Thu, 21 Aug 2008 07:05:07 BST CiteULike: happy_manatee's library [145 articles] http://www.citeulike.org/user/happy_manatee CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/happy_manatee/article/2531069 <i>J. Biol. Chem., Vol. 281, No. 45. (10 November 2006), pp. 34503-34514.</i><br /><br />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. 10.1074/jbc.M600071200 Characterization and Classification of ATP-binding Cassette Transporter ABCA3 Mutants in Fatal Surfactant Deficiency Yoshihiro Matsumura Nobuhiro Ban Kazumitsu Ueda Nobuya Inagaki doi:10.1074/jbc.M600071200 J. Biol. Chem., Vol. 281, No. 45. (10 November 2006), pp. 34503-34514. 2008-03-14T08:40:33-00:00 2006 J. Biol. Chem. 281 45 34503 34514 abca mutants http://www.citeulike.org/user/happy_manatee/article/205022 <i>Bioinformatics, Vol. 21, No. 10. (15 May 2005), pp. 2185-2190.</i> Prediction of the phenotypic effects of non-synonymous single nucleotide polymorphisms using structural and evolutionary information Lei Bao Yan Cui doi:10.1093/bioinformatics/bti365 Bioinformatics, Vol. 21, No. 10. (15 May 2005), pp. 2185-2190. 2005-05-19T18:11:44-00:00 2005 Bioinformatics 1367-4803 21 10 2185 2190 Oxford University Press abcpaper1 mutants prediction snppredict http://www.citeulike.org/user/happy_manatee/article/1218509 <i>Bioinformatics, Vol. 23, No. 6. (15 March 2007), pp. 664-672.</i> Deleterious SNP prediction: be mindful of your training data! Care A Matthew Needham J Chris Bulpitt J Andrew Westhead R David doi:10.1093/bioinformatics/btl649 Bioinformatics, Vol. 23, No. 6. (15 March 2007), pp. 664-672. 2007-04-09T23:47:22-00:00 2007 Bioinformatics 1367-4803 23 6 664 672 Oxford University Press abcpaper1 prediction uniprot variants http://www.citeulike.org/user/happy_manatee/article/674068 <i>Mol Pharmacol, Vol. 63, No. 6. (June 2003), pp. 1256-1272.</i><br /><br />The superfamily of G-protein-coupled receptors (GPCRs) is very diverse in structure and function and its members are among the most pursued targets for drug development. We identified more than 800 human GPCR sequences and simultaneously analyzed 342 unique functional nonolfactory human GPCR sequences with phylogenetic analyses. Our results show, with high bootstrap support, five main families, named glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin, forming the GRAFS classification system. The rhodopsin family is the largest and forms four main groups with 13 sub-branches. Positions of the GPCRs in chromosomal paralogons regions indicate the importance of tetraploidizations or local gene duplication events for their creation. We also searched for "fingerprint" motifs using Hidden Markov Models delineating the putative inter-relationship of the GRAFS families. We show several common structural features indicating that the human GPCRs in the GRAFS families share a common ancestor. This study represents the first overall map of the GPCRs in a single mammalian genome. Our novel approach of analyzing such large and diverse sequence sets may be useful for studies on GPCRs in other genomes and divergent protein families. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. R Fredriksson MC Lagerström LG Lundin HB Schiöth doi:10.1124/mol.63.6.1256 Mol Pharmacol, Vol. 63, No. 6. (June 2003), pp. 1256-1272. 2006-05-29T11:28:16-00:00 2003 Mol Pharmacol 0026-895X 63 6 1256 1272 csmp gpcrs superfamily http://www.citeulike.org/user/happy_manatee/article/1603017 <i>PNAS, Vol. 104, No. 35. (28 August 2007), pp. 13936-13941.</i><br /><br />Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Based on the analysis of 43 eukaryotic membrane proteins, we present a cost-effective high-throughput approach for rapidly screening membrane proteins that can be overproduced to levels of >1 mg per liter in Saccharomyces cerevisiae. We find that 70% of the well expressed membrane proteins tested in this system are stable, targeted to the correct organelle, and monodisperse in either Fos-choline 12 (FC-12) or n-dodecyl-[beta]-D-maltoside. We illustrate the advantage of such an approach, with the purification of monodisperse human and yeast nucleotide-sugar transporters to unprecedented levels. We estimate that our approach should be able to provide milligram quantities for at least one-quarter of all membrane proteins from both yeast and higher eukaryotic organisms. 10.1073/pnas.0704546104 High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae Simon Newstead Hyun Kim Gunnar von Heijne So Iwata David Drew doi:10.1073/pnas.0704546104 PNAS, Vol. 104, No. 35. (28 August 2007), pp. 13936-13941. 2007-08-29T03:48:12-00:00 2007 PNAS 104 35 13936 13941 csmp membrane yeast http://www.citeulike.org/user/happy_manatee/article/1816282 <i>Toxicon, Vol. 49, No. 4. (15 March 2007), pp. 531-549.</i><br /><br />The biological effects of Latrodectus spider venom are similar in animals from different phyla, but these symptoms are caused by distinct phylum-specific neurotoxins (collectively called latrotoxins) with molecular masses ranging from 110 to 140 kDa. To date, the venom has been found to contain five insecticidal toxins, termed [alpha], [beta], [gamma], [delta] and [epsilon]-latroinsectotoxins (LITs). There is also a vertebrate-specific neurotoxin, [alpha]-latrotoxin ([alpha]-LTX), and one toxin affecting crustaceans, [alpha]-latrocrustatoxin ([alpha]-LCT). These toxins stimulate massive release of neurotransmitters from nerve terminals and act (1) by binding to specific receptors, some of which mediate an exocytotic signal, and (2) by inserting themselves into the membrane and forming ion-permeable pores. Specific receptors for LITs have yet to be identified, but all three classes of vertebrate receptors known to bind [alpha]-LTX are also present in insects. All LTXs whose structures have been elucidated ([alpha]-LIT, [delta]-LIT, [alpha]-LTX and [alpha]-LCT) are highly homologous and have a similar domain architecture, which consists of a unique N-terminal sequence and a large domain composed of 13-22 ankyrin repeats. Three-dimensional (3D) structure analysis, so far done for [alpha]-LTX only, has revealed its dimeric nature and an ability to form symmetrical tetramers, a feature probably common to all LTXs. Only tetramers have been observed to insert into membranes and form pores. A preliminary 3D reconstruction of a [delta]-LIT monomer demonstrates the spatial similarity of this toxin to the monomer of [alpha]-LTX. Insecticidal toxins from black widow spider venom A Rohou J Nield YA Ushkaryov doi:10.1016/j.toxicon.2006.11.021 Toxicon, Vol. 49, No. 4. (15 March 2007), pp. 531-549. 2007-10-24T17:28:00-00:00 2007 Toxicon 49 4 531 549 evolution interesting ion toxin http://www.citeulike.org/user/happy_manatee/article/1785726 <i>Physiol Rev, Vol. 84, No. 1. (January 2004), pp. 41-68.</i><br /><br />The cone snails (genus Conus) are venomous marine molluscs that use small, structured peptide toxins (conotoxins) for prey capture, defense, and competitor deterrence. Each of the 500 Conus can express approximately 100 different conotoxins, with little overlap between species. An overwhelming majority of these peptides are probably targeted selectively to a specific ion channel. Because conotoxins discriminate between closely related subtypes of ion channels, they are widely used as pharmacological agents in ion channel research, and several have direct diagnostic and therapeutic potential. Large conotoxin families can comprise hundreds or thousands of different peptides; most families have a corresponding ion channel family target (i.e., omega-conotoxins and Ca channels, alpha-conotoxins and nicotinic receptors). Different conotoxin families may have different ligand binding sites on the same ion channel target (i.e., mu-conotoxins and delta-conotoxins to sites 1 and 6 of Na channels, respectively). The individual peptides in a conotoxin family are typically each selectively targeted to a diverse set of different molecular isoforms within the same ion channel family. This review focuses on the targeting specificity of conotoxins and their differential binding to different states of an ion channel. Conus venoms: a rich source of novel ion channel-targeted peptides. H Terlau BM Olivera doi:10.1152/physrev.00020.2003 Physiol Rev, Vol. 84, No. 1. (January 2004), pp. 41-68. 2007-10-18T20:59:11-00:00 2004 Physiol Rev 0031-9333 84 1 41 68 conotoxin http://www.citeulike.org/user/happy_manatee/article/876132 <i>Nature (27 September 2006)</i> Identification of a mammalian mitochondrial porphyrin transporter Partha Krishnamurthy Guoqing Du Yu Fukuda Daxi Sun Janardhan Sampath Kelly Mercer Junfeng Wang Beatriz Sosa-Pineda Gopal Murti John Schuetz doi:10.1038/nature05125 Nature (27 September 2006) 2006-09-28T09:21:49-00:00 2006 Nature 0028-0836 Nature Publishing Group abcb6 csmp membrane mitochondria http://www.citeulike.org/user/happy_manatee/article/1222474 <i>Nature, Vol. 419, No. 6907. (10 October 2002), pp. 587-593.</i><br /><br />AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel. Crystal structure of bacterial multidrug efflux transporter AcrB. S Murakami R Nakashima E Yamashita A Yamaguchi doi:10.1038/nature01050 Nature, Vol. 419, No. 6907. (10 October 2002), pp. 587-593. 2007-04-12T16:13:16-00:00 2002 Nature 0028-0836 419 6907 587 593 csmp membrane resistance http://www.citeulike.org/user/happy_manatee/article/1632806 <i>Science, Vol. 285, No. 5425. (9 July 1999), pp. 242-244.</i><br /><br />10.1126/science.285.5425.242 A Nucleoside Transporter from Trypanosoma brucei Involved in Drug Resistance Pascal Mäser Christine Sütterlin Anastasia Kralli Ronald Kaminsky doi:10.1126/science.285.5425.242 Science, Vol. 285, No. 5425. (9 July 1999), pp. 242-244. 2007-09-08T00:10:10-00:00 1999 Science 285 5425 242 244 csmp membrane multidrug resistance http://www.citeulike.org/user/happy_manatee/article/1242702 <i>Molecular Microbiology, Vol. 64, No. 2. (April 2007), pp. 407-420.</i> Differences in trans-stimulated chloroquine efflux kinetics are linked to PfCRT in Plasmodium falciparum Sanchez P Cecilia Rohrbach Petra Mclean E Jeremy Fidock A David Stein D Wilfred Lanzer Michael doi:10.1111/j.1365-2958.2007.05664.x Molecular Microbiology, Vol. 64, No. 2. (April 2007), pp. 407-420. 2007-04-22T00:44:29-00:00 2007 Molecular Microbiology 0950-382X 64 2 407 420 Blackwell Publishing csmp membrane resistance http://www.citeulike.org/user/happy_manatee/article/1632718 <i>Mol Pharmacol, Vol. 70, No. 5. (1 November 2006), pp. 1585-1592.</i><br /><br />A novel trypanocide, 2,5-bis(4-amidinophenyl)furan (DB75), in its prodrug amidoxime-derivative form, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), is in trials as the first orally administered drug for human African trypanosomiasis. DB75 is a diamidine. Resistance to some diamidines correlates to loss of uptake via the P2 aminopurine transporter. We show here that uptake of DB75 into Trypanosoma brucei also occurs principally via the P2 transporter. Uptake of tritiated DB75 occurred via a high-affinity (Km app, 3.2 microM) carriermediated route that was inhibited by adenosine, adenine, and pentamidine, all known substrates of the P2 transporter. Trypanosomes lacking the TbAT1 gene that encodes the P2 transporter demonstrated an 11-fold reduction in sensitivity to DB75 when measured under controlled in vitro conditions. These knockout cells were also less sensitive to DB75 than wild-type cells in mice. Initial uptake rates of DB75 into the Deltatbat1 knockout cell line were greatly reduced compared with rates in wild-type cells. A trypanosome cell line selected in vitro for DB75 resistance was shown to have lost P2-mediated DB75 uptake. The TbAT1 gene was mapped to chromosome V of the T. brucei genome and the DB75-resistant parasites were shown to have deleted both alleles of this gene. Fluorescence microscopy of DB75-treated trypanosomes revealed that DB75 fluorescence localizes rapidly within the DNA-containing organelles of wild-type trypanosomes, whereas no fluorescence was observed in Deltatbat1-null parasites or in the parasites selected for resistance to DB75. 10.1124/mol.106.024653 Roles for the Trypanosoma brucei P2 Transporter in DB75 Uptake and Resistance Charlotte Lanteri Mhairi Stewart Janice Brock Vincent Alibu Steven Meshnick Richard Tidwell Michael Barrett doi:10.1124/mol.106.024653 Mol Pharmacol, Vol. 70, No. 5. (1 November 2006), pp. 1585-1592. 2007-09-07T23:13:07-00:00 2006 Mol Pharmacol 70 5 1585 1592 csmp disease membrane http://www.citeulike.org/user/happy_manatee/article/445176 <i>Protein Sci, Vol. 13, No. 7. (July 2004), pp. 1948-1949.</i><br /><br />The rate of membrane protein (MP) structure determination has been examined for the 18-year period following the publication of the first high-resolution crystal structure. The growth is solidly exponential, but lags behind the rate for soluble proteins during the equivalent time period. The progress of membrane protein structure determination. SH White doi:10.1110/ps.04712004 Protein Sci, Vol. 13, No. 7. (July 2004), pp. 1948-1949. 2005-12-20T09:48:40-00:00 2004 Protein Sci 0961-8368 13 7 1948 1949 csmp membrane review http://www.citeulike.org/user/happy_manatee/article/507246 <i>Nat Rev Drug Discov, Vol. 1, No. 9. (2002), pp. 727-730.</i> THE DRUGGABLE GENOME Andrew Hopkins Colin Groom doi:10.1038/nrd892 Nat Rev Drug Discov, Vol. 1, No. 9. (2002), pp. 727-730. 2006-02-16T17:31:06-00:00 2002 Nat Rev Drug Discov 1 9 727 730 csmp membrane review http://www.citeulike.org/user/happy_manatee/article/825265 <i>ChemMedChem, Vol. 1, No. 8. (2006), pp. 760-782.</i><br /><br />Chemical biology approaches have a long history in the exploration of the G-protein-coupled receptor (GPCR) family, which represents the largest and most important group of targets for therapeutics. The analysis of the human genome revealed a significant number of new members with unknown physiological function which are today the focus of many reverse pharmacology drug-discovery programs. As the seven hydrophobic transmembrane segments are a defining common structural feature of these receptors, and as signaling through heterotrimeric G proteins is not demonstrated in all cases, these proteins are also referred to as seven transmembrane (7 TM) or serpentine receptors. This review summarizes important historic milestones of GPCR research, from the beginning, when pharmacology was mainly descriptive, to the age of modern molecular biology, with the cloning of the first receptor and now the availability of the entire human GPCR repertoire at the sequence and protein level. It shows how GPCR-directed drug discovery was initially based on the careful testing of a few specifically made chemical compounds and is today pursued with modern drug-discovery approaches, including combinatorial library design, structural biology, molecular informatics, and advanced screening technologies for the identification of new compounds that activate or inhibit GPCRs specifically. Such compounds, in conjunction with other new technologies, allow us to study the role of receptors in physiology and medicine, and will hopefully result in novel therapies. We also outline how basic research on the signaling and regulatory mechanisms of GPCRs is advancing, leading to the discovery of new GPCR-interacting proteins and thus opening new perspectives for drug development. Practical examples from GPCR expression studies, HTS (high-throughput screening), and the design of monoamine-related GPCR-focused combinatorial libraries illustrate ongoing GPCR chemical biology research. Finally, we outline future progress that may relate today?s discoveries to the development of new medicines. The 7 TM G-Protein-Coupled Receptor Target Family Edgar Jacoby Rochdi Bouhelal Marc Gerspacher Klaus Seuwen doi:10.1002/cmdc.200600134 ChemMedChem, Vol. 1, No. 8. (2006), pp. 760-782. 2006-09-01T14:09:28-00:00 2006 ChemMedChem 1 8 760 782 csmp membrane review http://www.citeulike.org/user/happy_manatee/article/407716 <i>The EMBO Journal, Vol. aop, No. current. (24 November 2005)</i> 3-D structural and functional characterization of the purified KATP channel complex Kir6.2[ndash]SUR1 Michael Mikhailov Jeff Campbell Heidi de Wet Kenju Shimomura Brittany Zadek Richard Collins Mark Sansom Robert Ford Frances Ashcroft doi:10.1038/sj.emboj.7600877 The EMBO Journal, Vol. aop, No. current. (24 November 2005) 2005-11-24T18:57:28-00:00 2005 The EMBO Journal 0261-4189 aop current Nature Publishing Group abcc9 channel csmp sur1 sur2 http://www.citeulike.org/user/happy_manatee/article/1538717 <i>J. Bacteriol., Vol. 187, No. 5. (1 March 2005), pp. 1552-1558.</i><br /><br />NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process. 10.1128/JB.187.5.1552-1558.2005 Identification of Essential Amino Acid Residues of the NorM Na+/Multidrug Antiporter in Vibrio parahaemolyticus Masato Otsuka Makoto Yasuda Yuji Morita Chie Otsuka Tomofusa Tsuchiya Hiroshi Omote Yoshinori Moriyama doi:10.1128/JB.187.5.1552-1558.2005 J. Bacteriol., Vol. 187, No. 5. (1 March 2005), pp. 1552-1558. 2007-08-06T21:05:11-00:00 2005 J. Bacteriol. 187 5 1552 1558 csmp mate_family membrane multidrug mutants http://www.citeulike.org/user/happy_manatee/article/1538682 <i>Antimicrob Agents Chemother, Vol. 50, No. 11. (November 2006), pp. 3717-3723.</i><br /><br />NorM, a putative efflux pump of Vibrio cholerae, is a member of the multidrug and toxic compound extrusion family of transporters. We demonstrate that NorM confers resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Inactivation of norM rendered V. cholerae hypersensitive towards these fluoroquinolones. Multiple sequence alignment of members of its family identified several regions of high sequence conservation. The topology of NorM was determined using beta-lactamase and chloramphenicol acetyltransferase fusions. The amino acid residues G(184), K(185), G(187), P(189), E(190), G(192), and G(195) in the periplasmic loops and L(381), R(382), G(383), Y(384), K(385), and D(386) in the cytoplasmic loops, as well as all the acidic and cysteine residues of NorM, were mutated. Mutants G184V, G184W, K185I, P189S, E190K, and E190A lost the norfloxacin resistance-imparting phenotype characteristic of NorM. Mutants E124V, D155V, G187V, G187R, C196S, Y384H, Y384S, and Y384F exhibited partial resistance to norfloxacin. Mutants with replacements of G(184) or G(187) by A, K(185) by R, and E(190) by D retained the norfloxacin resistance phenotype of NorM. Analysis of the accumulation of norfloxacin in intact cells of Escherichia coli expressing NorM or its mutants in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone supported the results obtained through susceptibility testing and argued in favor of NorM-mediated efflux as the determining factor in norfloxacin susceptibility in the genetically manipulated strains. Taken together, these results suggested that E(124), D(155), G(184), K(185), G(187), P(189), E(190), C(196), and Y(384) are likely involved in NorM-dependent norfloxacin efflux. Except for D(155), C(196), and Y(384), all of these residues are located in periplasmic loops. Analysis of the topology of Vibrio cholerae NorM and identification of amino acid residues involved in norfloxacin resistance. AK Singh R Haldar D Mandal M Kundu doi:10.1128/AAC.00460-06 Antimicrob Agents Chemother, Vol. 50, No. 11. (November 2006), pp. 3717-3723. 2007-08-06T20:08:17-00:00 2006 Antimicrob Agents Chemother 0066-4804 50 11 3717 3723 csmp mate_family membrane multidrug mutants http://www.citeulike.org/user/happy_manatee/article/1538650 <i>Biochemical Pharmacology, Vol. 74, No. 3. (1 August 2007), pp. 477-487.</i><br /><br />The mechanism of severe nephrotoxicity caused by cisplatin, but not carboplatin, oxaliplatin, and nedaplatin, is not fully understood. The renal accumulation and subsequent nephrotoxicity of platinum agents were examined in rats. Among these four drugs, only cisplatin induced nephrotoxicity at 2 days after its intraperitoneal administration. The urinary activity of N-acetyl-[beta]-d-glucosaminidase and expression of kidney injury molecule-1 mRNA and osteopontin were markedly enhanced in the cisplatin-treated rats. Although some markers were affected in the rats administered nedaplatin, only minor histological change was observed. The renal accumulation of cisplatin was much greater than that of the other drugs. In the in vitro study, the cellular accumulation of cisplatin and oxaliplatin was stimulated by the expression of rat (r) OCT2. Oxaliplatin was also transported by rOCT3. A luminal H+/organic cation antiporter, rMATE1 (multidrug and toxin extrusion) as well as human (h) MATE1 and hMATE2-K, stimulated the H+-gradient-dependent antiport of oxaliplatin, but not of cisplatin. Carboplatin and nedaplatin were not transported by these transporters. In conclusion, the nephrotoxicity of platinum agents was closely associated with their renal accumulation, which is determined by the substrate specificity of the OCT and MATE families. Differential contribution of organic cation transporters, OCT2 and MATE1, in platinum agent-induced nephrotoxicity Sachiko Yokoo Atsushi Yonezawa Satohiro Masuda Atsushi Fukatsu Toshiya Katsura Ken-Ichi Inui doi:10.1016/j.bcp.2007.03.004 Biochemical Pharmacology, Vol. 74, No. 3. (1 August 2007), pp. 477-487. 2007-08-06T19:24:53-00:00 2007 Biochemical Pharmacology 74 3 477 487 csmp mate_family membrane multidrug mutants http://www.citeulike.org/user/happy_manatee/article/1466158 <i>Pharmaceutical Research, Vol. 24, No. 7. (July 2007), pp. 1227-1251.</i> Polyspecific Organic Cation Transporters: Structure, Function, Physiological Roles, and Biopharmaceutical Implications Koepsell Hermann Lips Katrin Volk Christopher doi:10.1007/s11095-007-9254-z Pharmaceutical Research, Vol. 24, No. 7. (July 2007), pp. 1227-1251. 2007-07-19T00:03:00-00:00 2007 Pharmaceutical Research 0724-8741 24 7 1227 1251 Springer csmp mate_family membrane multidrug mutants http://www.citeulike.org/user/happy_manatee/article/1538529 <i>Nature, Vol. 415, No. 6870. (24 January 2002), pp. 447-450.</i> Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein Jonne Helenius Davis Ng Cristina Marolda Peter Walter Miguel Valvano Markus Aebi doi:10.1038/415447a Nature, Vol. 415, No. 6870. (24 January 2002), pp. 447-450. 2007-08-06T17:02:35-00:00 2002 Nature 415 6870 447 450 csmp membrane multidrug mutants rft1_family http://www.citeulike.org/user/happy_manatee/article/1538527 <i>Pharm Res, Vol. 23, No. 8. (August 2006), pp. 1696-1701.</i><br /><br />PURPOSE: Transport characteristics and tissue distribution of the rat H+/organic cation antiporter MATE1 (multidrug and toxin extrusion 1) were examined. METHODS: Rat MATE1 cDNA was isolated by polymerase chain reaction (PCR) cloning. Transport characteristics of rat MATE1 were assessed by HEK293 cells transiently expressing rat MATE1. The mRNA expression of rat MATE1 was examined by Northern blot and real-time PCR analyses. RESULTS: The uptake of a prototypical organic cation tetraethylammonium (TEA) by MATEI-expressing cells was concentration-dependent, and showed the greatest value at pH 8.4 and the lowest at pH 6.0-6.5. Intracellular acidification induced by ammonium chloride resulted in a marked stimulation of TEA uptake. MATE1 transported not only organic cations such as cimetidine and metformin but also the zwitterionic compound cephalexin. MATE1 mRNA was expressed abundantly in the kidney and placenta, slightly in the spleen, but not expressed in the liver. Real-time PCR analysis of microdissected nephron segments showed that MATE1 was primarily expressed in the proximal convoluted and straight tubules. CONCLUSIONS: These findings indicate that MATE1 is expressed in the renal proximal tubules and can mediate the transport of various organic cations and cephalexin using an oppositely directed H+ gradient. Molecular cloning, functional characterization and tissue distribution of rat H+/organic cation antiporter MATE1. T Terada S Masuda J Asaka M Tsuda T Katsura K Inui doi:10.1007/s11095-006-9016-3 Pharm Res, Vol. 23, No. 8. (August 2006), pp. 1696-1701. 2007-08-06T17:00:13-00:00 2006 Pharm Res 0724-8741 23 8 1696 1701 csmp mate_family membrane multidrug mutants http://www.citeulike.org/user/happy_manatee/article/1538519 <i>Proc Natl Acad Sci U S A, Vol. 102, No. 50. (13 December 2005), pp. 17923-17928.</i><br /><br />In mammals, toxic electrolytes of endogenous and exogenous origin are excreted through the urine and bile. Before excretion, these compounds cross numerous cellular membranes in a transporter-mediated manner. However, the protein transporters involved in the final excretion step are poorly understood. Here, we show that MATE1, a human and mouse orthologue of the multidrug and toxin extrusion family conferring multidrug resistance on bacteria, is primarily expressed in the kidney and liver, where it is localized to the luminal membranes of the urinary tubules and bile canaliculi. When expressed in HEK293 cells, MATE1 mediates H(+)-coupled electroneutral exchange of tetraethylammonium and 1-methyl-4-phenylpyridinium. Its substrate specificity is similar to those of renal and hepatic H(+)-coupled organic cations (OCs) export. Thus, MATE1 appears to be the long searched for polyspecific OC exporter that directly transports toxic OCs into urine and bile. A human transporter protein that mediates the final excretion step for toxic organic cations. M Otsuka T Matsumoto R Morimoto S Arioka H Omote Y Moriyama doi:10.1073/pnas.0506483102 Proc Natl Acad Sci U S A, Vol. 102, No. 50. (13 December 2005), pp. 17923-17928. 2007-08-06T16:52:42-00:00 2005 Proc Natl Acad Sci U S A 0027-8424 102 50 17923 17928 csmp mate_family membrane http://www.citeulike.org/user/happy_manatee/article/1538466 <i>Mol Pharmacol, Vol. 71, No. 6. (June 2007), pp. 1487-1493.</i><br /><br />Multidrug and toxin extrusion 1 (MATE1) has been isolated as an H(+)/organic cation antiporter located at the renal brush-border membranes. Previous studies using rat renal brush-border membrane vesicles indicated that cysteine and histidine residues played critical roles in H(+)/organic cation antiport activity. In the present study, essential histidine and cysteine residues of MATE1 family were elucidated. When 7 histidine and 12 cysteine residues of rat (r)MATE1 conserved among species were mutated, substitution of His-385, Cys-62, and Cys-126 led to a significant loss of tetraethylammonium (TEA) transport activity. Cell surface biotinylation and immunofluorescence analyses with confocal microscopy indicated that rMATE1 mutant proteins were localized at plasma membranes. Mutation of the corresponding residues in human (h)MATE1 and hMATE2-K also diminished the transport activity. The transport of TEA via rMATE1 was inhibited by the sulfhydryl reagent p-chloromercuribenzenesulfonate (PCMBS) and the histidine residue modifier diethyl pyrocarbonate (DEPC) in a concentration-dependent manner. The PCMBS-caused inhibition of the transport via rMATE1 was protected by an excess of various organic cations such as TEA, suggesting that cysteine residues act as substrate-binding sites. In the case of DEPC, no such protective effects were observed. These results suggest that histidine and cysteine residues are required for MATE1 to function and that cysteine residues may serve as substrate-recognition sites. Identification of essential histidine and cysteine residues of the H+/organic cation antiporter multidrug and toxin extrusion (MATE). J Asaka T Terada M Tsuda T Katsura K Inui doi:10.1124/mol.106.032938 Mol Pharmacol, Vol. 71, No. 6. (June 2007), pp. 1487-1493. 2007-08-06T16:27:27-00:00 2007 Mol Pharmacol 0026-895X 71 6 1487 1493 csmp mate_family membrane multidrug mutants http://www.citeulike.org/user/happy_manatee/article/1538351 <i>Cell Mol Life Sci, Vol. 63, No. 22. (November 2006), pp. 2597-2607.</i><br /><br />Structure-based drug discovery has proven useful in improving and shortening the drug development process. The approach of structural genomics to study a large number of targets in parallel has been commonly applied to protein families and even whole genomes. Paradoxically, although membrane proteins represent the largest type of drug targets, up to 70% today, determination of their structure has been modest compared to that of soluble proteins. Because membrane proteins are important for drug discovery an emphasis has been placed on developing technologies and methods to determine membrane protein structures. Several structural genomics initiatives have been established, focusing on the structural biology of membrane proteins. Structural genomics for membrane proteins. K Lundstrom doi:10.1007/s00018-006-6252-y Cell Mol Life Sci, Vol. 63, No. 22. (November 2006), pp. 2597-2607. 2007-08-06T15:27:01-00:00 2006 Cell Mol Life Sci 1420-682X 63 22 2597 2607 csmp membrane structure http://www.citeulike.org/user/happy_manatee/article/1122817 <i>Pflügers Archiv, Vol. 453, No. 5. (February 2007), pp. 621-641.</i> Portrait of multifaceted transporter, the multidrug resistance-associated protein 1 (MRP1/ABCC1) Bakos Eva Homolya Laszlo doi:10.1007/s00424-006-0160-8 Pflügers Archiv, Vol. 453, No. 5. (February 2007), pp. 621-641. 2007-02-26T12:50:23-00:00 2007 Pflügers Archiv 0031-6768 453 5 621 641 Springer abcc1 mrp1 review http://www.citeulike.org/user/happy_manatee/article/1145002 <i>Cytometry A, Vol. 62, No. 2. (December 2004), pp. 129-138.</i><br /><br />BACKGROUND: To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively. METHODS: ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates, and a series of potential inhibitors of the transporter. RESULTS: ABCG2 expression was detected in all cell lines. The cell lines effluxed mitoxantrone (MXR), but only the mutants effluxed rhodamine 123 (Rho123), SYTO13, doxorubicin, and daunorubicin. After incubation with MXR, intracellular accumulations were 9- and 22-fold higher in MXRA than in MXRT and MXRG cells, respectively, suggesting that ABCG2 also modulates the influx rate of the drug. Flow cytometry kinetic studies of MXR efflux showed that MXRG cells effluxed 50% of the drug at a faster rate than MXRA and MXRT cells (t50: 15.3 min vs. 27.8 and 44.5 min, respectively). MXRG cells also extruded Rho123 and SYTO13 at a faster rate than MXRT cells. ABCG2-mediated transport was inhibited by fumitremorgin C, cyclosporine A, and PSC-833, but not by verapamil or probenecid. MXRG cells displayed the highest level of resistance to MXR, doxorubicin, and daunorubicin in the cytotoxicity assays. CONCLUSIONS: Glycine mutations at position 482 have a significant impact on ABCG2 function by modifying its substrate specificity and its influx/efflux rates. This study also demonstrates that flow cytometry constitutes a powerful tool for the kinetic analysis of ABC transporters. Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs. M García-Escarp V Martínez-Muñoz I Sales-Pardo J Barquinero JC Domingo P Marin J Petriz doi:10.1002/cyto.a.20072 Cytometry A, Vol. 62, No. 2. (December 2004), pp. 129-138. 2007-03-07T02:18:11-00:00 2004 Cytometry A 1552-4922 62 2 129 138 482 abcg2 facs kinetics mutant http://www.citeulike.org/user/happy_manatee/article/1145001 <i>Br J Cancer, Vol. 89, No. 10. (17 November 2003), pp. 1971-1978.</i><br /><br />Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 microM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2. Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity. RW Robey Y Honjo K Morisaki TA Nadjem S Runge M Risbood MS Poruchynsky SE Bates doi:10.1038/sj.bjc.6601370 Br J Cancer, Vol. 89, No. 10. (17 November 2003), pp. 1971-1978. 2007-03-07T02:17:19-00:00 2003 Br J Cancer 0007-0920 89 10 1971 1978 482 abcg2 facs mutant http://www.citeulike.org/user/happy_manatee/article/822578 <i>Nature (30 August 2006)</i> Structure of a bacterial multidrug ABC transporter Roger Dawson Kaspar Locher doi:10.1038/nature05155 Nature (30 August 2006) 2006-08-31T02:39:46-00:00 2006 Nature 0028-0836 Nature Publishing Group 2hyd complete_stx locher sav1866 http://www.citeulike.org/user/happy_manatee/article/1124539 <i>Nature (25 February 2007)</i> Structure of an ABC transporter in complex with its binding protein Kaspar Hollenstein Dominik Frei Kaspar Locher doi:10.1038/nature05626 Nature (25 February 2007) 2007-02-27T01:54:47-00:00 2007 Nature 0028-0836 Nature Publishing Group complete_stx locher multidrug structure http://www.citeulike.org/user/happy_manatee/article/1142046 <i>Journal of Bioenergetics and Biomembranes, Vol. V33, No. 6. (1 December 2001), pp. 453-458.</i> Overview: ABC Transporters and Human Disease Michael Gottesman Suresh Ambudkar doi:10.1023/A:1012866803188 Journal of Bioenergetics and Biomembranes, Vol. V33, No. 6. (1 December 2001), pp. 453-458. 2007-03-05T18:19:08-00:00 2001 Journal of Bioenergetics and Biomembranes V33 6 453 458 disease review http://www.citeulike.org/user/happy_manatee/article/1134865 <i>Cancer Sci, Vol. 98, No. 2. (February 2007), pp. 231-239.</i><br /><br />Impacts of genetic polymorphisms of the ATP-binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp-In-293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Drug resistance profiles of Flp-In-293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN-38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2-mediated drug resistance without any influence from major non-synonymous polymorphisms. Re-evaluation and functional classification of non-synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2. A Tamura K Wakabayashi Y Onishi M Takeda Y Ikegami S Sawada M Tsuji Y Matsuda T Ishikawa Cancer Sci, Vol. 98, No. 2. (February 2007), pp. 231-239. 2007-03-02T01:26:32-00:00 2007 Cancer Sci 1347-9032 98 2 231 239 abcg2 bcrp functional variants http://www.citeulike.org/user/happy_manatee/article/1134789 <i>Drug Metab Dispos, Vol. 33, No. 6. (June 2005), pp. 697-705.</i><br /><br />Previous studies have shown that the V12M and Q141K variants of breast cancer resistance protein (BCRP) can affect expression and function of the transporter. In this study, the effects of the I206L, N590Y, and D620N variants on protein expression, plasma membrane localization, and transport activity of BCRP were investigated. Wild-type BCRP and the three variants were stably expressed in human embryonic kidney (HEK) cells. Confocal microscopy analysis showed that the three variants were predominantly routed to the plasma membrane of HEK cells. The expression level of I206L in the plasma membrane was approximately 45% of that of wild-type protein, whereas the N590Y and D620N levels were increased approximately 3.6-fold and 2.4-fold, respectively, as determined by immunoblotting. All three variants transported mitoxantrone, pheophorbide a, and BODIPY FL-prazosin. After normalization for differences in BCRP expression, I206L, N590Y, and D620N exhibited approximately 2-fold, 0.3-fold, and 0.5-fold wild-type efflux activities, respectively. The variants also conferred resistance to mitoxantrone and topotecan. Mitoxantrone and topotecan resistance by I206L and N590Y was approximately 2-fold and 0.3-fold of the wild-type BCRP resistance levels, respectively. Although D620N conferred a topotecan resistance similar to that of the wild-type protein, its level of mitoxantrone resistance was decreased by 50%. After normalization to BCRP expression levels, ATPase activities of I206L were not significantly different from those of wild-type protein, whereas N590Y and D620N exhibited approximately 30% and 50% of wild-type ATPase activities, respectively. These results suggest that I206L has the lowest protein expression and the highest activity, whereas N590Y and D620N display higher expression and lower activity, relative to wild-type BCRP. Functional analysis of the human variants of breast cancer resistance protein: I206L, N590Y, and D620N. RR Vethanayagam H Wang A Gupta Y Zhang F Lewis JD Unadkat Q Mao doi:10.1124/dmd.105.003657 Drug Metab Dispos, Vol. 33, No. 6. (June 2005), pp. 697-705. 2007-03-02T00:53:31-00:00 2005 Drug Metab Dispos 0090-9556 33 6 697 705 abcg2 bcrp functional variants http://www.citeulike.org/user/happy_manatee/article/1134784 <i>Pharm Res, Vol. 21, No. 10. (October 2004), pp. 1895-1903.</i><br /><br />PURPOSE: The aim of the current study was to identify the effect of single nucleotide polymorphisms (SNPs) in breast cancer resistance protein (BCRP/ABCG2) on its localization, expression level, and transport activity. METHODS: The cellular localization was identified using the wild type and seven different SNP variants of BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S, and S441N BCRP) after transfection of their cDNAs in plasmid vector to LLC-PK1 cells. Their expression levels and transport activities were determined using the membrane vesicles from HEK293 cells infected with the recombinant adenoviruses containing these kinds of BCRP cDNAs. RESULTS: Wild type and six different SNP variants of BCRP other than S441N BCRP were expressed on the apical membrane, whereas S441N BCRP showed intracellular localization. The expression levels of Q141K and S441N BCRP proteins were significantly lower compared with the wild type and the other five variants. Furthermore, the transport activity of E1S, DHEAS, MTX, and PAH normalized by the expression level of BCRP protein was almost the same for the wild type, V12M, Q141K, A149P, R163K, Q166E, and P269S BCRP. CONCLUSIONS: These results suggest that Q141K SNPs may associate with a lower expression level, and S441N SNPs may affect both the expression level and cellular localization. It is possible that subjects with these polymorphisms may have lower expression level of BCRP protein and, consequently, a reduced ability to export these substrates. Functional analysis of SNPs variants of BCRP/ABCG2. C Kondo H Suzuki M Itoda S Ozawa J Sawada D Kobayashi I Ieiri K Mine K Ohtsubo Y Sugiyama Pharm Res, Vol. 21, No. 10. (October 2004), pp. 1895-1903. 2007-03-02T00:49:29-00:00 2004 Pharm Res 0724-8741 21 10 1895 1903 abcg2 bcrp functional variants http://www.citeulike.org/user/happy_manatee/article/1122816 <i>Pflügers Archiv, Vol. 453, No. 5. (February 2007), pp. 643-659.</i> The apical conjugate efflux pump ABCC2 (MRP2) Nies Anne Keppler Dietrich doi:10.1007/s00424-006-0109-y Pflügers Archiv, Vol. 453, No. 5. (February 2007), pp. 643-659. 2007-02-26T12:50:22-00:00 2007 Pflügers Archiv 0031-6768 453 5 643 659 Springer abcc2 abcpaper1 functional mrp2 http://www.citeulike.org/user/happy_manatee/article/1134294 <i>Pharmacogenet Genomics, Vol. 15, No. 9. (September 2005), pp. 647-657.</i><br /><br />The 190-kDa ATP-binding cassette (ABC) multidrug resistance protein 1 (MRP1) encoded by the MRP1/ABCC1 gene mediates the active cellular efflux of glucuronide, glutathione and sulfate conjugates. It can also confer resistance to a diverse spectrum of chemotherapeutic agents and transport a variety of toxicants. In the present study, we examined 10 MRP1/ABCC1 missense genetic variants [non-synonymous single nucleotide polymorphisms (SNPs)] to determine whether or not they affect expression or function of the transporter. Variants 218C>T (Thr73Ile), 257C>T (Ser92Phe), 350C>T (Thr117Met), 689G>A (Arg230Gln), 1898G>A (Arg633Gln), 2168G>A (Arg723Gln), 2965G>A (Ala989Thr), 3140G>C (Cys1047Ser), 3173G>A (Arg1058Gln) and 4535C>T (Ser1512Leu) were recreated using site-directed mutagenesis and transfected into human embryonic kidney cells. Immunoblotting experiments showed that all mutant proteins were expressed at levels comparable to wild-type MRP1. Vesicular transport assays revealed that the Ala989Thr mutation caused a significant decrease in estradiol 17beta-glucuronide transport due to a decrease in apparent affinity (Km) for this organic anion. The transport properties of the other mutants were comparable to wild-type MRP1. When the MRP1/ABCC1 non-synonymous SNPs were evaluated by the SIFT algorithm using subsets of homologs and orthologs of MRP1/ABCC1, Arg230Gln, Val353Met, Arg433Ser, Gly671Val and Arg1058 mutations were predicted to be deleterious, whereas the PolyPhen algorithm predicted Ser92Phe and Gly671Val to be potentially damaging. Thus most predictions of these algorithms were not in accordance with our experimental results. In conclusion, our data suggest that none of the MRP1/ABCC1 variants studied are likely by themselves to have major deleterious effects in healthy individuals, and the SIFT and PolyPhen algorithms appear to be poor predictors of the phenotypic consequences of these MRP1 mutations at least in vitro. Functional characterization of non-synonymous single nucleotide polymorphisms in the gene encoding human multidrug resistance protein 1 (MRP1/ABCC1). IJ Létourneau RG Deeley SP Cole Pharmacogenet Genomics, Vol. 15, No. 9. (September 2005), pp. 647-657. 2007-03-01T22:55:10-00:00 2005 Pharmacogenet Genomics 1744-6872 15 9 647 657 abcc1 functional mrp1 mutant http://www.citeulike.org/user/happy_manatee/article/1061030 <i>Mol Pharmacol, Vol. 70, No. 1. (July 2006), pp. 287-296.</i><br /><br />The ATP-binding cassette (ABC) transporter ABCG2 has been implicated to play a significant role in the response of patients to medication and/or the risk of diseases. To clarify the possible physiological or pathological relevance of ABCG2 polymorphisms, we have functionally validated single nucleotide polymorphisms (SNP) of ABCG2. In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Because porphyrins are considered to be endogenous substrates for ABCG2, we have investigated the porphyrin transport activity of those variant forms in vitro. We herein provide evidence that the variants Q126stop, F208S, S248P, E334stop, and S441N are defective in porphyrin transport, whereas F489L exhibited impaired transport, approximately 10% of the activity observed for the wild type. Furthermore, Flp-In-293 cells expressing those variants were photosensitive. Thus, among those genetic polymorphisms of ABCG2, at least the hitherto validated alleles of Q126stop, S441N, and F489L are suggested to be of clinical importance related to the potential risk of porphyria. Functional validation of the genetic polymorphisms of human ATP-binding cassette (ABC) transporter ABCG2: identification of alleles that are defective in porphyrin transport. A Tamura M Watanabe H Saito H Nakagawa T Kamachi I Okura T Ishikawa doi:10.1124/mol.106.023556 Mol Pharmacol, Vol. 70, No. 1. (July 2006), pp. 287-296. 2007-01-22T22:25:43-00:00 2006 Mol Pharmacol 0026-895X 70 1 287 296 abcg2 atpase transmembrane transporter http://www.citeulike.org/user/happy_manatee/article/1046487 <i>J Gastroenterol, Vol. 40, No. 4. (April 2005), pp. 366-370.</i><br /><br />BACKGROUND: Recent studies have indicated that dysfunction or loss of the multidrug resistance protein 2 (MRP2) is the molecular basis of Dubin-Johnson syndrome (DJS). To clarify the genetic basis of the disease and the long-term stability of serum bilirubin levels, we conducted a mutational analysis of the MRP2 gene and followed up serum bilirubin levels in Japanese DJS patients 30 years after they were originally diagnosed, based on traditional criteria. METHODS: Patients were interviewed by telephone, and blood tests, including a genetic analysis of MRP2, were performed on the patients and family members who gave informed consent. RESULTS: Over the 30 years, hyperbilirubinemia remained unchanged in four of the five patients studied, while it worsened in 1 patient whose DJS was complicated by chronic hepatitis C. From an MRP2 gene mutational analysis, six mutations, including the novel mutation 1177C>T, were found. Three patients of a consanguineous family were homozygotes for three mutations (298C>T, 1967+2T>C, and 2439+2T>C). Two patients were compound heterozygotes (1177C>T/2302C>T and 1967+2T>C/2026G>C). A familial study showed no difference in serum bilirubin levels between mutant/wild heterozygotes and wild/wild homozygotes. CONCLUSIONS: The hyperbilirubinemia of four Japanese patients with DJS, one of whom had a novel mutation, 1177C>T, of the MRP2 gene, had not worsened with aging. Mutational analysis of the MRP2 gene and long-term follow-up of Dubin-Johnson syndrome in Japan. I Machida S Wakusawa F Sanae H Hayashi A Kusakabe H Ninomiya M Yano K Yoshioka doi:10.1007/s00535-004-1555-y J Gastroenterol, Vol. 40, No. 4. (April 2005), pp. 366-370. 2007-01-17T17:44:36-00:00 2005 J Gastroenterol 0944-1174 40 4 366 370 abcc2 mrp2 mutant r393w http://www.citeulike.org/user/happy_manatee/article/1046443 <i>J Biol Chem, Vol. 281, No. 40. (6 October 2006), pp. 29436-29440.</i><br /><br />Deletion of Phe-508 (DeltaF508) in cystic fibrosis transmembrane conductance regulator causes cystic fibrosis because of misfolding of the protein. P-glycoprotein (P-gp) containing the equivalent mutation (DeltaY490) is also misfolded but can be rescued with drug substrates. Whether rescue is due to direct binding of drug substrate to the transmembrane (TM) segments or to indirect effects on cellular protein folding pathways is still controversial. P-gp-drug substrate interactions likely involve hydrogen bonds. If the mechanism of drug rescue involves changes to TM packing then we should be able to identify suppressor mutations in the TM segments that can mimic the drug rescue effects. We predicted that an arginine residue in the TM segments predicted to line the drug-binding pocket of P-gp (I306(TM5) or F343(TM6)) might suppress DeltaY490 P-gp protein misfolding because it has the highest propensity to form hydrogen bonds. We show that R306(TM5) or R343(TM6) increased the relative amount of mature DeltaY490 P-gp by 6-fold. Most other changes to Ile-306 or Phe-343 did not enhance maturation of DeltaY490 P-gp. The I306R mutant also promoted maturation of misprocessed mutants that had mutations in the second nucleotide-binding domain (L1260A), the cytoplasmic loops (G251V, F804A), the linker region (P709A), or in TM segments (G300V, G722A). These results show that arginine residues in the TM domains can mimic the drug rescue effects and are effective suppressor mutations for processing mutations located throughout the molecule. Insertion of an arginine residue into the transmembrane segments corrects protein misfolding. TW Loo MC Bartlett DM Clarke doi:10.1074/jbc.C600209200 J Biol Chem, Vol. 281, No. 40. (6 October 2006), pp. 29436-29440. 2007-01-17T17:39:41-00:00 2006 J Biol Chem 0021-9258 281 40 29436 29440 abcb1 folding processing http://www.citeulike.org/user/happy_manatee/article/1030340 <i>Cell, Vol. 127, No. 4. (17 November 2006), pp. 803-815.</i><br /><br />The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, DeltaF508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of DeltaF508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the "chaperome") is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. X Wang J Venable P LaPointe DM Hutt AV Koulov J Coppinger C Gurkan W Kellner J Matteson H Plutner JR Riordan JW Kelly JR Yates WE Balch doi:10.1016/j.cell.2006.09.043 Cell, Vol. 127, No. 4. (17 November 2006), pp. 803-815. 2007-01-08T19:25:33-00:00 2006 Cell 0092-8674 127 4 803 815 cftr folding interaction interesting http://www.citeulike.org/user/happy_manatee/article/392495 <i>PLoS Biol, Vol. 3, No. 12. (15 November 2005)</i><br /><br />Changes in the cis-regulation of neural genes likely contributed to the evolution of our species' unique attributes, but evidence of a role for natural selection has been lacking. We found that positive natural selection altered the cis-regulation of human prodynorphin, the precursor molecule for a suite of endogenous opioids and neuropeptides with critical roles in regulating perception, behavior, and memory. Independent lines of phylogenetic and population genetic evidence support a history of selective sweeps driving the evolution of the human prodynorphin promoter. In experimental assays of chimpanzee-human hybrid promoters, the selected sequence increases transcriptional inducibility. The evidence for a change in the response of the brain's natural opioids to inductive stimuli points to potential human-specific characteristics favored during evolution. In addition, the pattern of linked nucleotide and microsatellite variation among and within modern human populations suggests that recent selection, subsequent to the fixation of the human-specific mutations and the peopling of the globe, has favored different prodynorphin cis-regulatory alleles in different parts of the world. Ancient and Recent Positive Selection Transformed Opioid cis-Regulation in Humans. Matthew V Rockman Matthew W Hahn Nicole Soranzo Fritz Zimprich David B Goldstein Gregory A Wray doi:10.1371/journal.pbio.0030387 PLoS Biol, Vol. 3, No. 12. (15 November 2005) 2005-11-14T19:50:57-00:00 2005 PLoS Biol 1545-7885 3 12 interesting regulation http://www.citeulike.org/user/happy_manatee/article/1012312 <i>Protein Sci, Vol. 15, No. 7. (1 July 2006), pp. 1597-1607.</i><br /><br />Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding. 10.1110/ps.051998406 The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding Karin Ejendal Ndeye Diop Linda Schweiger Christine Hrycyna Protein Sci, Vol. 15, No. 7. (1 July 2006), pp. 1597-1607. 2006-12-24T22:49:53-00:00 2006 Protein Sci 15 7 1597 1607 abcg2 http://www.citeulike.org/user/happy_manatee/article/1012311 <i>Physiol Rev, Vol. 86, No. 3. (July 2006), pp. 849-899.</i><br /><br />Multidrug Resistance Proteins (MRPs), together with the cystic fibrosis conductance regulator (CFTR/ABCC7) and the sulfonylurea receptors (SUR1/ABCC8 and SUR2/ABCC9) comprise the 13 members of the human "C" branch of the ATP binding cassette (ABC) superfamily. All C branch proteins share conserved structural features in their nucleotide binding domains (NBDs) that distinguish them from other ABC proteins. The MRPs can be further divided into two subfamilies "long" (MRP1, -2, -3, -6, and -7) and "short" (MRP4, -5, -8, -9, and -10). The short MRPs have a typical ABC transporter structure with two polytropic membrane spanning domains (MSDs) and two NBDs, while the long MRPs have an additional NH2-terminal MSD. In vitro, the MRPs can collectively confer resistance to natural product drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and, under certain circumstances, alkylating agents. The MRPs are also primary active transporters of other structurally diverse compounds, including glutathione, glucuronide, and sulfate conjugates of a large number of xeno- and endobiotics. In vivo, several MRPs are major contributors to the distribution and elimination of a wide range of both anticancer and non-anticancer drugs and metabolites. In this review, we describe what is known of the structure of the MRPs and the mechanisms by which they recognize and transport their diverse substrates. We also summarize knowledge of their possible physiological functions and evidence that they may be involved in the clinical drug resistance of various forms of cancer. Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. RG Deeley C Westlake SP Cole doi:10.1152/physrev.00035.2005 Physiol Rev, Vol. 86, No. 3. (July 2006), pp. 849-899. 2006-12-24T22:47:57-00:00 2006 Physiol Rev 0031-9333 86 3 849 899 abcc1 abcc2 abcc6 abcc9 mrp1 mrp2 review http://www.citeulike.org/user/happy_manatee/article/738801 <i>Molecular Microbiology, Vol. 61, No. 2. (July 2006), pp. 277-284.</i> Promiscuity in multidrug recognition and transport: the bacterial MFS Mdr transporters Lewinson Oded Adler Julia Sigal Nadejda Bibi Eitan doi:10.1111/j.1365-2958.2006.05254.x Molecular Microbiology, Vol. 61, No. 2. (July 2006), pp. 277-284. 2006-07-04T22:59:18-00:00 2006 Molecular Microbiology 0950-382X 61 2 277 284 Blackwell Publishing mdr promiscuity transmembrane transporter http://www.citeulike.org/user/happy_manatee/article/1012306 <i>J. Biol. Chem., Vol. 281, No. 41. (13 October 2006), pp. 31152-31163.</i><br /><br />Multidrug resistance is a serious problem in successful cancer chemotherapy. Studies using model cell lines have demonstrated that overexpression of some members of the ATP-binding cassette (ABC) transporter superfamily, such as ABCC1, causes enhanced efflux and, thus, decreased accumulation of multiple anticancer drugs, which leads to increased cell survival. Unlike most other ABC transporters, ABCC1 has an additional membrane-spanning domain (MSD0) with a putative extracellular amino terminus of 32 amino acids. However, the function of MSD0 and the role of the extracellular amino terminus are largely unknown. In this study, we examined the structural folding and the function of the amino terminus. We found that it has a U-shaped folding with the bottom of the U-structure facing cytoplasm and both ends in extracellular space. We also found that this U-shaped amino terminus probably functions as a gate to regulate the drug transport activity of human ABCC1. 10.1074/jbc.M603529200 The Amino Terminus of the Human Multidrug Resistance Transporter ABCC1 Has a U-shaped Folding with a Gating Function Qun Chen Youyun Yang Lang Li Jian-Ting Zhang doi:10.1074/jbc.M603529200 J. Biol. Chem., Vol. 281, No. 41. (13 October 2006), pp. 31152-31163. 2006-12-24T22:40:12-00:00 2006 J. Biol. Chem. 281 41 31152 31163 abcc1 transmembrane http://www.citeulike.org/user/happy_manatee/article/967338 <i>J Mol Biol, Vol. 358, No. 4. (12 May 2006), pp. 1060-1070.</i><br /><br />Certain prokaryotic transport proteins similar to the lactose permease of Escherichia coli (LacY) have been identified by BLAST searches from available genomic databanks. These proteins exhibit conservation of amino acid residues that participate in sugar binding and H(+) translocation in LacY. Homology threading of prokaryotic transporters based on the X-ray structure of LacY (PDB ID: 1PV7) and sequence similarities reveals a common overall fold for sugar transporters belonging to the Major Facilitator Superfamily (MFS) and suggest new targets for study. Evolution-based searches for sequence similarities also identify eukaryotic proteins bearing striking resemblance to MFS sugar transporters. Like LacY, the eukaryotic proteins are predicted to have 12 transmembrane domains (TMDs), and many of the irreplaceable residues for sugar binding and H(+) translocation in LacY appear to be largely conserved. The overall size of the eukaryotic homologs is about twice that of prokaryotic permeases with longer N and C termini and loops between TMDs III-IV and VI-VII. The human gene encoding protein FLJ20160 consists of six exons located on more than 60,000 bp of DNA sequences and requires splicing to produce mature mRNA. Cellular localization predictions suggest membrane insertion with possible proteolysis at the N terminus, and expression studies with the human protein FJL20160 demonstrate membrane insertion in both E.coli and Pichia pastoris. Widespread expression of the eukaryotic sugar transport candidates suggests an important role in cellular metabolism, particularly in brain and tumors. Homology is observed in the TMDs of both the eukaryotic and prokaryotic proteins that contain residues involved in sugar binding and H(+) translocation in LacY. Sequence alignment and homology threading reveals prokaryotic and eukaryotic proteins similar to lactose permease. VN Kasho IN Smirnova HR Kaback doi:10.1016/j.jmb.2006.02.049 J Mol Biol, Vol. 358, No. 4. (12 May 2006), pp. 1060-1070. 2006-11-29T20:16:10-00:00 2006 J Mol Biol 0022-2836 358 4 1060 1070 csmp http://www.citeulike.org/user/happy_manatee/article/965539 <i>PNAS, Vol. 99, No. 16. (6 August 2002), pp. 10347-10352.</i><br /><br />P-glycoprotein (P-gp) is a plasma membrane ATP-binding cassette transporter, responsible for multidrug resistance in tumor cells. P-gp catalyzes the ATP hydrolysis-dependent efflux of numerous amphiphilic compounds of unrelated chemical structures. In the absence of any identified substrate, P-gp exhibits an apparently futile, basal ATPase activity. By using native membrane vesicles containing high amounts of P-gp, we show here that (i) this basal ATPase activity is tightly dependent on the presence of cholesterol in the membrane; (ii) the stimulation of P-gp ATPase activity by drugs transported by P-gp is higher in the absence than in the presence of cholesterol and, conversely, the stimulation of P-gp ATPase activity by cholesterol is higher in the absence than in the presence of known P-gp substrates; (iii) P-gp mediates the ATP-dependent relocation of cholesterol from the cytosolic leaflet to the exoplasmic leaflet of the plasma membrane; and (iv) the decrease of the cholesterol dependence of P-gp ATPase activity induced by known P-gp substrates is correlated with the inhibition of the ATP-dependent cholesterol redistribution within the membrane. These data are highly evocative of a coupling between the basal ATPase activity of P-gp and its intramembrane cholesterol-redistribution function, and they are fully consistent with the possibility that P-gp may actively translocate cholesterol in the membrane. Finally, this P-gp-mediated cholesterol redistribution in the cell membrane makes it likely that P-gp contributes in stabilizing the cholesterol-rich microdomains, rafts and caveolae, and that it is involved in the regulation of cholesterol trafficking in cells. 10.1073/pnas.162366399 The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane Alexia Garrigues Alexandre Escargueil Stephane Orlowski PNAS, Vol. 99, No. 16. (6 August 2002), pp. 10347-10352. 2006-11-28T20:06:06-00:00 2002 PNAS 99 16 10347 10352 abcb1 mutants pgp pgpmuts http://www.citeulike.org/user/happy_manatee/article/965538 <i>J Biol Chem, Vol. 277, No. 46. (15 November 2002), pp. 44332-44338.</i><br /><br />The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 plus 681-1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2-12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding. Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein. TW Loo DM Clarke doi:10.1074/jbc.M208433200 J Biol Chem, Vol. 277, No. 46. (15 November 2002), pp. 44332-44338. 2006-11-28T20:02:45-00:00 2002 J Biol Chem 0021-9258 277 46 44332 44338 abcb1 mutants pgp pgpmuts http://www.citeulike.org/user/happy_manatee/article/965536 <i>J Biol Chem, Vol. 280, No. 4. (28 January 2005), pp. 2522-2528.</i><br /><br />P-glycoprotein (P-gp, ABCB1) actively transports a broad range of cytotoxic compounds out of the cell. The COOH terminus of P-gp contains a dileucine motif (Leu(1260)-Leu(1261)) and a conserved phenylalanine (Phe(1268)). Similar residues in SUR1 (ABCC8) were reported to be important plasma membrane-targeting signals (Sharma, N., Crane, A., Clement, J. P. t., Gonzalez, G., Babenko, A. P., Bryan, J., and Aguilar-Bryan, L. (1999) J. Biol. Chem. 274, 20628-20632). Here, we used alanine-scanning mutagenesis to test whether these residues were essential for trafficking of P-gp to the cell surface. Mutant L1260A expressed a 150-kDa immature protein that did not reach the cell surface and was sensitive to digestion by Endo H(f). By contrast, mutants L1261A, F1268A, and wild-type P-gps expressed the 170-kDa mature proteins at the cell surface. Mutation of Leu(1260) to Gly, Ile, Trp, Lys, or Glu also resulted in the expression of the 150-kDa immature protein. All of the mutants, however, expressed the 170-kDa protein in the presence of the drug substrate/specific chemical chaperone cyclosporin A. Mutant L1260A P-gp exhibited drug-stimulated ATPase activities similar to that of wild-type enzyme after rescue with cyclosporin A. Deletion of the last 22 amino acids (Q(1259)-Q(1280)) also caused misprocessing. The mutant, however, was rescued by expression in the presence of cyclosporin A and conferred resistance to colchicine in transfected cells. These results show that the dileucine motif is not a plasma membrane targeting signal. The COOH terminus is required for proper folding of P-gp but not for activity. The dileucine motif at the COOH terminus of human multidrug resistance P-glycoprotein is important for folding but not activity. TW Loo MC Bartlett DM Clarke doi:10.1074/jbc.M411483200 J Biol Chem, Vol. 280, No. 4. (28 January 2005), pp. 2522-2528. 2006-11-28T19:59:36-00:00 2005 J Biol Chem 0021-9258 280 4 2522 2528 abcb1 mutants pgp pgpmuts http://www.citeulike.org/user/happy_manatee/article/955715 <i>Biochemistry, Vol. 45, No. 24. (20 June 2006), pp. 7605-7616.</i><br /><br />Each nucleotide-binding domain (NBD) of mammalian P-glycoproteins (Pgps) and human ATP-binding cassette (ABC) B subfamily members contains a tyrosine residue approximately 25 residues upstream of the Walker A domain. To assess the role of the conserved Y401 and Y1044 residues of human Pgp, we substituted these residues with F, W, C, or A either singly or together. The mutant proteins were expressed in a Vaccinia virus-based transient expression system as well as in baculovirus-infected HighFive insect cells. The Y401F, Y401W, Y1044F, Y1044W, or Y401F/Y1004F mutants transported fluorescent substrates similar to the wild-type protein. On the other hand, Y401L and Y401C exhibited partial (30-50%) function, and transport was completely abolished in Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Similarly, in Y401A, Y1044A, and Y401A/Y1044A mutants, TNP-ATP binding, vanadate-induced trapping of nucleotide, and ATP hydrolysis were completely abolished. Thus, an aromatic residue upstream of the Walker A motif in ABC transporters is critical for binding of ATP. Additionally, the crystal structures of several NBDs in the nucleotide-bound form, data mining, and alignment of 18,514 ABC domains with the consensus conserved sequence in a database of all nonredundant proteins indicate that an aromatic residue is highly conserved in approximately 85% of ABC proteins. Although the role of this aromatic residue has previously been studied in a few ABC proteins, we provide evidence for a near-universal structural and functional role for this residue and recognize its presence as a conserved subdomain approximately 25 amino acids upstream of the Walker A motif that is critical for ATP binding. We named this subdomain the "A-loop" (aromatic residue interacting with the adenine ring of ATP). The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette. IW Kim XH Peng ZE Sauna PC FitzGerald D Xia M Müller K Nandigama SV Ambudkar doi:10.1021/bi060308o Biochemistry, Vol. 45, No. 24. (20 June 2006), pp. 7605-7616. 2006-11-21T19:00:26-00:00 2006 Biochemistry 0006-2960 45 24 7605 7616 abcb1 mutants pgp pgpmuts