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PubMed1988

ABC excinuclease incises both 5' and 3' to the CC-1065-DNA adduct and its incision activity is stimulated by DNA helicase II and DNA polymerase I.

CC-1065 is a large molecule that binds covalently to adenine residues of DNA in a sequence-specific manner and lies in the minor groove about four bases to the 5' side of the adducted residue. Using a reconstituted Escherichia coli nucleotide excision repair system, we have obtained data showing that the ABC excinuclease makes incisions both 5' and 3' to the CC-1065 adduct and that the incision activity is stimulated by the addition of helicase II and DNA polymerase I (and dNTPs). Our results with the CC-1065 adduct are consistent with the reported in vitro processing of other adducts (e.g., cisplatin, UV photoproducts) but do not agree with a recent study that reported anomalous processing of the CC-1065 adduct by ABC excinuclease and helicase II. Our results also imply that, in binding to damaged DNA, ABC excinuclease does not make important contacts in the minor groove four bases to the 5' side of the damaged residue.

Adenosine Triphosphatases↗

PubMed2001

Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP >> UTP. These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.

ATP-Binding Cassette Transporters↗

PubMed2005

Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: identification of a conserved motif.

DrrA and DrrB proteins confer resistance to the commonly used anticancer agents daunorubicin and doxorubicin in the producer organism Streptomyces peucetius. The drrAB locus has previously been cloned in Escherichia coli, and the proteins have been found to be functional in this host. DrrA, a soluble protein, belongs to the ABC family of proteins. It forms a complex with the integral membrane protein DrrB. Previous studies suggest that the function and stability of DrrA and DrrB are biochemically coupled. Thus, DrrA binds ATP only when it is in a complex with DrrB in the membrane. Further, DrrB is completely degraded if DrrA is absent. In the present study, we have characterized domains in DrrB that may be directly involved in interaction with DrrA. Several single-cysteine substitutions in DrrB were made. Interaction between DrrA and DrrB was studied by using a cysteine to amine chemical cross-linker that specifically cross-links a free sulfhydryl group in one protein (DrrB) to an amine in another (DrrA). We show here that DrrA cross-links with both the N- and the C-terminal ends of the DrrB protein, implying that they may be involved in interaction. Furthermore, this study identifies a motif within the N-terminal cytoplasmic tail of DrrB, which is similar to a motif recently shown by crystal structure analysis in BtuC and previously shown by sequence analysis to be also present in exporters, including MDR1. We propose that the motif present in DrrB and other exporters is actually a modified version of the EAA motif, which was originally believed to be present only in the importers of the ABC family. The present work is the first report where domains of interaction in the membrane component of an ABC drug exporter have been biochemically characterized.

ATP-Binding Cassette Transporters↗

PubMed2006

Abc amino acids: design, synthesis, and properties of new photoelastic amino acids.

Photoisomerizable amino acids provide a direct avenue to the experimental manipulation of bioactive polypeptides, potentially allowing real-time, remote control of biological systems and enabling useful applications in nanobiotechnology. Herein, we report a new class of photoisomerizable amino acids intended to cause pronounced expansion and contraction in the polypeptide backbone, i.e., to be photoelastic. These compounds, termed Abc amino acids, employ a photoisomerizable azobiphenyl chromophore to control the relative disposition of aminomethyl and carboxyl substituents. Molecular modeling of nine Abc isomers led to the identification of one with particularly attractive properties, including the ability to induce contractions up to 13 A in the backbone upon trans --> cis photoisomerization. This isomer, designated mpAbc, has substituents at meta and para positions on the inner (azo-linked) and outer rings, respectively. An efficient synthesis of Fmoc-protected mpAbc was executed in which the biaryl components were formed via Suzuki couplings and the azo linkage was formed via amine/nitroso condensation; protected forms of three other Abc isomers were prepared similarly. An undecapeptide incorporating mpAbc was synthesized by conventional solid-phase methods and displayed characteristic azobenzene photochemical behavior with optimal conversion to the cis isomer at 360 nm and a thermal cis --> trans half-life of 100 min at 80 degrees C.

Amino Acids↗

PubMed2001

Complete characterization of the human ABC gene family.

The human ATP-binding cassette (ABC) transporters comprise a large family of membrane transport proteins and play a vital role in many cellular processes. The genes provide functions as diverse as peptide transport, cholesterol and sterol transport, bile acid, retinoid, and iron transport. In addition some ABC genes play a role as regulatory elements. Many ABC genes play a role in human genetic diseases, and several are critical drug transport proteins overexpressed in drug resistant cells. Analysis of the gene products allows the genes to be grouped into seven different subfamilies.

ATP-Binding Cassette Transporters↗

PubMed2001

ABC proteins of Leishmania.

ABC proteins were first characterized in the protozoan parasite Leishmania while studying mechanisms of drug resistance. PGPA is involved in resistance to arsenite and antimonite and it most likely confers resistance by sequestering metal-thiol conjugates into an intracellular vesicle. PGPA is part of gene family with at least four more members which are in search of a function. Leishmania also contains a P-glycoprotein, homologous to the mammalian MDR1, that is involved in multidrug resistance. The ongoing genome project of Leishmania has pinpointed several novel ABC transporters and experiments are carried out to study the function of the ABC proteins in drug resistance and in host-pathogen interactions.

ATP-Binding Cassette Transporters↗

PubMed1997

Modeling of nucleotide binding domains of ABC transporter proteins based on a F1-ATPase/recA topology: structural model of the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR).

Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the three dimensional structure of no member of this super-family has been elucidated. To gain structural insight, the known structures of several other nucleotides binding proteins can be used as a framework for modeling these domains. We have modeled both nucleotide binding domains of the protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) using the two similar domains of mitochondrial F1-ATPase. The models obtained, provide useful insights into the putative functions of these domains and their possible interaction as well as a rationale for the basis of Cystic Fibrosis causing mutations. First, the two nucleotide binding domains (folds) of CFTR are each predicted to span a 240-250 amino acid sequence rather than the 150-160 amino acid sequence originally proposed. Second, the first nucleotide binding fold, is predicted to catalyze significant rates of ATP hydrolysis as a catalytic base (E504) resides near the y phosphate of ATP. This prediction has been verified experimentally [Ko, Y.H., and Pedersen, P.L. (1995) J. Biol. Chem. 268, 24330-24338], providing support for the model. In contrast, the second nucleotide binding fold is predicted at best to be a weak ATPase as the glutamic acid residue is replaced with a glutamine. Third, F508, which when deleted causes approximately 70% of all cases of cystic fibrosis, is predicted to lie in a cleft near the nucleotide binding pocket. All other disease causing mutations within the two nucleotide binding domains of CFTR either reside near the Walker A and Walker B consensus motifs in the heart of the nucleotide binding pocket, or in the C motif which lies outside but near the nucleotide binding pocket. Finally, the two nucleotide binding domains of CFTR are predicted to interact, and in one of the two predicted orientations, F508 resides near the interface. This is the first report where both nucleotide binding domains of an ABC transporter and their putative domain-domain interactions have been modeled in three dimensions. The methods and the template used in this work can be used to analyze the structures and function of the nucleotide binding domains of all other members of the ABC transporter super-family.

ATP-Binding Cassette Transporters↗

PubMed2003

New approach to vital analysis of functional activity in ABC transporters (markers for multidrug resistance) in solid tumors by the method of flow cytofluorometry.

We developed and described a new approach to vital analysis of functional activity of multidrug resistance markers (ABC transporters) in intact biopsy specimens from human solid tumors by the method of flow cytofluorometry. The algorithm of the study underwent revision, and the cell suspension was obtained in the final stage. Intensification of intracellular doxorubicin accumulation (fluorescence) and increase in the number of fluorescent cells and total fluorescence of doxorubicin-accumulating cells produced by ABC transporter inhibitor sodium azide served as the criteria of expression of these transporters in tumor tissue. Informative value of changes in various parameters of doxorubicin fluorescence is discussed. The increase in the count of fluorescent cells in the suspension of tumors cells after treatment with the inhibitor indicates the presence of tumor cells absolutely resistant to this preparation. The proposed method is technically simple, suitable for structurally different tumors, requires small amounts of the biopsy material and, therefore, can be used for routine analysis. The results of our analysis and spectrofluorometric assay of ABC transporters agree very closely, which suggest that this method is adequate for the purpose.

ATP-Binding Cassette Transporters↗

PubMed2004

Insights into ABC transport in archaea.

In archaea, ATP-binding cassette (ABC) transporters play a crucial role in substrate uptake, export, and osmoregulation. Archael substrate-binding-protein-dependent ABC transporters are equipped with a very high affinity for their cognate substrates which provide these organisms with the ability to efficiently scavenge substrates from their environment even when present only at low concentration. Further adaptations to the archaeal way of life are especially found in the domain organization and anchoring of the substrate-binding proteins to the membrane. Examination of the signal peptides of binding proteins of 14 archael genomes showed clear differences between euryarchaeotes and crenarchaeotes. Furthermore, a profiling and comparison of ABC transporters in the three sequenced pyrococcal strains was performed.

ATP-Binding Cassette Transporters↗

PubMed2006

The ABC protein turned chloride channel whose failure causes cystic fibrosis.

CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

ATP-Binding Cassette Transporters↗

PubMed1997

Complete inventory of the yeast ABC proteins.

The complete sequence of the yeast genome predicts the existence of 29 proteins belonging to the ubiquitous ATP-binding cassette (ABC) superfamily. Using binary comparison, phylogenetic classification and detection of conserved amino acid residues, the yeast ABC proteins have been classified in a total of six clusters, including ten subclusters of distinct predicted topology and presumed distinct function. Study of the yeast ABC proteins provides insight into the physiological function and biochemical mechanisms of their human homologues, such as those involved in cystic fibrosis, adrenoleukodystrophy, Zellweger syndrome, multidrug resistance and the antiviral activity of interferons.

ATP-Binding Cassette Transporters↗

PubMed2006

ABC transporters, neural stem cells and neurogenesis--a different perspective.

Stem cells intrigue. They have the ability to divide exponentially, recreate the stem cell compartment, as well as create differentiated cells to generate tissues. Therefore, they should be natural candidates to provide a renewable source of cells for transplantation applied in regenerative medicine. Stem cells have the capacity to generate specific tissues or even whole organs like the blood, heart, or bones. A subgroup of stem cells, the neural stem cells (NSCs), is characterized as a self-renewing population that generates neurons and glia of the developing brain. They can be isolated, genetically manipulated and differentiated in vitro and reintroduced into a developing, adult or a pathologically altered central nervous system. NSCs have been considered for use in cell replacement therapies in various neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Characterization of genes with tightly controlled expression patterns during differentiation represents an approach to understanding the regulation of stem cell commitment. The regulation of stem cell biology by the ATP-binding cassette (ABC) transporters has emerged as an important new field of investigation. As a major focus of stem cell research is in the manipulation of cells to enable differentiation into a targeted cell population; in this review, we discuss recent literatures on ABC transporters and stem cells, and propose an integrated view on the role of the ABC transporters, especially ABCA2, ABCA3, ABCB1 and ABCG2, in NSCs' proliferation, differentiation and regulation, along with comparisons to that in hematopoietic and other stem cells.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB.

The ABC transporter HlyB is a central element of the HlyA secretion machinery, a paradigm of Type I secretion. Here, we describe the crystal structure of the HlyB-NBD (nucleotide-binding domain) with H662 replaced by Ala in complex with ATP/Mg2+. The dimer shows a composite architecture, in which two intact ATP molecules are bound at the interface of the Walker A motif and the C-loop, provided by the two monomers. ATPase measurements confirm that H662 is essential for activity. Based on these data, we propose a model in which E631 and H662, highly conserved among ABC transporters, form a catalytic dyad. Here, H662 acts as a 'linchpin', holding together all required parts of a complicated network of interactions between ATP, water molecules, Mg2+, and amino acids both in cis and trans, necessary for intermonomer communication. Based on biochemical experiments, we discuss the hypothesis that substrate-assisted catalysis, rather than general base catalysis might operate in ABC-ATPases.

ATP-Binding Cassette Transporters↗

PubMed2006

ABC transporter expression profiling after ischemic reperfusion injury in mouse kidney.

Renal ATP binding cassette (ABC) transporters have an important role in the elimination of metabolic waste products and compounds foreign to the body. The kidney has the ability to tightly control the expression of these efflux transporters to maintain homeostasis, and as a major mechanism of adaptation to environmental stress. In the present study, we investigated the expression of 45 ABC transporter genes in the mouse kidney under basal conditions, after induction of ischemia and after regeneration. Two days after clamping, mice showed a 76% decrease in renal creatinine clearance, which improved clearly within 7 days. This was confirmed by histological examinations. Seven days after ischemia, real-time quantitative Polymerase chain reaction data showed that transcript abundance of abcb1, abcb11, and abcc4 was increased, and that of abca3, abcc2, and abcg2 decreased. Expression of all transporters returned to baseline after 14 days, except for abcb11, which was reduced. Abcb11 is the major liver canalicular bile salt export pump. Here we show for the first time expression in the kidney and localization of the transporter to the apical membrane of proximal tubules. The presence of another novel renal transporter, abca3, was confirmed by Western blotting. Immunohistochemistry showed that abca3 is localized to the peritubular capillaries and apical membrane of proximal tubules. In conclusion, after inducing ischemic reperfusion injury in the kidney, ABC transporters appear to be differentially regulated, which might be associated with the renal regeneration process. Furthermore, we showed for the first time expression and subcellular localization of abcb11 and abca3 in mouse kidney.

ATP Binding Cassette Transporter, Subfamily B↗

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

Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly771 of the ABC signature sequence but does not enhance the intrinsic ATPase activity.

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn642-Ser871) and NBD2 (Ser1286-Val1531) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10(-5) s(-1)) and NBD2 (6x10(-6) s(-1)) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 microM. Despite the fact that both monomers bind ATP, no stable NBD1.NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of 15N-NBD1 in the presence of NBD2. This apparent NBD1.NBD2 interaction only occurred in the presence of Mg2+ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly771 of the LSGGQ sequence is involved in NBD1.NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1.NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.

Adenosine Triphosphatases↗

PubMed2005

ABC transporters in the balance: is there a role in multidrug resistance?

Drug resistance can occur at several levels and is the major cause of treatment failure in oncology. The ABC (ATP-binding cassette) transporters, beginning with the discovery of P-glycoprotein (Pgp) almost 30 years ago, have been intensively studied as potential mediators of drug resistance. Although we understand that drug resistance is almost certainly multifactorial, investigators have attempted to link anticancer drug resistance to overexpression of ABC transporters and the consequent reduction in drug accumulation. A body of evidence implicated Pgp as being important in clinical outcome; however, critical studies aimed at proving the hypothesis using Pgp inhibitors in clinical trials have to date failed. Identification of the MRP (multidrug resistance protein)/ABCC subfamily expanded the possible mechanisms of reduced drug accumulation, and the discovery of ABCG2 added a new chapter in these investigations. Correlative studies examining ABCG2 and the ABCC subfamily members in clinical drug resistance have been less avidly pursued, while basic molecular studies of structure and function have proceeded briskly. Recently, studies have focused on how single nucleotide polymorphism in multidrug transporters might affect the pharmacokinetics and pharmacodynamics of anticancer agents. These studies suggest an important role for ABC transporters in pharmacology, independent of the ultimate determination of their role in multidrug resistance.

ATP-Binding Cassette Transporters↗

PubMed2005

A molecular understanding of the catalytic cycle of the nucleotide-binding domain of the ABC transporter HlyB.

The ABC transporter (ATP-binding-cassette transporter) HlyB (haemolysin B) is the central element of a type I secretion machinery, dedicated to the secretion of the toxin HlyA in Escherichia coli. In addition to the ABC transporter, two other indispensable elements are necessary for the secretion of the toxin across two membranes in a single step: the transenvelope protein HlyD and the outer membrane protein TolC. Despite the fact that the hydrolysis of ATP by HlyB fuels secretion of HlyA, the essential features of the underlying transport mechanism remain an enigma. Similar to all other ABC transporters, ranging from bacteria to man, HlyB is composed of two NBDs (nucleotide-binding domains) and two transmembrane domains. Here we summarize our detailed biochemical, biophysical and structural studies aimed at an understanding of the molecular principles of how ATP-hydrolysis is coupled to energy transduction, including the conformational changes occurring during the catalytic cycle, leading to substrate transport. We have obtained individual crystal structures for each single ground state of the catalytic cycle. From these and other biochemical and mutational studies, we shall provide a detailed molecular picture of the steps governing intramolecular communication and the utilization of chemical energy, due to ATP hydrolysis, in relation to resulting structural changes within the NBD. These data will be summarized in a general model to explain how these molecular machines achieve translocation of molecules across biological membranes.

Adenosine Triphosphate↗