共查询到20条相似文献,搜索用时 15 毫秒
1.
S Dey M Ramachandra I Pastan MM Gottesman SV Ambudkar 《Canadian Metallurgical Quarterly》1997,94(20):10594-10599
Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42-46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp. 相似文献
2.
EB Mechetner B Schott BS Morse WD Stein T Druley KA Davis T Tsuruo IB Roninson 《Canadian Metallurgical Quarterly》1997,94(24):12908-12913
The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp. 相似文献
3.
US Rao 《Canadian Metallurgical Quarterly》1998,37(42):14981-14988
P-glycoprotein (Pgp) mediates drug transport utilizing the energy released from ATP hydrolysis. However, the mechanism by which Pgp couples these two reactions remains unclear. The present work is undertaken to describe kinetically the first step, which is the interdependence of nucleotide and drug binding to the Pgp by the use of vanadate. Preincubation of human Pgp expressed in Sf9 insect cells with vanadate in the presence of Mg2+, ATP, and verapamil resulted in nearly complete and stable inhibition of the drug-stimulated ATPase function. In contrast, the Pgp ATPase function was nearly unaffected when Mg2+, ATP, or verapamil was omitted. Inhibition was highly specific for divalent cations that support ATP hydrolysis, for nucleotides that serve as substrates of hydrolysis, and for those drugs/compounds that interact with the drug-binding/transport sites of the Pgp. Kinetic analysis indicated that vanadate inhibition was MgATP concentration-dependent with an apparent Ki value similar to the apparent Km, suggesting that MgATP was bound to a similar ATP-binding site in both the ATPase inhibition and activation reactions. In support of this conclusion, vanadate, in the presence of Mg2+ and verapamil, caused selective trapping of 8-azido [alpha-32P] ATP and covalent labeling of ATP-binding site in the Pgp. Differences were observed in the vanadate-induced inhibition of wild-type and Val185 mutant Pgp's with different drug/compounds. These results suggested that the affinity of the interacting drug/compound is a constant and influences the overall stability of the inhibited Pgp species. Possible implications of these observations for the coupling of ATP hydrolysis to drug transport are discussed. 相似文献
4.
T Scheibel HI Siegmund R Jaenicke P Ganz H Lilie J Buchner 《Canadian Metallurgical Quarterly》1999,96(4):1297-1302
Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90. 相似文献
5.
FJ Sharom 《Canadian Metallurgical Quarterly》1997,160(3):161-175
Pgp is an atypical translocating ATPase, with low affinity for ATP and high constitutive ATPase activity. Pgp also has an unusually broad specificity for hydrophobic substrates, including many chemotherapeutic drugs. Transport studies in reconstituted systems indicate that drug transport requires ATP hydrolysis and is active, generating a drug concentration gradient. Binding of drugs and ATP to Pgp induces conformational changes in the protein, and the drug binding site is conformationally coupled to the NBDs. Evidence accumulated to date suggests that the transporter interacts directly with nonpolar substrates within the membrane environment, and may act as a drug flippase, moving drugs from the inner to the outer leaflet of the bilayer. Chemosensitizers that block the action of Pgp are proposed to act as alternative substrates, but their high rate of spontaneous flip-flop across the membrane results in futile cycling of the transporter. 相似文献
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7.
M Dong L Ladavière F Penin G Deléage LG Baggetto 《Canadian Metallurgical Quarterly》1998,1371(2):317-334
P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp. 相似文献
8.
CA Hrycyna M Ramachandra SV Ambudkar YH Ko PL Pedersen I Pastan MM Gottesman 《Canadian Metallurgical Quarterly》1998,273(27):16631-16634
Human P-glycoprotein (P-gp), an ATP-dependent efflux pump responsible for cross-resistance of human cancers to a variety of lipophilic compounds, is composed of two homologous halves, each containing six transmembrane domains and an ATP-binding/utilization domain. To determine whether each site can hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced ADP-trapping technique (P-gp.MgADP.Vi). In analogy with other ATPases, a photochemical peptide bond cleavage reaction occurs within the Walker A nucleotide binding domain consensus sequence (GX4GK(T/S)) when the molecule is trapped with Vi in an inhibited catalytic transition state (P-gp.MgADP.Vi) and incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from baculovirus-infected insect cells had drug-stimulated ATPase activity. Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the presence or absence of Vi under ultraviolet (365 nm) light on ice for 60 min. The resultant products were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with seven different human P-gp-specific antibodies covering the entire length of the molecule. Little to no degradation of P-gp was observed in the absence of Vi. In the presence of Vi, products of approximately 28, 47, 94, and 110 kDa were obtained, consistent with predicted molecular weights from cleavage at either of the ATP sites but not both sites. An additional Vi-dependent cleavage site was detected at or near the trypsin site in the linker region of P-gp. These results suggest that both the amino- and carboxyl-terminal ATP sites can hydrolyze ATP. However, there is no evidence that ATP can be hydrolyzed simultaneously by both sites. 相似文献
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P Hafkemeyer S Dey SV Ambudkar CA Hrycyna I Pastan MM Gottesman 《Canadian Metallurgical Quarterly》1998,37(46):16400-16409
P-glycoprotein (Pgp), the product of the MDR1 gene, confers multidrug resistance on cancer cells by ATP-dependent extrusion of anticancer drugs. Biochemical and genetic studies with Pgp have identified the putative transmembrane (TM) region 12 (residues 974-994) as a major region involved in drug interactions with amino acid residues conserved among Pgp family members shown to be essential for transport. To determine whether nonconserved residues might be involved in substrate specificity, seven amino acid residues were identified within TM 12 that were not strictly conserved among the MDR1 and MDR2 family of proteins from different mammalian species. We replaced all seven of these amino acid residues with alanine, one at a time and in combinations, and used a vaccinia virus based transient expression system to analyze function. None of the single replacements caused any alteration in transport function. However, when residues L975, V981, and F983 were replaced collectively, drug transport, drug-stimulated ATP hydrolysis, and photoaffinity labeling with the drug analogue, [125I]iodoarylazidoprazosin (IAAP), were abrogated, with little effect on [alpha-32P]-8-azido-ATP labeling and basal ATPase activity. Pairwise alanine substitutuions showed variable effects on function. Substitutions including L975A in combination with any one of the other two replacements had the least effect on Pgp function. The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. In contrast, alanine substitutions of all four nonconserved residues M986, V988, Q990, and V991 at the putative carboxy-terminal half of TM 12 showed no effect on drug transport except for a partial reduction in bodipy-verapamil extrusion. These results suggest that nonconserved residues in the putative amino-proximal half of TM 12 of Pgp play a more direct role in determining specificity of drug transport function than those in the putative carboxy-terminal half of TM 12. 相似文献
11.
NaeI is a remarkable type II restriction endonuclease. It must bind two recognition sequences to cleave DNA, forms a covalent protein-DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity. The latter activities apparently derive from reactivation of a cryptic DNA ligase active site. Here, we demonstrate that NaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa. The domains were purified by cloning. The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intact NaeI. Analytical ultracentrifugation showed this domain to be a monomer in solution. The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association. DNA greatly inhibited proteolysis of the linker region. The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure for NaeI. 相似文献
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13.
A peptide corresponding to the interdomain linker of chloroplast IF3 has been synthesized and its structure studied by NMR and CD as a function of temperature and pH. At low temperature and neutral pH the apparent helical content is 25%. pH and ionic strength dependent CD studies demonstrate that sidechain-sidechain interactions stabilize the structure observed at low temperature. The helicity decreases with temperature and above 25 degrees C the peptide is less than 15% helical. These results indicate that the peptide has little intrinsic tendency to form helical structure at physiologically relevant temperatures and strongly suggests that the linker region is flexible in intact chloroplast IF3. 相似文献
14.
MA Spear U Herrlinger N Rainov P Pechan R Weissleder XO Breakefield 《Canadian Metallurgical Quarterly》1998,4(2):133-147
Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous halves, each comprising 6 putative transmembrane alpha-helices and an ATP binding domain. Despite the plethora of data from site-directed, scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous data. The model comprises 2 membrane-embedded 16-strand beta-barrels, attached by short loops to two 6-helix bundles beneath each barrel. Each ATP binding domain contributes 2 beta-strands and 1 alpha-helix to the structure. This model, together with an analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data. 相似文献
15.
CD44 isoforms, such as CD44s (the standard form), contain at least one ankyrin-binding site within the 70-amino acid (aa) cytoplasmic domain and several hyaluronic acid (HA)-binding sites within the extracellular domain. To study the role of CD44s-ankyrin interaction in regulating human prostate tumor cells, we have constructed several CD44s cytoplasmic deletion mutants that lack the ankyrin-binding site(s). These truncated cDNAs were stably transfected into CD44-negative human prostate tumor cells (LNCaP). Our results indicate that a critical region of 15-amino acids (aa) between aa 304 and aa 318 of CD44s is required for ankyrin binding. Biochemical analyses, using competition binding assays with a synthetic peptide containing the 15 aa between aa 304 and aa 318 (NSGNGAVEDRKPSGL), further support the conclusion that this region contains the ankyrin-binding domain of CD44s. Deletion of this 15-aa ankyrin-binding sequence from CD44s results in a drastic reduction of HA-mediated binding/cell adhesion, Src p60 kinase(s) interaction and anchorage-independent growth in soft agar. These findings suggest that the binding of cytoskeletal proteins, such as ankyrin, to the cytoplasmic domain of CD44s plays a pivotal role in regulating HA-mediated functions as well as Src kinase activity and prostate tumor cell transformation. 相似文献
16.
AL Hughes 《Canadian Metallurgical Quarterly》1994,11(6):899-910
The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P-glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N-terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot. 相似文献
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18.
hTom20 is an outer mitochondrial membrane receptor involved in protein translocation. The cytosolic domain (aa30-145) and selected truncated versions of this domain were overexpressed and purified to study the structure-function relationship of this protein. Our studies reveal that the secondary structure of the cytosolic domain is very resistant to unfolding by guanidine-HCl and urea and is stabilized mainly by hydrophobic interactions. However, the tertiary structure of the N-terminal targeting signal binding domain (aa30-90) is more flexible. The first 30 amino acids of the cytosolic domain (aa30-60) are involved in recognizing N-terminal targeting signals and in stabilizing the cytosolic domain on the lipid surface. Moreover, we show that specifically aa30-48 interact with the membrane surface; a construct containing aa48-145 will only bind to the membrane surface in the presence of an N-terminal targeting signal peptide. The C-terminal region of hTom20 (aa141-145) interacts with the N-terminal region of hTom20, helping to stabilize the proper conformation of the N-terminal targeting signal binding domain. Finally, hTom20 interacts with the N-terminal targeting signal of preornithine carbamyl transferase fused to dihydrofolate reductase very weakly (Kd = 8 microM), as would be expected if this interaction was the first in a series orchestrated by the import receptor complex to draw the targeted protein into the mitochondrion. 相似文献
19.
K Alfthan K Takkinen D Sizmann H S?derlund TT Teeri 《Canadian Metallurgical Quarterly》1995,8(7):725-731
Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric. 相似文献
20.
The X-ray structures of the maltose bound forms of two insertion/deletion mutants of the Escherichia coli maltodextrin binding protein, MalE322 and MalE178, have been determined and refined. MalE322 involves a one residue deletion, two residue insertion in a hinge segment connecting the two (N and C) domains of the protein, an area already identified as being critical for the correct functioning of the protein. MalE178 involves a nine residue deletion and two residue insertion in a helix at the periphery of the C-domain. The function of both mutant proteins is similar to the wild-type, although MalE322 increases the ability to transport maltose and maltodextrin whilst inhibiting the ability of the cell to grow on dextrins. Both proteins exhibit very localized and conservative conformational changes due to their mutations. The structure of MalE322 shows some deformation of the third hinge strand, indicating the likely cause of change in its biochemistry. MalE178 is stable and its activity virtually unchanged from the wild-type. This is most likely due to the long distance of the mutation from the binding site and conservation of the number of interactions between the area around the deletion site and the main body of the protein. 相似文献