ABCMdb

ABCB1 p.Asp805Cys

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Publications

PMID: 24064216 [PubMed] Kapoor K et al: "Mutations in intracellular loops 1 and 3 lead to misfolding of human P-glycoprotein (ABCB1) that can be rescued by cyclosporine A, which reduces its association with chaperone Hsp70."

No. Sentence Comment

7 It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of P-gp, which thus failed to transport the drug substrates. Login to comment
29 2 The abbreviations used are: ABC, ATP binding cassette; P-gp, P-glycoprotein; CsA, cyclosporine A; CYH, cycloheximide; ER, endoplasmic reticulum; ICL, intracellular loop; NBD, nucleotide binding domain; NBD-CsA, NBD-cyclosporine A; Rh123, rhodamine 123; SNP, single-nucleotide polymorphism; TMD, transmembrane domain; Endo H, endo-beta-N-acetylgluco- saminidase H; PNGase F, peptide N-glycosidase F; FKBP, FK506-binding protein; DD, D164C/D805C. Login to comment
35 Our insect cell studies show that the double mutant D164C/D805C displays no change in Km for ATP binding, thus contradicting the suggested direct interaction of these residues with ATP (5). Login to comment
37 The conserved aspartates, when mutated to cysteine singly (D164C, D805C) or together (D164C/D805C), affected the processing and trafficking of P-gp to the cell membrane. Login to comment
38 We discovered that the maturation defect associated with the D164C/D805C mutant was sensitive to growth temperature. Login to comment
39 When cells expressing the D164C/ D805C mutant were incubated at 27 &#b0;C (similar to growth conditions for High-five insect cells), normal maturation of P-gp was observed. Login to comment
41 Subsequently, we observed that the incubation of cells expressing the D164C/ D805C mutant in the presence of pharmacological chaperones or substrates such as cyclosporine A (CsA) completely rescued the misfolded protein as a functional protein to the cell surface. Login to comment
60 Preparation of Crude Membranes from Baculovirus-infected High-Five Insect Cells-High-Five insect cells (Invitrogen) were infected at multiplicity of infection 10 with recombinant baculovirus carrying the human cysless-MDR1 cDNA (all seven cysteines replaced with alanine) and D164C/D805C (in a cysless background) with a His6 tag at the C terminus as described previously. Login to comment
71 Briefly, either cysless-WT, single (D164C, D805C), or double mutant (D164C/D805C) baculovirus was added to HeLa cells at a ratio of 50-60 viral particles per cell in 3 ml of media. Login to comment
84 Rescue of Mutant P-gp Using Cyclosporine A-HeLa cells were transduced with D164C, D805C, or D164C/D805C mutant P-gps as described above. Login to comment
87 For time course experiments, the cells were transduced with D164C/D805C mutant P-gp and grown at 37 &#b0;C for 24 h as described above. Login to comment
90 Also, to check if preformed protein was being rescued by CsA, HeLa cells were transduced with the D164C/D805C P-gp. Login to comment
94 Rescue of Mutant P-gp Using Various Substrates/Modulators or CFTR Correctors-HeLa cells were transduced with D164C/ D805C as described earlier. Login to comment
146 Mutant P-gps Display Lower Cell Surface Expression and Defective Transport Function When Expressed in HeLa Cells-The Baculovirus BacMam system was used to express single (D164C or D805C) and double (D164C/D805C) mutant P-gps in HeLa cells. Login to comment
153 The figure was prepared with PyMOL v1.4. Mechanism of the Rescue of Misfolded P-glycoprotein NOVEMBER 8, 2013ߦVOLUME 288ߦNUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32625 D805C, and D164C/D805C, respectively (Fig. 2, A-C). Login to comment
156 Fig. 2, D-F, shows a typical histogram for accumulation of daunorubicin in single (D164C, D805C) and double (D164C/ D805C) mutants. Login to comment
158 The ability of D164C, D805C, and D164C/ D805C mutant P-gp to transport other substrates such as Rh123, NBD-CsA, and calcein-AM was also analyzed. Login to comment
160 On the contrary, D805C completely fails to transport any of the substrates (Table 1). Login to comment
161 The double mutant D164C/D805C was defective in transport for almost all these substrates to varying degrees except Rh123, which was near normal. Login to comment
162 D164C/D805C was severely defective in the transport of NBD-CsA and daunorubicin (b03;30%), whereas calcein-AM was partially transported (b03;60%) (Table 1). Login to comment
164 Either a total loss or significantly lower cell surface expression of D164C, D805C, and D164C/D805C strongly suggests that the substitution of these negatively charged residues with cysteine has an effect on synthesis, translocation, proper folding, or trafficking of these mutant proteins. Login to comment
165 Total Expression of Single and Double Mutants in HeLa Cells Indicates Defective Maturation of P-gp-Lysates from D164C-, D805C-, and D164C/D805C-transduced HeLa cells were checked by Western blot to analyze the total expression of P-gp. Login to comment
166 Consistent with the MRK-16 antibody staining, we observed that D164C exhibited near normal total P-gp expression, whereas D805C showed no protein at all (data not shown). Login to comment
167 The D164C/D805C double mutant shows a very faint mature band but shows immature P-gp levels similar to cysless-WT, indicating that the mutant protein is trapped in the intracellular compartment, possibly in the ER. Login to comment
168 Growth at Lower Temperature Corrects the Folding Defect of D164C/D805C Double Mutant-As we knew that the èc;F508 CFTR mutant, which is linked to cystic fibrosis, can be functionally rescued to the cell surface when cells are grown at a lower temperature, we tried to retrieve the surface expression of these P-gp mutants by growing cells for a longer time at 27 &#b0;C. Login to comment
170 BacMam baculovirus-transduced HeLa cells exhibit altered cell surface expression and transport function of single (D164C and D805C) and double (D164C/D805C) mutant P-gp at 37 &#b0;C. P-gp-virus-transduced HeLa cells were incubated with UIC2 antibody (2 òe;g/100,000 cells) for 30 min at 37 &#b0;C followed by incubation with FITC-conjugated anti-mouse IgG2a secondary antibody. Login to comment
172 The cell surface localization of cysless-WT along with the three mutant P-gps (D164C, D805C, and D164C/D805C), as detected by UIC2 antibody, is shown in panels A-C, respectively. Login to comment
174 Panels D-F show typical histograms for accumulation of daunorubicin in HeLa cells expressing D164C, D805C, or D164C/D805C. Login to comment
181 It was found that growth at lower (27 &#b0;C) temperature rescues the activity of the D164C/D805C mutant up to 70-80% for all tested substrates (Table 1). Login to comment
183 Because D164C is near normal in function, whereas D805C is drastically defective in cell surface expression, these single mutants were not considered for further studies. Login to comment
185 We, therefore, decided to characterize the properties of D164C/D805C mutant P-gp expressed in both insect and mammalian cells. Login to comment
186 D164C/D805C Mutant P-gp Exhibits Km for ATP Similar to Cysless-WT-To test whether residues Asp-164 or Asp-805 are required for ATP binding and hydrolysis, the D164C/D805C mutant and cysless-WT were expressed in High-Five insect cells. Login to comment
187 The level of expression for D164C/D805C P-gp was found to be similar to cysless-WT (Fig. 3A), which corroborates with the results exhibited by transduced HeLa cells when grown at 27 &#b0;C. Login to comment
191 We further determined the affinity (Km) and rate of hydrolysis (Vmax) of ATP by D164C/D805C mutant P-gp. Login to comment
193 We found that the introduction of cysteine residues at both Asp-164 and Asp-805 (D164C/D805C) did not alter the Km of ATP (Cysless-WT, 0.3 afe; 0.03 mM; DD, 0.5 afe; 0.12 mM) but led to a considerable decrease in Vmax (Cys-less-WT, 65.1 afe; 1.3; DD, 19.4 afe; 1.2) compared with cys-less-WT (Fig. 3C). Login to comment
196 A Pharmacological Chaperone (CsA) and Small Molecules (VRT-325 and Corr-4a) Correct the Folding Defect of D164C/ D805C-It is well known that substrates and modulators of P-gp assist in protein folding and rescue the misprocessed mutants to the cell surface (23). Login to comment
197 Because D164C/D805C showed poor cell surface expression, we incubated transduced HeLa cells with 6.25 òe;M CsA, a P-gp substrate, for 18 h, as described under "Experimental Procedures". Login to comment
198 The percentage change in cell surface expression of D164C/D805C mutant P-gp was observed to be greater than 10-fold in comparison to cysless-WT (Fig. 4A). Login to comment
201 The single mutants were also grown in the presence of CsA, and it was observed that the cell surface expression of D164C was rescued to b03;80%, whereas for D805C it was rescued to 10-15% only. Login to comment
203 This may be due to the susceptibility of the cell surface D805C mutant P-gp to degradation by cell machinery. Login to comment
207 Although these correctors are known to rescue the misprocessed P-gp mutants, they could not completely rescue D164C/D805C to the plasma membrane. Login to comment
208 The percentage changes in cell surface expression of D164C/ D805C mutant P-gp were much less in the presence of VRT-325 (200%) or corr-4a (70%) as opposed to CsA (500%) (Fig. 4, A-C). Login to comment
220 of Repeats Cell surface expression (%) Transport function (%) Rh123 NBD-CsA Dauno Cal-AM D164C 37 &#b0;C n afd; 3 35-40 90-100 75-85 80-90 90-100 D805C n afd; 3 ND ND ND ND ND D164C/D805C n afd; 10 10-20 80-90 20-30 10-20 30-40 D164C 27 &#b0;C n afd; 3 40-50 90-100 70-80 80-90 95-100 D805C n afd; 3 ND ND ND ND ND D164C/D805C n afd; 5 70-80 90-100 70-80 80-90 95-100 D164C 37 &#b0;C af9; 6.25 òe;M CsA n afd; 3 70-80 90-100 80-90 90-100 95-100 D805C n afd; 3 10-15 5-10 ND ND ND D164C/D805C n afd; 10 90-100 90-100 90-100 95-100 90-100 Mechanism of the Rescue of Misfolded P-glycoprotein NOVEMBER 8, 2013ߦVOLUME 288ߦNUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32627 Treatment with C3 and C4 rescued the transport of daunorubicin to 70-75 and 50-55%, respectively (Fig. 4, E and F). Login to comment
221 Detection of Cell Surface Expression of D164C/D805C Mutant P-gp in HeLa Cells by Immunofluorescence Confocal Microscopy-The cell surface expression of mutant P-gp was also monitored using immunofluorescence, as described under "Experimental Procedures." Login to comment
226 D164C/D805C Transports Rhodamine 123 but at a Significantly Lower Efflux Rate-HeLa cells transduced with double (D164C/D805C) mutant P-gp expressed only 10-20% protein at the cell surface, thus resulting in decreased transport of almost all the substrates. Login to comment
228 To investigate further, we determined the rate of Rh123 efflux by cysless-WT and double mutant (D164C/D805C) P-gp-transduced cells when grown in the absence or presence of 6.25 òe;M CsA, as described under "Experimental Procedures." Login to comment
230 In the absence of CsA, the t1/2 for Rh123 efflux by cysless-WT was 1.8 min versus 15 min for D164C/D805C mutant P-gp (Fig. 6). Login to comment
233 Short Term Treatment with CsA Is Sufficient to Rescue Intracellularly Trapped D164C/D805C Mutant P-gp-We observed that the misprocessed mutant protein was localized to the cell surface when grown in the presence of CsA for 18 h (Figs. 4A and 5). Login to comment
236 The cell surface expression of D164C/D805C was checked at the indicated time points. Login to comment
237 As shown in Fig. 7A, the rescue of mislocalized D164C/D805C to the cell surface starts as early as 30 min. Login to comment
239 Expression profile and ATPase activity of D164C/D805C mutant P-gp expressed in insect cells. Login to comment
241 A, expression of cysless-WT and D164C/D805C were checked by separating crude membrane samples on 7% Tris-acetate gel (30 òe;g of protein/lane) followed by detection with Colloidal blue staining and by Western blot analysis (1 òe;g of protein/lane) using anti-P-gp antibody C219 at 1:2000 dilution. Login to comment
243 B, effect of selected compounds on the ATPase activity of cysless-WT and D164C/D805C mutant P-gp. Login to comment
247 Due to low basal activity of D164C/D805C, the measurements were performed in the presenceof30òe;M verapamil.Eachdatapointisthemeanafe;S.E.M.ofthreeindependentexperiments.ThedatawerefittedtotheMichaelis-Mentencurveusing GraphPad Prism 5.0. Login to comment
254 Short Term Treatment with Various P-gp Substrates and Modulators Rescues D164C/D805C Mutant P-gp to the Cell Surface-Because CsA, a well known substrate of P-gp, was able to rescue the misprocessed mutant to the cell surface, we further tested several other substrates and modulators for their ability to rescue this misfolded mutant. Login to comment
256 It was seen that various substrates/modulators at 15-25 òe;M concentration were able to rescue the D164C/D805C mutant to an extent ranging from 40 to 100% (Fig. 7C). Login to comment
257 Interestingly, compounds like cisplatin and camptothecin, which are not substrates or modulators of P-gp, were not able to rescue D164C/D805C mutant P-gp. Login to comment
260 To show that the rescue of D164C/ D805C was via an immunophilin-independent pathway, HeLa cells were transduced for 24 h with cysless-WT and D164C/ D805C mutant P-gp after which the virus was removed and the FIGURE 4. Login to comment
261 Cyclosporine A or small molecule correctors VRT-325 (C3) and corr-4a (C4) rescue the expression of D164C/D805C mutant P-gp at the cell surface. Login to comment
262 HeLa cells transduced with the D164C/D805C BacMam baculovirus were grown in the presence of CsA (6.25 òe;M) or CFTR correctors C3 or C4 (20 òe;M) for 16-18 h as described under "Experimental Procedures." Login to comment
263 Cell surface localization of cysless-WT and D164C/D805C was determined for cells incubated in the presence of CsA (A), corrector C3 (B), and corrector C4 (C) using MRK-16 antibody as described under "Experimental Procedures." Login to comment
265 Values are mean afe; S.E. Rescue of transport function by treatment with CsA (n afd; 13), corrector C3 (n afd; 3), or C4 (n afd; 3) is shown in panels D-F, which represent typical histograms for the accumulation of daunorubicin in HeLa cells expressing D164C/D805C in the presence of CsA, corrector C3 and C4. Login to comment
269 These results substantiate the fact that the rescue of D164C/D805C mutant P-gp in the presence of CsA/FK506 is not mediated via immunophilin-dependent pathway. Login to comment
270 Enzymatic Digestion Shows That the Misfolded Double (D164C/ D805C) Mutant Is Core-glycosylated and Is Trapped in the ER-The flow cytometry results were further supported by Western blot analysis of the total cell lysates from cysless-WT and D164C/D805C-expressing cells. Login to comment
272 Enzymatic digestion of cysless-WT or the D164C/D805C by PNGase F or Endo H showed that the mature protein (Band A) was susceptible to PNGase F digestion and thus corresponds to the complex glycosylated mature protein present on the plasma membrane. Login to comment
274 When digested with Endo H, Band A was found to be resistant, as seen in the cysless-WT as well as double (D164C/D805C) mutant samples, although the band is faint in the double mutant because of very low expression of this mature protein. Login to comment
278 Treatment with PNGase F or Endo H had no effect on the size of cysless-WT or double D164C/D805C mutant P-gp expressed in insect cells (Fig. 8C). Login to comment
279 The D164C/D805C Mutant Co-immunoprecipitates More with Hsp70 but Not with Hsc70 or Calnexin Antibodies-Some of the chaperones, including calnexin, Hsp70, and Hsc70 have been found to associate with P-gp (27). Login to comment
285 Importantly, in both cysless-WT and D164C/D805C, we observed less association of Hsp70 with immature P-gp when cells were treated with CsA (Fig. 9A). Login to comment
289 The level of associated P-gp was checked using the P-gp-specific antibody C219 in both CsA-treated and untreated cysless-WT and D164C/D805C mutant P-gp. Login to comment
290 As expected, a decreased association of immature P-gp with Hsp70 was observed in the presence of CsA for both cys-less-WT and D164C/D805C mutant P-gp (see weaker immature (lower) P-gp band in the presence of CsA in both control and mutant P-gp; left immunoblot in Fig. 10). Login to comment
292 Detection of cysless-WT and D164C/D805C mutant P-gp at the cell surface by immunofluorescence. Login to comment
293 HeLa cells transduced with cys-less-WT or D164C/D805C BacMam virus were fixed and stained using MRK-16 antibody followed by FITC-labeled IgG2a anti-mouse secondary antibody, and nuclei were stained with DAPI. Login to comment
295 The left panels show expression of cysless-WT and D164C/D805C in HeLa cells grown at 37 &#b0;C. Login to comment
296 The middle panels show cells grown at 27 &#b0;C for 39 h, and the right panels show level of cysless-WT and D164C/D805C in HeLa cells incubated in the presence of 6.25 òe;M CsA for 18 h at 37 &#b0;C. Login to comment
308 We expressed D164C, D805C, and D164C/D805C P-gp in HeLa cells for characterization of expression and function of these mutant P-gps. Login to comment
310 The D805C mutation drastically affects the protein expression (although the level of mRNA is normal) (data not shown), whereas the D164C/D805C double mutation results in retention of the predominantly immature protein in the ER; only 10-15% of double mutant protein is present at the cell surface (Fig. 2, Table 1). Login to comment
311 However, this misprocessing in the case of D164C and D164C/D805C could be completely corrected when the cells were grown either at lower temperature (27 &#b0;C) or in the presence of pharmacological chaperones or small molecule correctors at 37 &#b0;C. Login to comment
312 The D805C mutation is detrimental to the protein expression, and its expression could not be rescued. Login to comment
314 Single substitution of Asp-164 with Ala (D164A) or Asn (D164N) led to similar expression and function as D164C.3 The substitution of Asp-805 with Ala (D805A) or Asn (D805N) is less drastic as compared with D805C, but the protein still does not localize to the cell surface.3 All these substitutions with either Ala or Asn could be rescued to the cell surface in the presence of CsA (data not shown). Login to comment
318 Although only 10-25% of the D164C/D805C mutant P-gp was localized to the plasma membrane at 37 &#b0;C, at steady state 3 K. Kapoor, E. E. Chufan, and S. V. Ambudkar, unpublished data. Login to comment
323 The t1/2 of Rh123 efflux for cysless-WT is 1.8 min versus 15 min for D164C/D805C (absence of CsA), which is restored to levels similar to cysless-WT when the cells were grown in the presence of CsA. Login to comment
324 The right panel shows efflux of NBD-CsA; t1/2 of NBD-CsA efflux for cysless-WT is 4.2 min, whereas the rate cannot be determined for D164C/D805C as the total efflux was b0d;50% (in the absence of CsA). Login to comment
341 Similarly, the D164C and D805C mutations might FIGURE 7. Login to comment
343 A, time course of recovery of cell surface expression of D164C/D805C mutant P-gp upon short term treatment with CsA is plotted. Login to comment
345 The cell surface expression of D164C/D805C was monitored over regular time intervals for 8 h. Login to comment
346 The expression of double (D164C/D805C) mutant at the cell surface after 18 h treatment with CsA during growth was taken as 100%. Login to comment
348 B, recovery of cell surface expression of D164C/D805C mutant-Pgp by CsA in the presence of CYH. Login to comment
353 C, rescue of D164C/D805C to the cell surface by incubation with 15-25 òe;M of several substrates and modulators of P-gp was measured at 4 h. Login to comment
356 D, rescue of D164C/D805C when treated with lower concentrations (25, 250, 350 nM) of either CsA (filled bars) or FK506 (empty bars) for 4 h. Login to comment
367 Rather, they show increased association of the D164C/D805C mutant protein with other members of the Hsp70 family (Fig. 9). Login to comment
372 Failure to ever reach its properly folded state likely results in continued interaction with and retention by FIGURE8.Susceptibilityofmatureandimmatureformsofcysless-WTandD164C/D805CmutantP-gptodigestionbyPNGaseFandEndoH.A,Western blot analysis of total cell lysates of HeLa cells transduced with cysless-WT or D164C/D805C mutant P-gp when grown in the absence or presence of 6.25 òe;M CsA orCFTRcorrectorsC3andC4(20òe;M).Celllysateproteins(3òe;g)wereloadedineachlane,andimmunoblottingwithC219antibodywasperformedasdescribed under "Experimental Procedures." Login to comment
373 Lane 1, cysless-WT; lane 2, cysless-WT treated with 6.25 òe;M CsA; lane 3, cysless-WT treated with 20 òe;M corrector C3, lane 4, cysless-WT treated with 20 òe;M corrector C4; lane 5, D164C/D805C; lane 6, D164C/D805C treated with 6.25 òe;M CsA; lane 7, D164C/D805C treated with 20 òe;M C3; lane 8, D164C/D805C treated with 20 òe;M corrector C4. Login to comment
377 Cell lysates from HeLa cells transduced with cysless-WT (lanes 1, 2, and 3) and D164C/D805C (lanes 4, 5, and 6) and grown in the absence of CsA were subjected to Endo H or PNGase F digestion as described under "Experimental Procedures" and processed for immunoblotting using C219 antibody. Login to comment
379 C, glycosidase digestion of crude membranes from High-Five insect cells (0.5 òe;g of protein) expressing cysless-WT (lanes 1, 2, and 3) and D164C/D805C (lanes 4, 5, and 6) P-gp. Login to comment
386 NC, cysless-WT; DD, D164C/D805C; NCCsA, cysless-WT treated with 6.25 òe;M CsA for 18 h; DDCsA, D164C/D805C treated with 6.25 òe;M CsA for 18 h. Login to comment
390 It was observed that association of the immature P-gp with Hsp70 decreases when grown in the presence of CsA in both cysless-WT and D164C/D805C double mutant P-gp (Fig. 10). Login to comment
392 Furthermore, even though D164C/ D805C is misfolded, it does not seem to degrade rapidly. Login to comment

PMID: 24275649 [PubMed] Loo TW et al: "Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein."

No. Sentence Comment

212 In support of this prediction, it has been reported recently that a P-gp mutant containing the D805C mutation reduced verapamil-stimulated ATPase activity by about 70% but had no effect on the Km for ATP (49). Login to comment