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Kazumitsu Ueda

Publications and source records attributed to Kazumitsu Ueda.

At least 19 recordsLinked to original sources
PubMed2007

Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol.

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2007

Human MDR1 and MRP1 recognize berberine as their transport substrate.

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency.

The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.

ATP-Binding Cassette Transporters↗
PubMed2006

Platelet derived growth factor regulates ABCA1 expression in vascular smooth muscle cells.

The ATP-binding cassette transporter A1 (ABCA1) regulates lipid efflux from peripheral cells to High-density lipoprotein. The platelet-derived growth factor (PDGF) is a potent mitogen that enables vascular smooth muscle cells to participate in atherosclerosis. In this report, we showed that PDGF suppressed endogenous expression of ABCA1 in cultured vascular smooth muscle cells. Exposure of CRL-208 cells to PDGF elicited a rapid phosphorylation of a kinase downstream from PI3-K, Akt. The constitutively active form of both p110, a subunit of PI3-K, and Akt inhibited activity of the ABCA1 promoter. In conclusion, PI3-K-Akt pathways participate in PDGF-suppression of ABCA1 expression.

ATP Binding Cassette Transporter 1↗
PubMed2006

Abl kinase interacts with and phosphorylates vinexin.

Non-receptor tyrosine kinase Abl is a well known regulator of the actin-cytoskeleton, including the formation of stress fibers and membrane ruffles. Vinexin is an adapter protein consisting of three SH3 domains, and involved in signal transduction and the reorganization of actin cytoskeleton. In this study, we found that vinexin alpha as well as beta interacts with c-Abl mainly through the third SH3 domain, and that vinexin and c-Abl were colocalized at membrane ruffles in rat astrocytes. This interaction was reduced by latrunculin B, suggesting an F-actin-mediated regulatory mechanism. We also found that vinexin alpha but not beta was phosphorylated at tyrosine residue when c-Abl or v-Abl was co-expressed. A mutational analysis identified tyrosine 127 on vinexin alpha as a major site of phosphorylation by c- or v-Abl. These results suggest that vinexin alpha is a novel substrate for Abl.

Actins↗
PubMed2006

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1.

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner.

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.

ATP Binding Cassette Transporter 1↗
PubMed2006

Purification and ATPase activity of human ABCA1.

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.

ATP Binding Cassette Transporter 1↗
PubMed2006

Heterologous expression of a mammalian ABC transporter in plant and its application to phytoremediation.

Mammalian ATP-binding cassette (ABC) transporters involved in the multidrug-resistance of cancer cells can efflux cytotoxic compounds that show a wide variety of chemical structures and biological activities. Human multidrug resistance-associated protein (hMRP1) is one of the most intensively studied ABC transporters and many substrates have been identified, including both organic and inorganic compounds. In an attempt at novel 'transport engineering' using hMRP1 as a molecular pump, we established transgenic tobacco plants that showed clear resistance to cadmium and daunorubicin, although they were not resistant to etoposide, another known substrate of hMRP1. When expressed in tobacco cells, hMRP1 protein was localized at vacuolar membrane, while members of the MRP family are localized at plasma membrane in mammalian cells to reduce the cellular accumulation of various drugs. Thus, the hMRP1-expressing tobacco cells were able to take up these substrates across the tonoplast and sequestrate them in the vacuolar matrix. These results suggest that it may be possible to use the transgenic tobacco in phytoremediation, where a single transformation with an ABC transporter with broad substrate specificity should be effective for extracting various environmental pollutants including both organic and inorganic compounds, and accumulate them in the plant body. This should be advantageous for the remediation of a complex polluted environment, which is commonly found in the real world.

Antibiotics, Antineoplastic↗
PubMed2006

Protein kinase A-dependent increase in WAVE2 expression induced by the focal adhesion protein vinexin.

The focal adhesion protein vinexin is a member of a family of adaptor proteins that are thought to participate in the regulation of cell adhesion, cytoskeletal reorganization, and growth factor signaling. Here, we show that vinexin beta increases the amount of and reduces the mobility on SDS-PAGE of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 protein, which is a key factor modulating actin polymerization in migrating cells. This mobility retardation disappeared after in vitro phosphatase treatment. Co-immunoprecipitation assays revealed the interaction of vinexin beta with WAVE2 as well as WAVE1 and N-WASP. Vinexin beta interacts with the proline-rich region of WAVE2 through the first and second SH3 domains of vinexin beta. Mutations disrupting the interaction impaired the ability of vinexin beta to increase the amount of WAVE2 protein. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin beta. Inhibition of protein kinase A (PKA) activity suppressed the vinexin-induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin beta. These results suggest that vinexin beta regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.

Actins↗
PubMed2006

Vinexin beta regulates the phosphorylation of epidermal growth factor receptor on the cell surface.

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration and oncogenesis. In this study, we found that exogenous expression of vinexin beta enhanced the phosphorylation of 180-kDa proteins in an EGF-dependent manner in Cos-7 cells. Western blot analysis using phospho-specific antibodies against EGFR identified EGFR as a phosphorylated 180-kDa protein. Vinexin beta did not stimulate the phosphorylation of EGFR but suppressed the dephosphorylation, resulting in a sustained phosphorylation. Mutational analyses revealed that both the first and third SH3 domains were required for a sustained phosphorylation of EGFR. Small interfering RNA-mediated knockdown of vinexin beta reduced the phosphorylation of EGFR on the cell surface in HeLa cells. The sustained phosphorylation of EGFR induced by vinexin beta was completely abolished by adding the EGFR-specific inhibitor AG1478 even after EGF stimulation, suggesting that the kinase activity of EGFR is required for the sustained phosphorylation induced by vinexin beta. We also found that E3 ubiquitin ligase c-Cbl is a binding partner of vinexin beta through the third SH3 domain. Expression of wild-type vinexin beta but not a mutant containing a mutation in the third SH3 domain decreased the cytosolic pool of c-Cbl and increased the amount of membrane-associated c-Cbl. Furthermore, over-expression of c-Cbl suppressed the sustained phosphorylation of EGFR induced by vinexin beta. These results suggest that vinexin beta plays a role in maintaining the phosphorylation of EGFR on the plasma membrane through the regulation of c-Cbl.

Adaptor Proteins, Signal Transducing↗
PubMed2006

A T3587G germ-line mutation of the MDR1 gene encodes a nonfunctional P-glycoprotein.

The human multidrug resistance gene 1 (MDR1) encodes a plasma membrane P-glycoprotein (P-gp) that functions as an efflux pump for various structurally unrelated anticancer agents. We have identified two nonsynonymous germ-line mutations of the MDR1 gene, C3583T MDR1 and T3587G MDR1, in peripheral blood cell samples from Japanese cancer patients. Two patients carried the C3583T MDR1 allele that encodes H1195Y P-gp, whereas a further two carried T3587G MDR1 that encodes I1196S P-gp. Murine NIH3T3 cells were transfected with pCAL-MDR-IRES-ZEO constructs carrying either wild-type (WT), C3583T, or T3587G MDR1 cDNA and selected with zeocin. The resulting zeocin-resistant mixed populations of transfected cells were designated as 3T3/WT, 3T3/H1195Y, and 3T3/I1196S, respectively. The cell surface expression of I1196S P-gp in 3T3/I1196S cells could not be detected by fluorescence-activated cell sorting, although low expression of I1196S P-gp was found by Western blotting. H1195Y P-gp expression levels in 3T3/H1195Y cells were slightly lower than the corresponding WT P-gp levels in 3T3/WT cells. By immunoblotting analysis, both WT P-gp and H1195Y P-gp were detectable as a 145-kDa protein, whereas I1196S P-gp was visualized as a 140-kDa protein. 3T3/I1196S cells did not show any drug resistance unlike 3T3/H1195Y cells. Moreover, a vanadate-trap assay showed that the I1196S P-gp species lacks ATP-binding activity. Taken together, we conclude from these data that T3587G MDR1 expresses a nonfunctional P-gp and this is therefore the first description of such a germ-line mutation. We contend that the T3587G MDR1 mutation may affect the pharmacokinetics of MDR1-related anticancer agents in patients carrying this allele.

3T3 Cells↗
PubMed2005

ABCA7, a molecule with unknown function.

Many ABC proteins are functional in cellular lipid transport in various different manners. ABCA7 is a full-size ABC transporter, the physiological function of which is unknown to date. This is a protein that shows the highest homology known to ABCA1, an essential molecule for producing of plasma high-density lipoprotein (HDL), and in fact it mimics ABCA1 to mediate the production of HDL from cellular lipid when transfected in vitro. It is therefore rational to assume that ABCA7 plays a relevant role in regulating of lipid metabolism. However, the ABCA7 expression profile is distinct from that of ABCA1, with respect to tissue-specific distribution and response to some reagents, presumably because of different transcriptional and posttranscriptional regulation. Potential roles and functions of ABCA7 in lipid homeostasis are discussed, especially in relation to HDL metabolism, based on available publications.

ATP-Binding Cassette Transporters↗
PubMed2005

Role of interaction with vinculin in recruitment of vinexins to focal adhesions.

Although vinexin was originally identified as a protein binding to the proline-rich hinge region of vinculin, the functions and biochemical properties of the vinexin-vinculin interaction are not known. Here, we determined the affinity of the vinexin-vinculin interaction using surface plasmon resonance measurements and found that vinexin beta interacts with the C-terminal half of vinculin, which mimics an activated "open" form, with a threefold higher affinity than with the full-length "closed" vinculin. Coimmunoprecipitation experiments showed that cell adhesion on fibronectin enhances the vinexin-vinculin interaction. We also show that the interaction with vinculin is necessary for the efficient localization of vinexin alpha and beta at focal adhesions. These observations suggest a model that "activated" vinculin localized at focal adhesions recruits vinexins to focal adhesions.

Animals↗
PubMed2005

Cloning of ABCA17, a novel rodent sperm-specific ABC (ATP-binding cassette) transporter that regulates intracellular lipid metabolism.

The A subclass of the ABC (ATP-binding cassette) transporter superfamily has a structural feature that distinguishes it from other ABC transporters, and is proposed to be involved in the transmembrane transport of endogenous lipids. Here we have cloned mouse and rat full-length cDNAs of ABCA17, a novel ABC transporter belonging to the A subclass. Mouse and rat ABCA17 proteins comprise 1733 and 1773 amino acid residues respectively, having 87.3% amino acid identity; mouse ABCA17 has amino acid identities of 55.3% and 36.7% with mouse ABCA3 and sea urchin ABCA respectively. RNA blot and quantitative real-time PCR analyses showed that ABCA17 mRNA is expressed exclusively in the testis. Examination of testis by in situ hybridization showed that ABCA17 mRNA is expressed in germ cells, mainly spermatocytes, in the seminiferous tubule. Immunoblot analysis using a specific antibody showed that ABCA17 is a protein of 200 kDa, and immunohistochemical analysis demonstrated that the protein is detected in the anterior head of sperm and elongated spermatids. ABCA17 was localized in the endoplasmic reticulum in transiently transfected HEK293 cells. Metabolic labelling analysis showed that intracellular esterified lipids, including cholesteryl esters, fatty acid esters and triacylglycerols, were significantly decreased in HEK293 cells stably expressing ABCA17 compared with untransfected cells. These results suggest that ABCA17 may play a role in regulating lipid composition in sperm.

ATP-Binding Cassette Transporters↗
PubMed2005

Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7.

The assembly of HDL by helical apolipoprotein and cellular lipid was studied using HEK293 cells to which ecdysone-inducible human ABCA1 or human ABCA7 was transfected. Expression of both ABCA1 and ABCA7 was induced linearly proportional to ponasterone A concentration in the medium. In the experimental conditions used, the ABC protein expression levels limited the rate of lipid release when the apolipoprotein concentration was high, and the apolipoprotein concentration was rate-limiting when the ABC protein expression levels were high. When ABCA1 expression increased in conditions in which it was rate-limiting, relative cholesterol content to phospholipid increased in the HDL produced. In contrast, it was constant when ABCA7 expression increased. To investigate the background mechanism, the HDL particles were analyzed by density gradient ultracentrifugation and high performance lipid chromatography. The ABCA1-mediated reaction produced two distinct HDLs, large cholesterol-rich and small cholesterol-poor particles, and the ABCA7-mediated reaction generated mostly small cholesterol-poor particles. The increase of HDL assembly with the increase of ABCA1 expression was predominant in large cholesterol-rich particles, whereas only small cholesterol-poor HDL increased as ABCA7 expression increased. We conclude that ABCA1 generates cholesterol-rich and cholesterol-poor HDL and that the former is more prominently dependent on the increase of ABCA1 expression. ABCA7 produces this HDL subfraction only as a very minor component.

ATP Binding Cassette Transporter 1↗
PubMed2005

Fenofibric acid, an active form of fenofibrate, increases apolipoprotein A-I-mediated high-density lipoprotein biogenesis by enhancing transcription of ATP-binding cassette transporter A1 gene in a liver X receptor-dependent manner.

OBJECTIVE: Fibrates are widely used drugs to reduce plasma triglyceride and increase high-density lipoprotein. Their active forms, fibric acids, are peroxisome proliferator-activated receptor-alpha activators, but no direct evidence has been demonstrated for their activation of ATP-binding cassette transporter A1 (ABCA1) in relation to clinically used fibrates. We investigated the reaction of fenofibric acid in this regard. METHODS AND RESULTS: Fenofibric acid was examined for the effect of increase of ABCA1 activity. It enhanced ABCA1 gene transcription and its protein level in macrophage cell line cells and fibroblasts and increased apolipoprotein A-I-mediated cellular lipid release, all in a dose-dependent manner. Enhancement of the gene transcription was examined by using a reporter assay system for liver X receptor responsive element (LXRE) and its inactive mutant. The results demonstrated that the effect of fenofibric acid is dependent on active LXRE. CONCLUSIONS: Fenofibric acid increased transcription of ABCA1 gene in a liver X receptor-dependent manner.

ATP Binding Cassette Transporter 1↗