-induced downregulation of Mrp2, but not that of Bsep, at both mRNA and protein levels. LPS induced endocytic internalization of both transporters, visualized by immunofluorescence followed by confocal microscopy, and SL partially prevented this relocalization. SL did not prevent the increase in IL-1ß, IL-6, and TNF-α plasma levels. SIGNIFICANCE: SL prevents the impairment in Mrp2 expression and localization, and the resulting recovery of Mrp2 function normalizes the BSIBF by improving glutathione excretion.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colestase/tratamento farmacológico , Espironolactona/uso terapêutico , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bile/metabolismo , Colestase/sangue , Colestase/metabolismo , Citocinas/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Masculino , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
6.
PLoS Pathog ; 16(8): e1008697, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776976

RESUMO

The diamondback moth, Plutella xylostella, is a cosmopolitan pest and the first species to develop field resistance to toxins from the gram-positive bacterium Bacillus thuringiensis (Bt). Although previous work has suggested that mutations of ATP-binding cassette transporter subfamily C2 (ABCC2) or C3 (ABCC3) genes can confer Cry1Ac resistance, here we reveal that P. xylostella requires combined mutations in both PxABCC2 and PxABCC3 to achieve high-level Cry1Ac resistance, rather than simply a mutation of either gene. We identified natural mutations of PxABCC2 and PxABCC3 that concurrently occurred in a Cry1Ac-resistant strain (Cry1S1000) of P. xylostella, with a mutation (RA2) causing the mis-splicing of PxABCC2 and another mutation (RA3) leading to the premature termination of PxABCC3. Genetic linkage analysis showed that RA2 and RA3 were tightly linked to Cry1Ac resistance. Introgression of RA2 and RA3 enabled a susceptible strain (G88) of P. xylostella to obtain high resistance to Cry1Ac, confirming that these genes confer resistance. To further support the role of PxABCC2 and PxABCC3 in Cry1Ac resistance, frameshift mutations were introduced into PxABCC2 and PxABCC3 singly and in combination in the G88 strain with CRISPR/Cas9 mediated mutagenesis. Bioassays of CRISPR-based mutant strains, plus genetic complementation tests, demonstrated that the deletion of PxABCC2 or PxABCC3 alone provided < 4-fold tolerance to Cry1Ac, while disruption of both genes together conferred >8,000-fold resistance to Cry1Ac, suggesting the redundant/complementary roles of PxABCC2 and PxABCC3. This work advances our understanding of Bt resistance in P. xylostella by demonstrating mutations within both PxABCC2 and PxABCC3 genes are required for high-level Cry1Ac resistance.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Bacillus thuringiensis , Proteínas de Insetos/química , Proteínas de Insetos/genética , Mariposas/química , Mariposas/genética , Mariposas/metabolismo , Mutação , Alinhamento de Sequência
7.
Gene ; 759: 145000, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32717310

RESUMO

Upregulation of the ATP-binding cassette (ABC) transporter is one of the most important factors leading to multidrug resistance (MDR) in several types of cancer. In the present study, we investigated the ability of rucaparib, a Poly (ADP-ribose) polymerase (PARP) inhibitor which is currently in clinical development, on overcoming ABC transporters-mediated MDR in cervical cancer cell lines. Rucaparib significantly enhanced the cytotoxic effects of a series of conventional chemotherapeutic drugs in drug resistance cervical cancer cell lines. Moreover, rucaparib significantly increased the accumulation of rhodamine 123 in doxorubicin- and paclitaxel-resistance cervical cancer cell lines. In addition, rucaparib significantly increased the accumulation of tritium-labeled chemotherapeutic drugs in drug resistance cervical cancer cells, and decrease the efflux of tritium-labeled chemotherapeutic drugs. Molecular docking study indicated that rucaparib could bind to the active site of the ABC transporters. The present study indicated that rucaparib could antagonize MDR in cervical cancer cells by blocking the function of ABC transporters. The results obtained in the present study provide the potential possibilities that the combination of rucaparib with other chemotherapeutic agents may benefit patients with cervical cancer.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Indóis/farmacologia , Simulação de Acoplamento Molecular , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias do Colo do Útero/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Ligação Proteica
8.
Life Sci ; 257: 118079, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32668326

RESUMO

PURPOSE: Obesity affecting drug pharmacokinetics results in the risk of the therapeutic failure or toxic side effects of drugs increasing. Unfortunately, the pharmacokinetic data in obese patients still lack for majority of drugs. Therefore, our study principally investigated the effect of obesity induced by high fat-diet (HFD) on the pharmacokinetics of rosuvastatin and explored the underlying mechanism via the hepatic pregnane X receptor (Pxr)- organic anion transporting polypeptide 2 (Oatp2) signaling pathway and multidrug resistance-associated protein 2 (Mrp2) in rats. MAIN METHODS: Rats with obesity was induced by HFD for 4 weeks, and subsequently, the effect of obesity on the blood concentration, pharmacokinetic parameters and biliary excretion of rosuvastatin administrated intravenously and the hepatic uptake of rosuvastatin in the rat primary hepatocytes were evaluated. Additionally, in order to illuminate the underlying mechanism, the alterations of the mRNA expressions of Oatp2, Mrp2 and Pxr and the concentrations of lithocholic acid (LCA), glycine-LCA (GLCA) and taurine-LCA (TLCA) in liver were determined. KEY FINDINGS: The blood concentration of rosuvastatin that has great relationship with the muscle toxicity increased in rats with HFD-induced obesity, which could be principally ascribed to the decreased hepatic uptake of rosuvastatin that was mainly resulted from the inhibition of hepatic Pxr-Oatp2 pathway. SIGNIFICANCE: The decreased hepatic uptake of rosuvastatin causing the increase of the rosuvastatin concentration in blood under the condition of HFD-induced obesity provides a cue for clinicians to reduce the rosuvastatin dose for obese patients to avoid the occurrence risk of the muscle toxicity of rosuvastatin.


Assuntos
Fígado/metabolismo , Obesidade/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Receptor de Pregnano X/metabolismo , Rosuvastatina Cálcica/farmacocinética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Anticolesterolemiantes/farmacocinética , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Masculino , Ratos , Ratos Wistar
9.
Life Sci ; 257: 118131, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32710948

RESUMO

AIMS: ATP-binding cassette (ABC) transporters constitute one of the largest families of membrane proteins in most organisms; however, their functions in hepatocellular carcinoma (HCC) remain unclear. MAIN METHODS: A set of bioinformatic tools was integrated to analyze the expression of 49 members of the ABC transporter family. The function of members which had prognostic values in HCC was explored by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. KEY FINDINGS: ABCA8 and ABCA9 were significantly down-regulated in HCC. Prognostic analysis indicated that HCC patients with low expression of ABCA8 and ABCA9 had significantly shorter survival time. On the contrary, ABCB6 was over-expressed in the disease and high expression of ABCB6 was associated with worse prognosis. Co-expression analysis, and subsequently GO and KEGG analysis indicated that ABCA8 and ABCA9 might participate in the catabolic processes of multiple metabolites, while ABCB6 might regulate ferroptosis. SIGNIFICANCE: This study reveals a previously unrecognized function of ABCB6 in HCC, by regulating ferroptosis. Since ABCB6 is over-expressed in HCC and ferroptosis involves in cancer development, ABCB6 might be a promising therapeutic target in the disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carcinoma Hepatocelular/metabolismo , Ferroptose , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/mortalidade , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/mortalidade , Prognóstico , Mapas de Interação de Proteínas , Análise de Sobrevida
10.
Proc Natl Acad Sci U S A ; 117(32): 19228-19236, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703810

RESUMO

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Sphingomonadaceae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Glutationa/química , Glutationa/metabolismo , Ferro/metabolismo , Domínios Proteicos , Sphingomonadaceae/química , Sphingomonadaceae/genética
11.
Chemosphere ; 260: 127627, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32673864

RESUMO

Nickel is the most prevailing metal allergen with the highest sensitization rate among the "TOP 25" contact allergens and can affect about 15% of the human population. It is an essential trace metal in plants, animals, and humans. However, the environmental levels of nickel are considerably higher than what is needed for human life. Exposure to high levels of nickel can lead to skin allergies, lung fibrosis, and carcinogenesis. Few existing studies have closely examined the toxicity of nickel, let alone investigated the effective detoxification pathways. Here, we developed a high-throughput screening platform to comprehensively evaluate the nickel toxicity in wild-type C. elegans and explore the underlying detoxification mechanisms in transgenic nematodes. We demonstrated that nickel exerted multiple toxic effects on growth, brood size, feeding, and locomotion in C. elegans. Of which, brood size is the most sensitive endpoint. Nickel was found to first bind to phytochelatin (PC) after entering the worms' body and this PC-Ni complex was further transported by the ABC transporter, CeHMT-1, into the coelomocytes for further detoxification. Our study also demonstrated that the high-throughput screening platform is a promising system for evaluation and investigation of the ecological risks of heavy metals.


Assuntos
Caenorhabditis elegans/fisiologia , Níquel/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Caenorhabditis elegans/metabolismo , Locomoção , Metais Pesados/toxicidade , Nematoides , Níquel/toxicidade , Fitoquelatinas/metabolismo
12.
Toxicology ; 441: 152527, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32553669

RESUMO

Multidrug resistance-associated protein 2 (Mrp2), expressed at the brush border membrane (BBM) of the enterocyte, is an ABC transporter with relevant intestinal barrier function. Its toxicological relevance lies in preventing absorption and tissue accumulation of dietary contaminants, drugs, and potentially harmful endogenous metabolites. Expression and activity of intestinal Mrp2 is downregulated in LPS-induced endotoxemia. In addition, confocal microscopy studies demonstrated internalization of the transporter to endocytic vesicles. Since IL-1ß plays an important role as early mediator of LPS-inflammatory responses, we evaluated whether IL-1ß mediates LPS-induced impairment of Mrp2 function. Two protocols were used: I) In vivo administration of LPS (5 mg/kg b.wt., i.p., single dose) to rats in simultaneous with administration of anti-IL-1ß (25 µg/kg b.wt., i.p., 4 doses), followed by studies of Mrp2 expression, localization and activity, 24 h after LPS administration; II) In vitro incubation of isolated intestinal sacs with IL-1ß (10 ng/mL) for 30 min, followed by analysis of Mrp2 activity and localization. We found that in vivo immunoneutralization of IL-1ß partially prevented the decrease of Mrp2 protein expression and activity as well as its internalization to intracellular domains induced by LPS. Involvement of IL-1ß in the alteration of Mrp2 localization and activity was more directly demonstrated in isolated intestinal sacs, as incubation with IL-1ß resulted in detection of Mrp2 in intracellular regions of the enterocyte in simultaneous with alteration of transport activity. In conclusion, IL-1ß induces early internalization of intestinal Mrp2, which could partially explain loss of expression at the BBM under conditions of experimental endotoxemia. Concomitant impairment of Mrp2-dependent barrier function may have pathophysiological relevance since IL-1ß mediates the effect of many local and systemic inflammatory processes.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Endotoxemia/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/metabolismo , Animais , Western Blotting , Endotoxemia/patologia , Feminino , Mucosa Intestinal/ultraestrutura , Microscopia Confocal , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
13.
Food Chem ; 331: 127360, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32585548

RESUMO

The influence of food components on nanoparticle (NP) internalization indicates a need to investigate the behaviors of NPs in a complex system. This study measured the changes of TiO2 NP colloidal stability and quenching of anthocyanin fluorescence to indicate NP-anthocyanin interactions, and cytotoxicity, oxidative stress, expression of ABC transporters and intracellular Ti concentrations in 3D Caco-2 spheroids co-exposed to NPs and anthocyanins to indicate the influence of anthocyanins on NP bio-effects. The anthocyanins were observed to have minimal impacts on colloidal properties of TiO2 NPs. Meanwhile, NP-anthocyanin co-exposure did not induce cytotoxicity or oxidative stress. The fluorescence quenching study indicated the binding of anthocyanins onto TiO2 NPs, and the binding affinity was inversely correlated with NP internalization into 3D Caco-2 spheroids. This may be partially related with the up-regulation of ABC transporters. Our results may provide novel insights into understanding the interactions of NPs and anthocyanins with human intestinal cells.


Assuntos
Antocianinas/farmacologia , Nanopartículas Metálicas/química , Titânio/farmacocinética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antocianinas/química , Células CACO-2 , Coloides/farmacocinética , Depuradores de Radicais Livres/farmacologia , Humanos , Estresse Oxidativo/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Titânio/química
14.
Proc Natl Acad Sci U S A ; 117(19): 10476-10483, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32354992

RESUMO

Cholesterol-laden macrophage foam cells are a hallmark of atherosclerosis. For that reason, cholesterol metabolism in macrophages has attracted considerable scrutiny, particularly the mechanisms by which macrophages unload surplus cholesterol (a process referred to as "cholesterol efflux"). Many studies of cholesterol efflux in macrophages have focused on the role of ABC transporters in moving cholesterol onto high-density lipoproteins (HDLs), but other mechanisms for cholesterol efflux likely exist. We hypothesized that macrophages have the capacity to unload cholesterol directly onto adjacent cells. To test this hypothesis, we used methyl-ß-cyclodextrin (MßCD) to load mouse peritoneal macrophages with [13C]cholesterol. We then plated the macrophages (in the absence of serum or HDL) onto smooth muscle cells (SMCs) that had been metabolically labeled with [15N]choline. After incubating the cells overnight in the absence of HDL or serum, we visualized 13C and 15N distribution by nanoscale secondary ion mass spectrometry (NanoSIMS). We observed substantial 13C enrichment in SMCs that were adjacent to [13C]cholesterol-loaded macrophages-including in cytosolic lipid droplets of SMCs. In follow-up studies, we depleted "accessible cholesterol" from the plasma membrane of [13C]cholesterol-loaded macrophages with MßCD before plating the macrophages onto the SMCs. After an overnight incubation, we again observed substantial 13C enrichment in the SMCs adjacent to macrophages. Thus, macrophages transfer cholesterol to adjacent cells in the absence of serum or HDL. We suspect that macrophages within tissues transfer cholesterol to adjacent cells, thereby contributing to the ability to unload surplus cholesterol.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Transporte Biológico , Células Espumosas/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas HDL/metabolismo , Macrófagos/fisiologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Soro/metabolismo , beta-Ciclodextrinas/metabolismo
15.
Xenobiotica ; 50(11): 1380-1392, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32421406

RESUMO

Pesticides are now recognised to interact with drug transporters, but only few data are available on this issue for carbamate pesticides, a widely used class of agrochemicals, to which humans are highly exposed. The present study was therefore designed to determine whether four representative carbamate pesticides, i.e. the insecticides aminocarb and carbofuran, the herbicide chlorpropham and the fungicide propamocarb, may impair activities of main drug transporters implicated in pharmacokinetics. The interactions of carbamates with solute carrier and ATP-binding cassette transporters were investigated using cultured transporter-overexpressing cells, reference substrates and spectrofluorimetry-, liquid chomatography/tandem mass spectrometry- or radioactivity-based methods. Aminocarb and carbofuran exerted no or minimal effects on transporter activities, whereas chlorpropham inhibited BCRP and OAT3 activities and propamocarb decreased those of OCT1 and OCT2, but cis-stimulated that of MATE2-K. Such alterations of transporters however required chlorpropham/propamocarb concentrations in the 5-50 µM range, likely not relevant to environmental exposure. Trans-stimulation assays and propamocarb accumulation experiments additionally suggested that propamocarb is not a substrate for OCT1, OCT2 and MATE2-K. These data indicate that some carbamate pesticides can interact in vitro with some drug transporters, but only when used at concentrations higher than those expected to occur in environmentally exposed humans.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Carbamatos/metabolismo , Praguicidas/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Interações Medicamentosas , Humanos , Inseticidas , Proteínas de Neoplasias , Proteínas de Transporte de Cátions Orgânicos
16.
PLoS One ; 15(4): e0232476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353073

RESUMO

P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Biocatálise , Cloreto de Cálcio/metabolismo , Ensaios Enzimáticos , Hidrólise , Mutação , Naftóis , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Triazinas
17.
Nature ; 580(7803): 409-412, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296172

RESUMO

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis1-3. Although Mtb can synthesize vitamin B12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis2. Mtb does not encode any characterized cobalamin transporter4-6; however, the gene rv1819c was found to be essential for uptake of cobalamin1. This result is difficult to reconcile with the original annotation of Rv1819c as a protein implicated in the transport of antimicrobial peptides such as bleomycin7. In addition, uptake of cobalamin seems inconsistent with the amino acid sequence, which suggests that Rv1819c has a bacterial ATP-binding cassette (ABC)-exporter fold1. Here, we present structures of Rv1819c, which reveal that the protein indeed contains the ABC-exporter fold, as well as a large water-filled cavity of about 7,700 Å3, which enables the protein to transport the unrelated hydrophilic compounds bleomycin and cobalamin. On the basis of these structures, we propose that Rv1819c is a multi-solute transporter for hydrophilic molecules, analogous to the multidrug exporters of the ABC transporter family, which pump out structurally diverse hydrophobic compounds from cells8-11.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Bleomicina/metabolismo , Mycobacterium tuberculosis/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
18.
Nat Commun ; 11(1): 1763, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273501

RESUMO

Energy coupling factor (ECF) transporters are responsible for the uptake of micronutrients in bacteria and archaea. They consist of an integral membrane unit, the S-component, and a tripartite ECF module. It has been proposed that the S-component mediates the substrate transport by toppling over in the membrane when docking onto an ECF module. Here, we present multi-scale molecular dynamics simulations and in vitro experiments to study the molecular toppling mechanism of the S-component of a folate-specific ECF transporter. Simulations reveal a strong bending of the membrane around the ECF module that provides a driving force for toppling of the S-component. The stability of the toppled state depends on the presence of non-bilayer forming lipids, as confirmed by folate transport activity measurements. Together, our data provide evidence for a lipid-dependent toppling-based mechanism for the folate-specific ECF transporter, a mechanism that might apply to other ECF transporters.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Ácido Fólico/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Membrana Celular/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Conformação Proteica
19.
PLoS One ; 15(4): e0224643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348310

RESUMO

Grey mould is caused by the ascomycetes Botrytis cinerea in a range of crop hosts. As a biological control agent, the nucleoside antibiotic wuyiencin has been industrially produced and widely used as an effective fungicide. To elucidate the effects of wuyiencin on the transcriptional regulation in B. cinerea, we, for the first time, report a genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin. 2067 genes were differentially expressed, of them, 886 and 1181 genes were significantly upregulated and downregulated, respectively. Functional categorization indicated that transcript levels of genes involved in amino acid metabolism and those encoding putative secreted proteins were altered in response to wuyiencin treatment. Moreover, the expression of genes involved in protein synthesis and energy metabolism (oxidative phosphorylation) and of those encoding ATP-binding cassette transporters was markedly upregulated, whereas that of genes participating in DNA replication, cell cycle, and stress response was downregulated. Furthermore, wuyiencin resulted in mycelial malformation and negatively influenced cell growth rate and conidial yield in B. cinerea. Our results suggest that this nucleoside antibiotic regulates all aspects of cell growth and differentiation in B. cinerea. To summarize, some new candidate pathways and target genes that may related to the protective and antagonistic mechanisms in B. cinerea were identified underlying the action of biological control agents.


Assuntos
Antifúngicos/farmacologia , Botrytis/genética , Farmacorresistência Fúngica , Transcriptoma , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Botrytis/efeitos dos fármacos , Botrytis/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Regulação para Cima
20.
Ecotoxicol Environ Saf ; 197: 110606, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32304921

RESUMO

ATP binding cassette (ABC) transporters, types C, G, and B were monitored via qPCR in order to investigate the influence of heavy metal (HM) contamination of post-industrial and post-agricultural soils and the effects of its supplementation with sewage sludge, on Sinapis alba plants. Five house-keeping genes were selected and validated to ensure the best reference points. The relative expression of ABC types C and G genes was profoundly affected by experimental conditions and included their upregulation after plants exposure to heavy metals and downregulation after supplementation with sewage sludge. However, ABC type C was more responsive then type G. The experimental conditions altered the expression of ABC type C gene faster than ABC type G and thus, the expression of ABC type C can therefore potentially be used as a bioindicator during assisted phytoremediation of degraded sites. In clean soil, supplementation with sewage sludge with a slight content of heavy metals still caused an upregulation in the expression of ABC types C and G, which showed that proper toxicity assessments are necessary to ensure safe application of sewage sludge into soils. Results showed that the analysed genes take a significant part in plants metal detoxification and that their expression is regulated at transcriptional level after exposure to soil contaminated with heavy metals by both, industrial activities and by sewage sludge supplementation. Thus, their expression can potentially be used as an early-warning biomarker when soil supplementation with sewage sludge is incorporated into the soil-management process.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Metais Pesados/metabolismo , Esgotos , Sinapis/metabolismo , Poluentes do Solo/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Biodegradação Ambiental , Biomarcadores Ambientais , Metais Pesados/toxicidade , Sinapis/efeitos dos fármacos , Sinapis/genética , Solo/química , Poluentes do Solo/toxicidade
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