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1.
Mol Ther Nucleic Acids ; 7: 429-438, 2017 Jun 16.
Article in English | MEDLINE | ID: mdl-28624219

ABSTRACT

A three-nucleotide deletion in cystic fibrosis transmembrane conductance regulator/ATP-binding cassette transporter C7 (CFTR/ABCC7) resulting in the absence of phenylalanine at 508 leads to mis-fold of the mutated protein and causes cystic fibrosis. We have used a comparable three-nucleotide deletion mutant in another ABCC family member, multidrug resistance-associated protein (MRP1)/ABCC1, to determine whether CRISPR-Cas9-mediated recombination can safely and efficiently knock in three-nucleotide to correct the mutation. We have found that the rate of homology-directed recombination mediated by guideRNA (gRNA) complementary to the deletion mutant is significantly higher than the one mediated by gRNA complementary to the wild-type (WT) donor. In addition, the rate of homology-directed recombination mediated by gRNA complementary to the WT donor is significantly higher than that of gRNAs complementary to the 5' or 3' side of the deletion mutant. Interestingly, the frequency of mutations introduced by gRNA complementary to the deletion mutant is significantly higher than with gRNA complementary to WT donor. However, combination of gRNAs complementary to both WT donor and deletion mutant decreased the rate of homology-directed recombination, but did not significantly decrease the mutation rate introduced by this system. Thus, the data presented here provide guidance for designing of gRNA and donor DNA to do genome editing, especially to correct the mutations with three mismatched nucleotides, such as three-nucleotide deletion or insertion.

2.
Clin Genitourin Cancer ; 15(2): 196-202.e1, 2017 04.
Article in English | MEDLINE | ID: mdl-27771244

ABSTRACT

BACKGROUND: Prostate tissue expresses 2 estrogen receptor (ER) isoforms, ER-α and ER-ß, and estrogen-based therapies have shown activity in preclinical studies. Raloxifene, a selective ER modulator, has inhibited the growth of prostate cancer xenograft models and was tested in a phase II trial of castration-resistant prostate cancer (CRPC), with some patients achieving stable disease. However, no studies have examined the safety of the combination of bicalutamide plus raloxifene for CRPC. Therefore, we investigated the safety of treatment with bicalutamide plus raloxifene in patients with CRPC in an initial study. MATERIALS AND METHODS: We conducted a study to evaluate the toxicity (primary endpoint) of the combination of bicalutamide (50 mg) and raloxifene (60 mg) in 28-day cycles (maximum, 6 cycles) in men with progressive CRPC. The secondary endpoint, quality of life (QOL), was assessed by patients using a 6-item linear analog self-assessment or hormonal domain scale of the Expanded Prostate Cancer Index Composite. RESULTS: We enrolled 18 patients with CRPC in the study to evaluate the safety of, and patient assessment of QOL (mental, physical, social, emotional, and spiritual) with, bicalutamide plus raloxifene therapy. No grade 3 or 4 adverse events occurred. None of the 18 patients required dose reductions. The patient assessment of QOL showed no statistically significant changes after 2 treatment cycles. The median progression-free survival with bicalutamide plus raloxifene was 1.9 months (95% confidence interval, 1.8-2.8 months). CONCLUSION: The results of the present study have shown that bicalutamide/raloxifene treatment is well tolerated. However, limited clinical activity occurred in men with CRPC who had previously undergone secondary hormonal therapy or chemotherapy.


Subject(s)
Anilides/adverse effects , Estrogen Antagonists/adverse effects , Nitriles/adverse effects , Prostatic Neoplasms, Castration-Resistant/drug therapy , Raloxifene Hydrochloride/adverse effects , Tosyl Compounds/adverse effects , Aged , Aged, 80 and over , Anilides/administration & dosage , Drug Combinations , Estrogen Antagonists/administration & dosage , Humans , Male , Middle Aged , Nitriles/administration & dosage , Quality of Life , Raloxifene Hydrochloride/administration & dosage , Survival Analysis , Tosyl Compounds/administration & dosage , Treatment Outcome
3.
BMC Cancer ; 16: 297, 2016 May 03.
Article in English | MEDLINE | ID: mdl-27142104

ABSTRACT

BACKGROUND: Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity. METHODS: Cereblon binding partners were identified from a MM cell line expressing histidine-tagged cereblon by pulling down cereblon and its binding partners and verified by co-immunoprecipitation. IMiD effects were determined by western blot analysis, cell viability assay, microRNA array and apoptosis analysis. RESULTS: We identified argonaute 2 (AGO2) as a cereblon binding partner and found that the steady-state levels of AGO2 were regulated by cereblon. Upon treatment of IMiD-sensitive MM cells with lenalidomide, the steady-state levels of cereblon were significantly increased, whereas levels of AGO2 were significantly decreased. It has been reported that AGO2 plays a pivotal role in microRNA maturation and function. Interestingly, upon treatment of MM cells with lenalidomide, the steady-state levels of microRNAs were significantly altered. In addition, silencing of AGO2 in MM cells, regardless of sensitivity to IMiDs, significantly decreased the levels of AGO2 and microRNAs and massively induced cell death. CONCLUSION: These results support the notion that the cereblon binding partner AGO2 plays an important role in regulating MM cell growth and survival and AGO2 could be considered as a novel drug target for overcoming IMiD resistance in MM cells.


Subject(s)
Argonaute Proteins/biosynthesis , Cell Proliferation/genetics , Multiple Myeloma/genetics , Peptide Hydrolases/metabolism , Adaptor Proteins, Signal Transducing , Apoptosis/genetics , Argonaute Proteins/antagonists & inhibitors , Argonaute Proteins/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lenalidomide , MicroRNAs/biosynthesis , Multiple Myeloma/pathology , Peptide Hydrolases/genetics , Protein Binding , Thalidomide/administration & dosage , Thalidomide/analogs & derivatives , Ubiquitin-Protein Ligases
4.
Channels (Austin) ; 10(3): 247-51, 2016.
Article in English | MEDLINE | ID: mdl-26645934

ABSTRACT

The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Cell Line , Cricetinae , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Electrophysiological Phenomena , Enzyme Activation , Mutagenesis, Site-Directed , Mutation
5.
Urol Oncol ; 32(1): 45.e23-30, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24055426

ABSTRACT

PURPOSE: Currently, there is no reliable tool to predict response to intravesical bacillus Calmette-Guérin (BCG). Based on the fact that BCG is a Th1-polarizing immunotherapy, we attempt to correlate the pretreatment immunologic tumor microenvironment (Th1 or Th2) with response to therapy. MATERIALS AND METHODS: Bladder cancer patients with initial diagnosis of carcinoma in situ (Tis) were stratified based on their response to BCG treatment. A total of 38 patients met inclusion criteria (20 patients who responded and 18 patients who did not respond). Immunohistochemical (IHC) methods known to assess the type of immunologic microenvironment (Th1 vs. Th2) were performed on tumor tissue obtained at initial biopsy/resection: the level of tumor eosinophil infiltration and degranulation (Th2 response); the number of tumor-infiltrating GATA-3(+) (Th2-polarized) lymphocytes; and the number of tumor-infiltrating T-bet(+) (Th1-polarized) lymphocytes. Results obtained from these metrics were correlated with response to treatment with BCG immunotherapy. RESULTS: The IHC metrics of the tumor immune microenvironment prior to BCG treatment were each statistically significant predictors of responders (R) vs. nonresponders (NR). Eosinophil infiltration and degranulation was higher for R vs. NR: 1.02 ± 0.17 vs. 0.5 ± 0.12 (P = 0.01) and 1.1 ± 0.15 vs. 0.56 ± 0.15 (P = 0.04), respectively. Ratio of GATA-3(+) (Th2-polarized) lymphocytes to T-bet(+) (Th1-polarized) lymphocytes was higher for R vs. NR: 4.85 ± 0.94 vs. 0.98 ± 0.19 (P<0.001). The 3 markers were combined to create a Th2 signature biomarker, which was a statistically significant (P<0.0001) predictor of R vs. NR. All IHC markers demonstrated that a preexisting Th1 immunologic environment within the tumor was predictive of BCG failure. CONCLUSION: The Th1 vs. Th2 polarization of bladder tumor immune microenvironment prior to treatment with BCG represents a prognostic metric of response to therapy. If a patient has a preexisting Th1 immunologic response within the tumor, there is no value in using a therapy intended to create a Th1 immunologic response. An algorithm integrating 3 IHC methods provided a sensitive and specific technique that may become a useful tool for pathologists and urologists to predict response to BCG in patients with carcinoma in situ of the bladder.


Subject(s)
BCG Vaccine/immunology , Carcinoma in Situ/immunology , Immunity, Active/immunology , Urinary Bladder Neoplasms/immunology , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/therapeutic use , Administration, Intravesical , Aged , BCG Vaccine/administration & dosage , BCG Vaccine/therapeutic use , Biomarkers, Tumor/immunology , Biomarkers, Tumor/metabolism , Carcinoma in Situ/metabolism , Carcinoma in Situ/therapy , Cell Degranulation/immunology , Eosinophils/drug effects , Eosinophils/immunology , Eosinophils/physiology , Female , GATA3 Transcription Factor/immunology , GATA3 Transcription Factor/metabolism , Humans , Immunity, Active/drug effects , Immunohistochemistry , Immunotherapy/methods , Lymphocytes/drug effects , Lymphocytes/immunology , Lymphocytes/metabolism , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Male , Prognosis , T-Box Domain Proteins/immunology , T-Box Domain Proteins/metabolism , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/therapy
6.
Biochim Biophys Acta ; 1808(7): 1790-6, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21315686

ABSTRACT

Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the K(m) (Mg·ATP) and V(max) values in Mg·ATP-dependent leukotriene-C4 transport. However, the V(max) values of the double mutants Q713N/Q1375N, Q713M/Q1375M and Q713L/Q1375L were lower than that of wtMRP1, implying that the double mutants cannot efficiently bind Mg·ATP. Interestingly, MRP1 has higher affinity for Mn·ATP than for Mg·ATP and the Mn·ATP-dependent leukotriene-C4 transport activities of Q713N/Q1375N and Q713M/Q1375M are significantly higher than that of wtMRP1. All these results suggest that: 1) the glutamine residues in Q-loops contribute to ATP-binding via interaction with a metal cofactor; 2) it is most unlikely that these glutamine residues would play crucial roles in ATP hydrolysis and in transmitting the conformational changes between nucleotide-binding-domains and transmembrane-domains.


Subject(s)
Adenosine Triphosphate/metabolism , Drug Resistance, Multiple , Glutamine/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , DNA Primers , Glutamine/chemistry , Humans , Hydrolysis , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/genetics , Mutagenesis, Site-Directed , Photoaffinity Labels , Spodoptera
7.
Biochemistry ; 48(38): 9122-31, 2009 Sep 29.
Article in English | MEDLINE | ID: mdl-19691360

ABSTRACT

ABCG2 is a half-ATP binding cassette (ABC) drug transporter that consists of a nucleotide binding domain (NBD) followed by a transmembrane domain. This half-ABC transporter is thought to form a homodimer in the plasma membrane where it transports anticancer drugs across the biological membranes in an ATP-dependent manner. Substitution of the putative catalytic residue E211 with a nonacidic amino acid glutamine (E211Q) completely abolished its ATPase activity and ATP-dependent methotrexate transport, suggesting that ATP hydrolysis is required for the ATP-dependent solute transport. However, whether one ATP hydrolysis or two ATP hydrolyses in the homodimer of ABCG2 with the NBD.ATP.ATP.NBD sandwich structure is/are required for the ATP-dependent solute transport is not known yet. To address this question, we have made an YFP/ABCG2 fusion protein and expressed this 99 kDa fusion protein alone or along with the 70 kDa E211Q-mutated ABCG2 in BHK cells. Although membrane vesicles prepared from BHK cells expressing YFP/ABCG2 exert higher ATPase activity than that of wt ABCG2, the dATP-dependent methotrexate transport activities of these two proteins are the same. Interestingly, membrane vesicles prepared from BHK cells expressing both YFP/ABCG2 and E211Q-mutated ABCG2 (with a ratio of 1:1) form homodimers and heterodimer and exert 55% of wt ABCG2 ATPase activity that can be further enhanced by anticancer drugs, suggesting that the wt NBD in the heterodimer of YFP/ABCG2 and E211Q may be able to hydrolyze ATP. Furthermore, the membrane vesicles containing both YFP/ABCG2 and E211Q exert approximately 79% of wt ABCG2-mediated methotrexate transport activity, implying that the heterodimer harboring YFP/ABCG2 and E211Q may be able to transport the anticancer drug methotrexate across the biological membranes.


Subject(s)
ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Methotrexate/pharmacokinetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/chemistry , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Substitution , Animals , Antimetabolites, Antineoplastic/pharmacokinetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Biological Transport, Active/genetics , Catalytic Domain/genetics , Cell Line , Cricetinae , DNA Primers/genetics , Dimerization , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Humans , In Vitro Techniques , Kinetics , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mutagenesis, Site-Directed , Neoplasm Proteins/chemistry , Protein Structure, Quaternary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
8.
J Cell Sci ; 121(Pt 17): 2814-23, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18682497

ABSTRACT

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/biosynthesis , Oligosaccharides/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Calnexin/metabolism , Cell Line , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Endoplasmic Reticulum/metabolism , Glycosylation , Humans , Lectins/metabolism , Models, Biological , Molecular Chaperones/metabolism , Molecular Sequence Data , Mutagenesis , Mutant Proteins/metabolism , Protein Binding , Protein Transport
9.
J Mol Biol ; 365(4): 981-94, 2007 Jan 26.
Article in English | MEDLINE | ID: mdl-17113596

ABSTRACT

The dimerization of their two nucleotide binding domains (NBDs) in a so-called "nucleotide-sandwich" is the hallmark of ATP cassette binding (ABC) proteins and the basis of their catalytic activities. The major disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7), deletion of Phe508 in NBD1, does not grossly alter the structure of that domain but prevents conformational maturation of the whole CFTR protein, possibly by disrupting the native interaction between NBD1 and NBD2. However, the role of inter-domain interactions in CFTR folding has been brought into question by a recent report that all CFTR domains fold independently. Here we show that in addition to domain folding, correct inter-domain assembly is essential to form a stable unit that satisfies endoplasmic reticulum (ER) quality control. N-terminal domains depend on their more C-terminal neighbors, most essentially the second membrane-spanning domain (MSD2) but significantly, not NBD2. Wild-type C-terminal truncation constructs, completely devoid of NBD2 are transported out of the ER and to the cell surface where they form characteristic CFTR chloride channels with low open probability. The DeltaNBD2 wild-type protein matures and has similar stability as its full-length counterpart. Therefore, the catalytically crucial inter-NBD associations are not required to satisfy ER quality control mechanisms. The DeltaF508 mutation arrests the maturation of DeltaNBD2 just as it does full-length CFTR, indicating that DeltaF508 perturbs other portions of the molecule in addition to NBD2. We find that the mutation prevents formation of a compact MSD1, reflected in its susceptibility to protease digestion. This perturbation of MSD1 may in turn prevent its normal integration with MSD2. The dispensability of NBD2 in the folding of more N-terminal domains stands in contrast to the known hypersensitivity to proteolysis of NBD2 in the DeltaF508 protein.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Adenosine Triphosphate/chemistry , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Dimerization , Epitopes/chemistry , Glycoside Hydrolases/metabolism , Humans , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Trypsin/chemistry , Trypsin/pharmacology
10.
Biochem J ; 397(1): 121-30, 2006 Jul 01.
Article in English | MEDLINE | ID: mdl-16551273

ABSTRACT

MRP1 (multidrug resistance protein 1) couples ATP binding/hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L> or =CFTR (cystic fibrosis transmembrane conductance regulator)>K684L>>>wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.


Subject(s)
Adenosine Triphosphate/metabolism , Drug Resistance, Multiple/genetics , Lysine/chemistry , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/genetics , Protein Structure, Tertiary , Animals , Biological Transport , Cells, Cultured , Cricetinae , Hydrolysis , Hydrophobic and Hydrophilic Interactions , Kidney/cytology , Multidrug Resistance-Associated Proteins/physiology , Mutagenesis, Site-Directed
11.
J Physiol ; 572(Pt 2): 347-58, 2006 Apr 15.
Article in English | MEDLINE | ID: mdl-16484308

ABSTRACT

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Ion Channel Gating/physiology , Phenylalanine/analysis , Action Potentials/physiology , Adenosine Triphosphate/physiology , Animals , Blotting, Western , Cell Line , Chloride Channels/physiology , Cricetinae , Cysteine/analysis , Cystic Fibrosis/etiology , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation , Hydrolysis , Microscopy, Fluorescence , Mutation , Protein Binding , Protein Structure, Tertiary
12.
Biochim Biophys Acta ; 1668(2): 248-61, 2005 Mar 01.
Article in English | MEDLINE | ID: mdl-15737336

ABSTRACT

MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear. We now report that ATP hydrolysis at NBD1 may not be essential for transport, but that the dissociation of the NBD1-bound nucleotide facilitates ATP-dependent LTC4 transport. These conclusions are supported by the following results. The substitution of the putative catalytic E1455 with a non-acidic residue in NBD2 greatly decreases the ATPase activity of NBD2 and the ATP-dependent LTC4 transport, indicating that E1455 participates in ATP hydrolysis. The mutation of the corresponding D793 residue in NBD1 to a different acidic residue has little effect on ATP-dependent LTC4 transport. The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Along with their higher transport activities, their Michaelis constant Kms (ATP) are also higher than that of wild-type. Coincident with their higher Kms (ATP), their Kds derived from ATP binding are also higher than that of wild-type, implying that the rate of dissociation of the bound nucleotide from the mutated NBD1 is faster than that of wild-type. Therefore, regardless of whether the bound ATP at NBD1 is hydrolyzed or not, the release of the bound nucleotide from NBD1 may bring the molecule back to its original conformation and facilitate the protein to start a new cycle of ATP-dependent solute transport.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Adenosine Triphosphate/metabolism , Nucleotides/metabolism , Protein Transport/physiology , Spodoptera/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Solubility , Structure-Activity Relationship
13.
J Biol Chem ; 278(33): 30764-71, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-12783859

ABSTRACT

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. The two NBDs possess different properties (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) and may play different roles during solute transport. We now report that NBD1 has moderately higher affinity for ATP than NBD2. The consequence of this difference is that the overall Kd value for wild-type MRP1 is mainly determined by ATP binding at NBD1. This conclusion is supported by the following: 1) mutation of the cysteine residue at 682 to alanine (C682A) in Walker A motif in NBD1 decreases the Kd value, indicating increased affinity for ATP; 2) mutation of the alanine residue at 1331 to cysteine (A1331C) in the Walker A motif of NBD2 does not have an effect on the Kd value; and 3) photolabeling of the protein with a cysteine residue in the Walker A motif of NBD1 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD2. In contrast, the Km for ATP in support of LTC4 transport is mainly determined by ATP hydrolysis at NBD2. This conclusion is supported by the following: 1) although mutation of A1331C does not have an effect on the Kd value, the Km values measured from LTC4 transport by proteins with this mutation in NBD2 are much higher than the proteins with wild-type NBD2, implying that the A1331C mutation affects ATP binding/hydrolysis at NBD2; and 2) ATP-dependent LTC4 transport by the protein with a cysteine residue in the Walker A motif of NBD2 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD1. Our previous results indicated that ATP binding at NBD1 at low concentration enhanced ATP binding/hydrolysis at NBD2. All of these results support the notion that ATP binding at NBD1 at low concentration plays a more important regulatory role than the binding at high ATP concentration and that ATP hydrolysis at NBD2 plays a dominant role in the ATP-dependent LTC4 transport.


Subject(s)
Adenosine Triphosphate/metabolism , Leukotriene C4/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Biological Transport/physiology , Cell Line , Cricetinae , Cysteine/genetics , Enzyme Inhibitors/pharmacology , Ethylmaleimide/pharmacology , Humans , Hydrolysis , Kidney/cytology , Mutagenesis , Protein Structure, Tertiary
14.
J Biol Chem ; 278(6): 3599-605, 2003 Feb 07.
Article in English | MEDLINE | ID: mdl-12458196

ABSTRACT

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP-dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. We found that ATP binding to the first NBD of MRP1 increases binding and trapping of ADP at the second domain (Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2002) J. Biol. Chem. 277, 5110-5119). These results were interpreted as indicating that the binding of ATP at NBD1 causes a conformational change in the molecule and increases the affinity for ATP at NBD2. However, we did not distinguish between the possibilities that the enhancement of ADP trapping might be caused by either ATP binding alone or hydrolysis. We now report the following. 1) ATP has a much lesser effect at 0 degrees C than at 37 degrees C. 2) After hexokinase treatment, the nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate), does not enhance ADP trapping. 3) Another nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene)triphosphate, whether hexokinase-treated or not, causes a slight enhancement. 4) In contrast, the hexokinase-treated poorly hydrolyzable ATP analogue, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), enhances ADP trapping to a similar extent as ATP under conditions in which ATPgammaS should not be hydrolyzed. We conclude that: 1) ATP hydrolysis is not required to enhance ADP trapping by MRP1 protein; 2) with nucleotides having appropriate structure such as ATP or ATPgammaS, binding alone can enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping is greatly diminished when the MRP1 protein is in a "frozen state" (0 degrees C); and 4) the steric structure of the nucleotide gamma-phosphate is crucial in determining whether binding of the nucleotide to NBD1 of MRP1 protein can induce the conformational change that influences nucleotide trapping at NBD2.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Cell Line , Cricetinae , Hydrolysis , Photoaffinity Labels
15.
J Biol Chem ; 278(5): 3347-56, 2003 Jan 31.
Article in English | MEDLINE | ID: mdl-12424247

ABSTRACT

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4). The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.


Subject(s)
Glutathione/pharmacology , Multidrug Resistance-Associated Proteins/chemistry , Pharmaceutical Preparations/metabolism , Adenosine Triphosphate/pharmacology , Amides/chemistry , Animals , Cell Line , Cell Membrane/ultrastructure , Cricetinae , Drug Resistance, Multiple , Humans , Kidney , Ligands , Models, Molecular , Multidrug Resistance-Associated Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Protein Structure, Secondary , Recombinant Proteins/chemistry , Spectroscopy, Fourier Transform Infrared , Transfection , Trypsin
16.
J Biol Chem ; 277(7): 5110-9, 2002 Feb 15.
Article in English | MEDLINE | ID: mdl-11741902

ABSTRACT

Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.


Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Multidrug Resistance-Associated Proteins , Adenosine Triphosphate/pharmacology , Adenylyl Imidodiphosphate/pharmacology , Allosteric Site , Amino Acid Motifs , Animals , Anions , Aspartic Acid/chemistry , Blotting, Western , Catalysis , Cell Membrane/metabolism , Cricetinae , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Immunoblotting , Light , Lysine/chemistry , Mice , MutS Homolog 3 Protein , Mutation , Protein Binding , Protein Structure, Tertiary , Temperature , Vanadates/pharmacology
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