共查询到20条相似文献,搜索用时 0 毫秒
1.
《Journal of molecular biology》2021,433(13):166980
ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E1S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates. 相似文献
2.
《Journal of molecular biology》2021,433(21):167218
ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport. 相似文献
3.
Akiyoshi Nakayama Hirotaka Matsuo Tappei Takada Kimiyoshi Ichida Takahiro Nakamura Yuki Ikebuchi 《Nucleosides, nucleotides & nucleic acids》2013,32(12):1091-1097
The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3′-azido-3′-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10?5). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion. 相似文献
4.
Human neutrophil antigen 2 (HNA-2) deficiency is a common phenotype as 3–5% humans do not express HNA-2. HNA-2 is coded by CD177 gene that associates with human myeloproliferative disorders. HNA-2 deficient individuals are prone to produce HNA-2 alloantibodies that cause a number of disorders including transfusion-related acute lung injury and immune neutropenia. In addition, the percentages of HNA-2 positive neutrophils vary significantly among individuals and HNA-2 expression variations play a role in human diseases such as myelodysplastic syndrome, chronic myelogenous leukemia, and gastric cancer. The underlying genetic mechanism of HNA-2 deficiency and expression variations has remained a mystery. In this study, we identified a novel CD177 nonsense single nucleotide polymorphism (SNP 829A>T) that creates a stop codon within the CD177 coding region. We found that all 829TT homozygous individuals were HNA-2 deficient. In addition, the SNP 829A>T genotypes were significantly associated with the percentage of HNA-2 positive neutrophils. Transfection experiments confirmed that HNA-2 expression was absent on cells expressing the CD177 SNP 829T allele. Our data clearly demonstrate that the CD177 SNP 829A>T is the primary genetic determinant for HNA-2 deficiency and expression variations. The mechanistic delineation of HNA-2 genetics will enable the development of genetic tests for diagnosis and prognosis of HNA-2-related human diseases. 相似文献
5.
Madhumita Pradhan Leslie A. Bembinster Sarah C. Baumgarten Jonna Frasor 《The Journal of biological chemistry》2010,285(41):31100-31106
6.
Divya K. Rao Haiyan Liu Suresh V. Ambudkar Michael Mayer 《The Journal of biological chemistry》2014,289(45):31397-31410
This paper introduces a strategy to kill selectively multidrug-resistant cells that express the ABCG2 transporter (also called breast cancer resistance protein, or BCRP). The approach is based on specific stimulation of ATP hydrolysis by ABCG2 transporters with subtoxic doses of curcumin combined with stimulation of ATP hydrolysis by Na+,K+-ATPase with subtoxic doses of gramicidin A or ouabain. After 72 h of incubation with the drug combinations, the resulting overconsumption of ATP by both pathways inhibits the efflux activity of ABCG2 transporters, leads to depletion of intracellular ATP levels below the viability threshold, and kills resistant cells selectively over cells that lack ABCG2 transporters. This strategy, which was also tested on a clinically relevant human breast adenocarcinoma cell line (MCF-7/FLV1), exploits the overexpression of ABCG2 transporters and induces caspase-dependent apoptotic cell death selectively in resistant cells. This work thus introduces a novel strategy to exploit collateral sensitivity (CS) with a combination of two clinically used compounds that individually do not exert CS. Collectively, this work expands the current knowledge on ABCG2-mediated CS and provides a potential strategy for discovery of CS drugs against drug-resistant cancer cells. 相似文献
7.
We have studied heritability of the concentration of each glycolytic intermediate and adenine nucleotide in the cytosol of human erythrocytes obtained from a random sample of apparently healthy young individuals. Preliminary to analysis of heritability, each trait was statistically described and the effects attributable to variation in measured concomitants were removed by regression. Heritability was estimated using the family-set method. This method removes covariances between the index case, sibling and first cousin, due to those environmental determinants of the phenotypic values that are shared with a matched, unrelated control member of the family set. It also removes covariances due to environments that are shared by siblings and first cousins. Heritability was estimated by employing the fact that the variance of differences between first cousins minus the variance of differences between full siblings estimates three-fourths of the additive genetic variance. The heritability estimates for G6P†, F6P, ATP and some other metabolite concentrations are high and significantly greater than zero. The heritabilities of G6P and F6P are likely attributable to genetic variation in the in vivo activity of HK and/or PFK, because the concentrations of these metabolites are tightly controlled by the two regulatory enzymes. Statistically significant heritability estimates for HK and PFK mass action ratios strongly suggest genes are responsible for a portion of the quantitative variation in these enzyme activities. Since HK and PFK regulate glycolysis and the production of ATP, genetic variation in their activities might be causally related to the heritability of ATP concentration. 相似文献
8.
9.
10.
11.
12.
人钠依赖性二羧酸转运蛋白2(hSDCT2)的组织表达谱分析 总被引:1,自引:0,他引:1
利用DNA重组技术 ,构建重组表达质粒pGEX hSDCT2 .IPTG诱导其表达后 ,采用谷胱甘肽 Sepharose 4B亲和层析 ,获得纯化的谷胱甘肽S 转移酶 (GST) hSDCT2重组融合蛋白 .以此为免疫原免疫兔制备GST hSDCT2融合蛋白抗体 .多组织Northern印迹法结果显示 ,3 6kb的hSDCT2基因转录产物 ,在心、骨骼肌、胸腺、小肠、肺和外周血白细胞等组织中几乎不表达 ,在脑、结肠、脾、肝和胎盘中仅有少量表达 ,但在肾脏中大量表达 ;并且在肾脏和脾脏中还存在着另一种约 4 3kb的转录产物 .Western印迹法证实 ,hSDCT2蛋白以类似方式于上述组织表达 .免疫组化双重染色结果发现 ,与分布于近端肾小管刷状缘的hSDCT1不同 ,hSDCT2主要分布于近端肾小管的基底膜侧 .这些结果为进一步研究人钠依赖性二羧酸转运蛋白 2的结构和功能奠定了基础 . 相似文献
13.
Marcus Rattray Sonia Baldessari †Marco Gobbi †Tiziana Mennini Rosario Samanin Caterina Bendotti 《Journal of neurochemistry》1996,67(2):463-472
Abstract: After a single intraperitoneal injection of the irreversible tryptophan hydroxylase inhibitor p -chlorophenylalanine (PCPA; 300 mg/kg), there was a rapid down-regulation of serotonin (5-HT) transporter mRNA levels in cell bodies. This change was significant at 1 and 2 days after PCPA administration within the ventromedial but not the dorsomedial portion of the dorsal raphe nucleus. Seven days after PCPA treatment, 5-HT transporter mRNA levels were significantly elevated compared with controls in both regions of the dorsal raphe nucleus. PCPA administration produced no change in the [3 H]-citalopram binding and synaptosomal [3 H]5-HT uptake in terminal regions at 2 and 7 days after treatment but significantly reduced both these parameters by ∼20% in the hippocampus and in cerebral cortex 14 days after PCPA administration. The striatum showed a lower sensitivity to this effect. No significant changes were observed in the levels of [3 H]citalopram binding to 5-HT cell bodies in the dorsal raphe nucleus. In the same animals used for 5-HT transporter mRNA level measurements, levels of tryptophan hydroxylase mRNA in neurons of the ventromedial and dorsomedial portions of the dorsal raphe nucleus were increased 2 days after PCPA administration and fell to control levels 7 days after injection in the ventromedial region but not in the dorsomedial portion of the dorsal raphe nucleus, where they remained significantly higher than controls. Altogether, these results show that changes in 5-HT transporter mRNA are not temporally related to changes in 5-HT transporter protein levels. In addition, our results suggest that the 5-HT transporter and tryptophan hydroxylase genes are regulated by different mechanisms. We also provide further evidence that dorsal raphe 5-HT neurons are differentially regulated by drugs, depending on their location. 相似文献
14.
Shinya Nagamatsu Hiroki Sawa Akira Wakizaka Takao Hoshino 《Journal of neurochemistry》1993,61(6):2048-2053
15.
16.
Naoki Kobayashi Nobuyoshi Kobayashi Akihito Yamaguchi Tsuyoshi Nishi 《The Journal of biological chemistry》2009,284(32):21192-21200
Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5′-(β,γ-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent Km value of 21 μm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.Sphingosine 1-phosphate (S1P),2 a bioactive lipid molecule present in the blood, plays an important role in diverse cellular responses, such as migration, proliferation, and differentiation (1, 2). These processes are triggered by the binding of S1P to its specific receptors (3), of which five subtypes (S1P1-S1P5) have been identified in endothelial and immune cells (4). Studies using S1P1 receptor-deficient mice showed abnormalities in lymphocyte egress from lymph nodes, spleen, and thymus (5, 6). Whereas blood plasma contains a basal level of S1P from the nanomolar to the micromolar range (7–12), lymphoid tissues maintain a low S1P environment through the activity of S1P lyase (13). It has been proposed that a higher concentration of S1P in the blood plasma than in the lymphoid organs establishes an essential gradient along which lymphocytes expressing the S1P1 receptor on cell surfaces migrate (2, 5, 6, 13–15).The source of plasma S1P remains unclear despite its importance in the cellular responses of endothelial cells and lymphocytes. Unlike most cells, blood cells, astrocytes, and vascular endothelial cells are reported to release S1P (8, 16–18). These cells contain sphingosine kinase, which synthesizes S1P through the phosphorylation of sphingosine (16, 18, 19). Whereas platelets and mast cells release S1P in a stimulus-dependent manner (17, 20), erythrocytes, neutrophils, and mononuclear cells release S1P in a stimulus-independent manner (16). The roles of S1P derived from erythrocytes, the most abundant of these blood cells, have not been elucidated. However, recent reports suggest that S1P released from erythrocytes is a major source of plasma S1P (7, 9) and promotes lymphocyte egress to blood (9).Previously, we showed that S1P is released from rat platelets upon stimulation by thrombin or Ca2+ (21). We proposed that an ATP-dependent transporter plays a key role in S1P release from platelets (21). However, the detailed mechanism of S1P release is unclear because there is no way to assay the transport of S1P across the membrane. In this study we compared the properties of S1P release from erythrocytes with that of platelets and showed that S1P release from erythrocytes does not require any stimuli. We then established an assay to measure the ATP-dependent S1P uptake into inside-out membrane vesicles (IOVs) prepared from rat erythrocytes and characterized S1P transport in erythrocytes. 相似文献
17.
Polyphenols contained within plant tissues are consumed in significant amounts in the human diet and are known to influence a number of biological processes. This study investigated the effects of an anthocyanin-rich berry-extract on glucose uptake by human intestinal Caco-2 cells. Acute exposure (15 min) to berry extract (0.125%, w/v) significantly decreased both sodium-dependent (Total uptake) and sodium-independent (facilitated uptake) 3H-D-glucose uptake. In longer-term studies, SGLT1 mRNA and GLUT2 mRNA expression were reduced significantly. Polyphenols are known to interact directly with glucose transporters to regulate the rate of glucose absorption. Our in vitro data support this mechanism and also suggest that berry flavonoids may modulate post-prandial glycaemia by decreasing glucose transporter expression. Further studies are warranted to investigate the longer term effects of berry flavonoids on the management of glycaemia in human volunteers. 相似文献
18.
Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter 总被引:16,自引:2,他引:14
下载免费PDF全文
![点击此处可从《Molecular biology of the cell》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Ralf Egner Friederike E. Rosenthal Anastasia Kralli Dominique Sanglard Karl Kuchler 《Molecular biology of the cell》1998,9(2):523-543
Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility. 相似文献
19.
Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.Download video file.(67M, mov) 相似文献