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1.
The yeast (Saccharomyces cerevisiae) multidrug transporter Pdr5p effluxes a broad range of substrates that are variable in structure and mode of action. Previous work suggested that molecular size and ionization could be important parameters. In this study, we compared the relative sensitivity of isogenic PDR5 and pdr5 strains toward putative substrates that are similar in chemical structure. Three series were used: imidazole-containing compounds, trialkyltin chlorides, and tetraalkyltin compounds. We demonstrate that the Pdr5p transporter is capable of mediating transport of substrates that neither ionize nor have electron pair donors and that are much simpler in structure than those transported by the human MDR1-encoded P-glycoprotein. Furthermore, the size of the substrate is critical and independent of any requirement for hydrophobicity. Substrates have surface volumes greater than 90 A(3) with an optimum response at approximately 200-225 A(3) as determined by molecular modeling. Assays measuring the efflux from cells of [(3)H]chloramphenicol and [(3)H]tritylimidazole were used. A concentration-dependent inhibition of chloramphenicol transport was observed with imidazole derivatives but not with either the organotin compounds or the antitumor agent doxorubicin. In contrast, several of the organotin compounds were potent inhibitors of tritylimidazole efflux, but the Pdr5p substrate tetrapropyltin was ineffective in both assays. This argues for the existence of at least three substrate-binding sites on Pdr5p that differ in behavior from those of the mammalian P-glycoprotein. Evidence also indicates that some substrates are capable of interacting at more than one site. The surprising observation that Pdr5p mediates resistance to tetraalkyltins suggests that one of the sites might use only hydrophobic interactions to bind substrates.  相似文献   

2.
P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity. However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance. The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity. The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site. In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction. A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells. The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction. The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors. The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.  相似文献   

3.
The yeast Pdr5p transporter is a 160 kDa protein that effluxes a large variety of xenobiotic compounds. In this study, we characterize its ATPase activity and demonstrate that it has biochemical features reminiscent of those of other ATP-binding cassette multidrug transporters: a relatively high Km for ATP (1.9 mM), inhibition by orthovanadate, and the ability to specifically bind an azidoATP analogue at the nucleotide-binding domains. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is also known to be a potent transport substrate. Our results indicate, however, that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. Curiously, Pdr5p-mediated transport of clotrimazole continues at intracellular concentrations of substrate that should eliminate all ATPase activity. Significantly, however, we observed that the Pdr5p has GTPase and UTPase activities that are relatively resistant to clotrimazole. Furthermore, the Km(GTPase) roughly matches the intracellular concentrations of the nucleotide reported for yeast. Using purified plasma membrane vesicles, we demonstrate that Pdr5p can use GTP to fuel substrate transport. We propose that Pdr5p increases its multidrug transport substrate specificity by using more than one nucleotide as an energy source.  相似文献   

4.
5.
P-glycoprotein is an ATP-dependent drug-efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane. Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy. The most commonly used photoreactive substrate is iodoarylazidoprazosin. Its binding domains within the P-glycoprotein have so far been inferred from indirect methods such as epitope mapping. In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides. P-glycoprotein-containing plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity-labeled with iodoarylazidoprazosin. After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125I-labeled peptides were separated by tricine/PAGE and HPLC and subjected to Edman sequencing. The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino-acid regions 248-312 [transmembrane segment (TM)4 to TM5], 758-800 (beyond TM7 to beyond TM8) and 1160-1218 (after the Walker A motif of the second nucleotide-binding domain). Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.  相似文献   

6.
In Saccharomyces cerevisiae several members of the ATP-binding cassette transporter superfamily efflux a broad range of xenobiotic substrates from cells. The vacuole also plays a critical role in multidrug resistance. Mutations in genes such as VPS3 that are essential for vacuolar acidification and carboxypeptidase Y vacuolar protein-sorting are multidrug sensitive. A similar phenotype is also observed with deletions of VPS15, VPS34, and VPS38, which encode essential members of the carboxypeptidase Y vacuolar protein-sorting pathway. Prior to the work described herein, detoxification by transporters and the vacuole were presumed to function independently. We demonstrate that this is not the case. Significantly, Vps3 has an epistatic relationship with Pdr5, a major yeast multidrug transporter. Thus, a double pdr5, vps3 deletion mutant is no more multidrug sensitive than its isogenic single-mutant counterparts. Subcellular fractionation experiments and analysis of purified plasma membrane vesicles indicate, however, that a vps3 mutation does not affect the membrane-localization or ATPase activity of Pdr5 even though rhodamine 6G efflux is reduced significantly. This suggests that Vps3 and probably other members of the carboxypeptidase Y vacuolar protein-sorting pathway are required for relaying xenobiotic compounds to transporters in the membrane.  相似文献   

7.
To find novel drugs for effective antifungal therapy in candidiasis, we examined disulfiram, a drug used for the treatment of alcoholism, for its role as a potential modulator of Candida multidrug transporter Cdr1p. We show that disulfiram inhibits the oligomycin-sensitive ATPase activity of Cdr1p and 2.5mM dithiothreitol reverses this inhibition. Disulfiram inhibited the binding of photoaffinity analogs of both ATP ([alpha-(32)P]8-azidoATP; IC(50)=0.76 microM) and drug-substrates ([(3)H]azidopine and [(125)I]iodoarylazidoprazosin; IC(50) approximately 12 microM) to Cdr1p in a concentration-dependent manner, suggesting that it can interact with both ATP and substrate-binding site(s) of Cdr1p. Furthermore, a non-toxic concentration of disulfiram (1 microM) increased the sensitivity of Cdr1p expressing Saccharomyces cerevisiae cells to antifungal agents (fluconazole, miconazole, nystatin, and cycloheximide). Collectively these results demonstrate that disulfiram reverses Cdr1p-mediated drug resistance by interaction with both ATP and substrate-binding sites of the transporter and may be useful for antifungal therapy.  相似文献   

8.
9.
ATP-binding cassette (ABC) transporters play important roles in drug efflux, but some may also function in cellular detoxification. The Pdr15p ABC protein is the closest homologue of the multidrug efflux transporter Pdr5p, which mediates pleiotropic drug resistance to hundreds of unrelated compounds. In this study, we show that the plasma membrane protein Pdr15p displays limited drug transport capacity, mediating chloramphenicol and detergent tolerance. Interestingly, Pdr15p becomes most abundant when cells exit the exponential growth phase, whereas its closest homologue, Pdr5p, disappears after exponential growth. Furthermore, in contrast to Pdr5p, Pdr15p is strongly induced by various stress conditions including heat shock, low pH, weak acids, or high osmolarity. PDR15 induction bypasses the Pdr1p/Pdr3p regulators but requires the general stress regulator Msn2p, which directly decorates the stress response elements in the PDR15 promoter. Remarkably, however, Pdr15p induction bypasses upstream components of the high osmolarity glycerol (HOG) pathway including the Hog1p and Pbs2p kinases as well as the dedicated HOG cell surface sensors. Our data provide evidence for a novel upstream branch of the general stress response pathway activating Msn2p. In addition, the results demonstrate a cross-talk between stress response and the pleiotropic drug resistance network.  相似文献   

10.
The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr5(1157) variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5(L183P), contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5(L183P) variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5(L183P) was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr5(1157) protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5(L183P) was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.  相似文献   

11.
ATP-binding cassette multidrug efflux pumps transport a wide range of substrates. Current models suggest that a drug binds relatively tightly to a transport site in the transmembrane domains when the protein is in the closed inward facing conformation. Upon binding of ATP, the transporter can switch to an outward facing (drug off or drug releasing) structure of lower affinity. ATP hydrolysis is critically important for remodeling the drug-binding site to facilitate drug release and to reset the transporter for a new transport cycle. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5. This substitution created broad, severe drug hypersensitivity, although drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. Several different rhodamine 6G efflux and accumulation assays yielded evidence consistent with the possibility that Ser-1368 prevents reentry of the excluded drug.  相似文献   

12.
The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.  相似文献   

13.
P-Glycoprotein (Pgp) is an important transport enzyme composed of two homologous domains and transports a wide range of structurally diverse xenobiotics from the cell. Recent studies have indicated that allosteric interactions occur between the nucleotide binding domains and between the substrate binding domains of the two halves, but the extent of this interaction as well as the means by which the enzyme can transport such a wide variety of substrates has not been elucidated. Herein, the Pgp-mediated transport of a marker substrate, daunorubicin (DNR), out of viable cells was examined in the presence of a variety of other known substrates of Pgp. For most of the typical Pgp substrates examined, the relationship between inhibition of DNR efflux and competing substrate concentration was sigmoidal and therefore not a simple mutually exclusive competitive inhibition of transport. The Hill coefficient ranged from about 3 to 5 for the inhibition of transport of DNR. This negative cooperativity in combination with recent evidence, including several examples of noncompetitive inhibition between the homologous halves of Pgp, indicates a "half-of-the-sites" reactivity. Our data support the mechanistic proposal that substrate binding at one putative transport binding site precludes activity at another unequal site; many of the substrates examined exert a negative allosteric effect on the other transport site (and vice versa). A half-of-the-sites reactivity model would account for many of these observations and may be critical to the efficiency of Pgp substrate transport of a broad spectrum of compounds.  相似文献   

14.
Both ATP sites of human P-glycoprotein are essential but not symmetric.   总被引:5,自引:0,他引:5  
Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.  相似文献   

15.
Pdr5p is one of the major multidrug efflux pumps whose overexpression confers multidrug resistance (MDR) in Saccharomyces cerevisiae. By using our original assay system, a fungal strain producing inhibitors for Pdr5p was obtained and classified as Fusarium sp. Y-53. The purified inhibitors were identified as ionophore antibiotics, enniatin B, B1, and D, respectively. A non-toxic concentration of each enniatin (5 microg/ml, approximately 7.8 microM) strongly inhibited a Pdr5p-mediated efflux of cycloheximide or cerulenin in Pdr5p-overexpressing cells. The enniatins accumulated a fluorescent dye rhodamine 123, a substrate of Pdr5p, into yeast cells. The mode of Pdr5p inhibition of enniatin was competitive against FK506, and its inhibitory activity was more potent with less toxicity than that of FK506. The enniatins showed similar inhibitory profile as FK506 against S1360 mutants (S1360A and S1360F) of Pdr5p. The enniatins did not inhibit the function of Snq2p, a homologue of Pdr5p. Thus, it was found that enniatins are potent and specific inhibitors for Pdr5p, with less toxicities than that of FK506.  相似文献   

16.
5'-Fluorosulfonylbenzonyl 5'-adenosine (FSBA) is an ATP analogue that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases, and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC(50 )= 0.21 mM) and the binding of 8-azido[α-(32)P]ATP (IC(50) = 0.68 mM). In addition, (14)C-FSBA cross-links to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photo-cross-linking of P-gp with [(125)I]iodoarylazidoprazosin (IAAP; IC(50) = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA cross-links to residues within or nearby the NBDs but not in the transmembrane domains, and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analogue that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated cross-linking is observed only at the NBDs.  相似文献   

17.
Abstract

The increasing number of multidrug-resistant pathogenic microorganisms is a serious public health issue. Among the multitude of mechanisms that lead to multidrug resistance, the active extrusion of toxic compounds, mediated by MDR efflux pumps, plays an important role. In our study we analyzed the inhibitory capability of 26 synthesized zosuquidar derivatives on three ABC-type MDR efflux pumps, namely Saccharomyces cerevisiae Pdr5 as well as Lactococcus lactis LmrA and LmrCD. For Pdr5, five compounds could be identified that inhibited rhodamine 6G transport more efficiently than zosuquidar. One of these is a compound with a new catechol acetal structure that might represent a new lead compound. Furthermore, the determination of IC50 values for rhodamine 6G transport of Pdr5 with representative compounds reveals values between 0.3 and 0.9 μM. Thus the identified compounds are among the most potent inhibitors known for Pdr5. For the ABC-type efflux pumps LmrA and LmrCD from L. lactis, seven and three compounds, which inhibit the transport activity more than the lead compound zosuquidar, were found. Interestingly, transport inhibition for LmrCD was very specific, with a drastic reduction by one compound while its diastereomers showed hardly an effect. Thus, the present study reveals new potent inhibitors for the ABC-type MDR efflux pumps studied with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria.  相似文献   

18.
We have previously shown that the synthetic nonsteroidal ecdysone agonist tebufenozide (RH-5992) is actively excluded by resistant cells of insects. To identify the transporter that could be involved in the efflux of RH-5992, the role of three ATP binding cassette transporters, Pdr5p, Snq2p and Ycf1p, has been studied using transporter-deletion mutants of yeast Saccharomyces cerevisiae. PDR5 (pleiotropic drug resistance 5) deletion mutants (Deltapdr5 and Deltapdr5Deltasnq2) retained significantly higher levels of 14C-radiolabeled RH-5992 within the cells when compared to wild-type strain or single deletion mutants of SNQ2 (Deltasnq2) and YCF1 (Deltaycf1). Introduction of an expression vector containing the PDR5 gene into the PDR5 single deletion mutant reversed the effect, resulting in the active exclusion of [14C]RH-5992 from these cells as efficiently as the wild-type cells. These results demonstrated that the ABC transporter Pdr5p but not Snq2p or Ycf1p was responsible for the active exclusion of [14C]RH-5992 in yeast. This exclusion was temperature-dependent and was blocked by the ATPase inhibitors oligomycin and vanadate, indicating that the efflux was an active process. The mutants with the PDR5 deletion can also selectively accumulate [14C]RH-0345 and [14C]RH-2485, but not [14C]RH-5849, indicating that these three compounds share the same transporter Pdr5p for efflux.  相似文献   

19.
Shukla S  Robey RW  Bates SE  Ambudkar SV 《Biochemistry》2006,45(29):8940-8951
The human ATP-binding cassette transporter, ABCG2, confers resistance to multiple chemotherapeutic agents and also affects the bioavailability of different drugs. [(125)I]Iodoarylazidoprazosin (IAAP) and [(3)H]azidopine were used for photoaffinity labeling of ABCG2 in this study. We show here for the first time that both of these photoaffinity analogues are transport substrates for ABCG2 and that [(3)H]azidopine can also be used to photolabel both wild-type R482-ABCG2 and mutant T482-ABCG2. We further used these assays to screen for potential substrates or modulators of ABCG2 and observed that 1,4-dihydropyridines such as nicardipine and nifedipine, which are clinically used as antihypertensive agents, inhibited the photolabeling of ABCG2 with [(125)I]IAAP and [(3)H]azidopine as well as the transport of these photoaffinity analogues by ABCG2. Furthermore, [(3)H]nitrendipine and bodipy-Fl-dihydropyridine accumulation assays showed that these compounds are transported by ABCG2. These dihydropyridines also inhibited the efflux of the known ABCG2 substrates, mitoxantrone and pheophorbide-a, from ABCG2-overexpressing cells, and nicardipine was more potent in inhibiting this transport. Both nicardipine and nifedipine stimulated the ATPase activity of ABCG2, and the nifedipine-stimulated activity was inhibited by fumitremorgin C, suggesting that these agents might interact at the same site on the transporter. In addition, nontoxic concentrations of dihydropyridines increased the sensitivity of ABCG2-expressing cells to mitoxantrone by 3-5-fold. In aggregate, results from the photoaffinity labeling and efflux assays using [(125)I]IAAP and [(3)H]azidopine demonstrate that 1,4-dihydropyridines are substrates of ABCG2 and that these photolabels can be used to screen new substrates and/or inhibitors of this transporter.  相似文献   

20.
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