ABCMdb

ABCG2 p.Tyr413Ser

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Publications

PMID: 25445676 [PubMed] Gal Z et al: "Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter."

No. Sentence Comment

6 We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. Login to comment
112 We found that mutation of Tyr to Ser at position 469 or 645 resulted in the loss of ATP hydrolysis (even if the activity is corrected for the lower expression level of the Y469S mutant); while mutations in the other two positions apparently did not alter ABCG2 functionality, as both the Y413S and Y570S mutants showed a high level of ATPase activity, which could be inhibited by a general ATPase inhibitor vanadate or the specific ABCG2 inhibitor Ko143 (Fig. 2B). Login to comment
123 Conversely, we found that in the presence of cholesterol, the ATPase activity of ABCG2-Y413S was distinct from that of the wild-type transporter. Login to comment
124 In the case of Y413S, cholesterol resulted in a major (50 +/- 8%; p b 0.01) enhancement of the basal ATP hydrolysis, which could be only slightly stimulated by the addition of prazosin or quercetin (Figs. 2B and C, and 4B). Login to comment
125 Next, we investigated whether other compounds which are known substrates of wtABCG2 also differently modify the Y413S-ATPase in the presence of cholesterol (Fig. 3). Login to comment
127 In contrast, the Y413S mutant had a significantly altered ATPase modulation pattern. Login to comment
129 For a detailed analysis of the effects of cholesterol loading, the basal and drug-stimulated ATPase activities of the wild-type and Y413S ABCG2 mutants were compared at increasing membrane cholesterol concentrations. Login to comment
130 As shown in Fig. 4A, the baseline ATPase of Y413S is activated by lower cholesterol concentrations than that of the wild-type protein, with a lower apparent half-maximum cholesterol activation concentration (KA value of 0.37 +/- 0.27 mM) as compared to the apparent affinity of the wild-type protein for cholesterol (KA for cholesterol of 0.70 +/- 0.09 mM). Login to comment
131 In contrast, in the case of the substrate-stimulated ATPase activities, wtABCG2 showed higher sensitivity to cholesterol loading (Fig. 4B): in the presence of quercetin the calculated KA value for cholesterol activation of the wild-type protein was 0.22 +/- 0.20 mM, while, due to the low level of cholesterol activation a reliable KA for the Y413S protein could not be determined. Login to comment
133 Altered bile acid sensitivity of the ABCG2-Y413S mutant The experiments described above showed that both the baseline and the drug-stimulated ATPase activity of the Y413S mutant differ significantly from that of the wild-type protein when excess cholesterol is present. Login to comment
144 bile acids on the ATPase activity of the Y413S mutant. Login to comment
146 Therefore the net result of bile acid effects was only a slight increase in the relative substrate stimulation of the Y413S mutant (Fig. 5B-D). Login to comment
159 As documented in Fig. 6, all the examined ABCG2 mutants, including Y413S, exhibited a methotrexate transport activity that, similarly to the wild-type transporter, was significantly (3- to 4-fold) enhanced by cholesterol loading of the membranes. Login to comment
161 Expression and transport activity of ABCG2 CRAC mutants in HEK 293 cells - effect of cholesterol depletion According to the presented results the baseline ATPase activity of the ABCG2-Y413S mutant showed a sterol sensitivity that significantly differed from that of the wild-type protein, while in the vesicular transport measurement the effect of cholesterol on this mutant did not differ from that measured for wtABCG2. Login to comment
163 Effect of known wtABCG2 substrates on the ATPase activity of wtABCG2 and the Y413S mutant. Login to comment
168 Effect of cholesterol on the wtABCG2 and Y413S-ATPase. Login to comment
174 generated mammalian HEK 293 cells stably expressing the Y413S, Y469F, Y570S and Y645F mutants. Login to comment
181 As shown in Fig. 7, all ABCG2 mutants, including Y413S, were able to actively transport Hoechst 33342, and cholesterol depletion significantly decreased the Hoechst 33342 transport activity of all the CRAC mutants similarly to wild-type ABCG2. Login to comment
183 Putative CRAC mutants of ABCG2, including Y413S, effectively transport substrate drugs and protect HEK 293 cells against cytotoxic substrates The differential effect of the transported substrates on the wild-type and the Y413S ABCG2 ATPase activity may reflect different transport properties. Login to comment
185 In order to further explore the relationship of altered drug-stimulated ATPase and transport activities, the transport of various fluorescent ABCG2 substrates was compared in HEK 293 cells overexpressing wtABCG2 and the Y413S mutant, respectively. Login to comment
187 Effect of bile acids on the wtABCG2 and Y413S-ATPase. Login to comment
199 Interestingly, in spite of its different behavior in the ATPase assay, the Y413S mutant also did not differ significantly from the wild-type protein, with respect to the transport of the above mentioned substrates. Login to comment
200 Among the cytotoxic drugs, the Y413S mutant ABCG2 protein (similarly to the wild-type) was able to transport doxorubicin (Fig. 8A) and provided protection against doxorubicin, SN-38, flavopiridol and topotecan, strongly indicating an active transport of these compounds by this mutant (Fig. 8B and Supplementary Fig. S3). Login to comment
239 However, the Y413S mutant exhibited a distinct ATPase activity. Login to comment
250 BODIPY-prazosin Pheophorbide A Hoechst 33342 mitoxantrone wtABCG2 + + + + Y413S + + + + Y469F + + + + Y570S + + + + Y645F + + + + Fig. 8. Login to comment
257 HEK 293 parental cells or stably expressing wtABCG2 or its Y413S mutant were incubated with 50 nM SN-38, 50 nM topotecan (TOP), 60 nM doxorubicin (DOX) or 500 nM flavopiridol (FLP) for 72 hours. Login to comment
262 Moreover, potential substrates that are known to activate the wild-type ABCG2-ATPase, rather inhibited the ATPase activity of the Y413S mutant (Fig. 3). Login to comment
263 In addition to this altered cholesterol-sensing, the ATPase activity of the Y413S mutant also showed an altered bile acid sensitivity, as this mutant was less sensitive to bile acids than the wild-type protein (Fig. 5). Login to comment
267 In the case of the Y413S mutant, cholesterol may promote "uncoupling" of the ATP hydrolysis in the ABCG2 protein. Login to comment
268 Alternatively, if we consider the possibility that cholesterol is transported by ABCG2, the results could be explained by a higher affinity to cholesterol by the ABCG2-Y413S mutant, also observed in the case of the R482G mutant [15]. Login to comment
270 If ABCG2 can transport cholesterol, our data can be interpreted to suggest that the Y413S mutant is a more efficient cholesterol transporter. Login to comment
273 Since ABCG2-mediated cholesterol transport has not been experimentally confirmed, we speculate that the "cholesterol-stimulated" ATPase activity of the ABCG2-Y413S mutant is a result of its differential sensitivity to the modulatory effect of cholesterol. Login to comment
274 In contrast to these findings related to the ABCG2-ATPase activity, we did not find any significant alteration in the cholesterol sensitivity of the ABCG2-Y413S mediated transport either in insect cell vesicular transport, or mammalian intact cell transport assays. Login to comment
275 Active substrate transport by the Y413S mutant was clearly activated by cholesterol in both systems, and by all the compounds examined (Figs. 6 and 7). Login to comment
276 Moreover, the Y413S mutant protected the cells against toxic drugs similarly to the wild-type ABCG2. Login to comment
277 All these experimental data suggest that although the ABCG2-Y413S mutant has an altered cholesterol interaction, it is rather manifested in a partial uncoupling of the ATPase activity of the transporter in the absence of transported substrates. Login to comment