The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy TIP W. LOO AND DAVID M. CLARKE1 MRC Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada ABSTRACT The human multidrug resistance Pglycoprotein (P-gp) contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy. A potential novel strategy to circumvent the effects of P-gp during chemotherapy is to prevent maturation of P-gp during biosynthesis so that the transporter does not reach the cell surface. Here we report that immature, core-glycosylated P-gp that is prevented from reaching the cell surface by processing mutations or by proteasome inhibitors such as lactacystin or MG-132 exhibited no detectable drug-stimulated ATPase activity. Disulfide crosslinking analysis also showed that the immature P-gp did not exhibit ATP-induced conformational changes as found in the mature enzyme. In addition, the immature P-gp was more sensitive to trypsin than the mature enzyme. These results suggest that P-gp is unlikely to be functional immediately after synthesis. These differences in the structural and enzymatic properties of the mature and core-glycosylated, immature P-gp could potentially be used during chemotherapy, and should result in the search for compounds that can specifically inhibit the maturation of P-gp.—Loo, T. W., Clarke, D. M. The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy. FASEB J. 13, 1724 –1732 (1999)
Key Words: multidrug transporter z protein folding z proteasome inhibitor z endoplasmic reticulum
Chemotherapy is the most important form of treatment in many cancers, such as Hodgkin’s disease, testicular cancer, and many pediatric cancers. Treatment with a combination of cytotoxic agents that have different intracellular targets is in vogue. It is unfortunate that the majority of cancers are either resistant to chemotherapy (e.g., renal and colon cancers) or acquire resistance (e.g., lymphoma, lung, breast cancers) during treatment (1). The intrinsic or acquired ability of tumor cells to show resistance to multiple chemotherapeutic agents (multidrug resistance) is a major obstacle to successful chemotherapy. 1724
The first protein implicated in the phenomenon of multidrug resistance was the human multidrug resistance P-glycoprotein (P-gp),2 the product of the human MDR1 gene (2). Cell lines overexpressing P-gp were initially selected for resistance to a single cytotoxic drug, but were subsequently found to exhibit cross-resistance to the effects of other structurally unrelated cytotoxic drugs (3–5). Analysis of P-gp levels in tumor cells obtained from patients showed that P-gp is expressed at biologically significant levels in ;50% of human cancers (1, 6 – 8). P-gp also interferes in the treatment of AIDS patients with protease inhibitors. Oral absorption and penetration of FDA-approved protease inhibitors into the central nervous system are quite poor. This is due to the presence of relatively high amounts of P-gp in the intestine and at the blood– brain barrier (9, 10). There has been an intense effort to understand how P-gp is able to confer multidrug resistance. Its physiological role is likely to protect us from endogenous and exogenous cytotoxic compounds (11–13). P-gp is a member of the ABC (ATP binding cassette) superfamily of transport proteins (14). Its 1280 amino acids are arranged in two tandem repeats joined by a linker region of 60 amino acids. Each repeat consists of an NH2-terminal hydrophobic domain containing six potential transmembrane segments, followed by a hydrophilic domain containing a nucleotide binding fold (15–17). An important goal is to devise strategies to ‘shut down’ P-gp during chemotherapy. The current protocols of using combined chemotherapy rely to some extent on the varying affinities of each drug for P-gp. 1 Correspondence: Department of Medicine, University of Toronto, Room 7342, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada. E-mail:
[email protected] 2 Abbreviations: ABC, ATP binding cassette; CFTR, cystic fibrosis transmembrane conductance regulator; MG-132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; P-gp, P-glycoprotein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TM, transmembrane; TPCK, L-1-tosylamido-2phenylethylchloromethyl ketone.
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In these mixtures, it is expected that drugs with a higher affinity for P-gp would act as ‘pseudo-inhibitors’, thereby allowing those with lower affinities to enter the cell. This form of therapy has been used successfully to treat pediatric tumors such as retinoblastoma and neuroblastoma (6). A potential novel strategy to bypass the effects of P-gp would be to prevent P-gp from reaching the cell surface by interfering with its maturation after synthesis in the endoplasmic reticulum. P-gp appears to be quite susceptible to such a strategy, since it was previously shown that its maturation can be manipulated by point mutations (18) and drug substrates (19). Many point mutations have been identified that result in the synthesis of 150 kDa core-glycosylated proteins as the major product (20). These core-glycosylated intermediates are retained in the endoplasmic reticulum in association with the chaperones calnexin (21) and hsc70 (22) and are rapidly degraded. These processing mutants, however, can be converted to the mature 170 kDa enzyme, which is trafficked to the cell surface if synthesis is carried out in the presence of drug substrates (19). By contrast, maturation of P-gp can be inhibited by carrying out expression in the presence of proteasome inhibitors such as lactacystin or MG-132 (23). It is generally believed that polytopic membrane proteins and those destined to be trafficked to the cell surface are fully active immediately after synthesis in the endoplasmic reticulum. Indeed, with CFTR, another member of the ABC superfamily of transporters, it has been reported that the protein is functional when still in the endoplasmic reticulum (24). In this study, we tested whether P-gp was active in the initial stages of biosynthesis. An inactive coreglycosylated P-gp intermediate would suggest that inhibition of P-gp maturation could be exploited as a potential novel method for circumventing the problem of multidrug resistance, thus resulting in the use of lower doses of chemotherapeutic drugs.
MATERIALS AND METHODS
codon for the three consensus glycosylation sites (N91, N94, and N99) to code for alanine (23). Purification of P-gp mutants and measurement of ATPase activity HEK293 cells transfected with cDNAs coding for the histidine-tagged P-gps were solubilized with 1% (w/v) n-dodecylb-D-maltoside, and the mutant P-gps were purified by nickel-chelate chromatography using Ni-NTA spin columns (Qiagen, Chatsworth, Calif.), as described previously (26). For isolation of the processing mutants of P-gp, the concentration of imidazole in the wash buffers was reduced from the standard 80 mM to 20 mM. Drug-stimulated ATPase activity of the isolated protein was determined after reconstitution with lipid as described previously (26). Sulfhydryl cross-linking analysis For each mutant P-gp, 10 (10 cm diameter) culture plates of HEK293 cells were transfected with the mutant MDR1 cDNA. After 16 h, the media was replaced with fresh media or fresh media containing 2 mM MG-132. The cells were harvested after another 24 h and membranes were prepared as before (28). The membranes were suspended in 200 ml of Trisbuffered saline, pH 7.4. A sample of the membrane suspension (12 ml) was mixed with 15 ml of 23 ATPase buffer (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2) containing none or 10 mM ATP. Cross-linking was initiated by addition of 3 ml of 20 mM Cu21(phenanthrolene)3. The samples were incubated for 5 min at 37°C and the reaction was stopped by addition of 50 ml of 23 sodium dodecyl sulfate (SDS) sample buffer [125 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS] containing 50 mM EDTA and no reducing agent. The samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto a sheet of nitrocellulose, and immunoblotted with a rabbit polyclonal antibody against P-gp, followed by detection with enhanced chemiluminescence (22). TPCK-trypsin digestion HEK293 cells were transfected with various MDR1 cDNAs and treated with or without MG-132, as described above. Membranes were then prepared and suspended in Tris-buffered saline. The membranes (5 mg/ml protein) were treated for 5 min at 22°C with various amounts of TPCK-trypsin [Sigma (St. Louis, Mo.) 12,000 BAEE units per mg], and the reaction was stopped by addition of lima bean trypsin inhibitor (Worthington, Freehold, N.J.) (20).
Generation of constructs
RESULTS
Full-length human MDR1 cDNA, modified to encode the epitope for monoclonal antibody A52 at the COOH-terminal end, was inserted into the mammalian expression vector pMT21, as described previously (25). For purification purposes, a full-length MDR1 cDNA was modified to encode for 10 histidine residues at the COOH terminus of the protein (26). The sequence at the COOH terminus of full-length P-gp, which would normally end as TKRQ, now became TKRA(His)10LDPRQ. Construction of Cys-less P-gp in which all cysteine residues were changed to alanine has been described (16). The Cys-less mutant was also modified to contain mutations L332C in TM6 and L975C in TM12 (27). A glycosylation-deficient mutant was made by changing the
Drug-simulated ATPase activity of P-gp processing mutants
BLOCKING THE MATURATION OF P-GLYCOPROTEIN
P-gp is synthesized in HEK293 cells as two major products: a 170 kDa fully glycosylated (mature) protein and a 150 kDa core-glycosylated biosynthetic intermediate. In P-gp processing mutants, the 150 kDa protein accumulates in the cell and is rapidly degraded (21). We have been able to purify the mature form of the histidine-tagged P-gp by nickel chelate chromatography for measurement of drug1725
contrast, there was no detectable basal or drugstimulated ATPase activity in the 150 kDa coreglycosylated proteins from the processing mutants. These results suggest that the core-glycosylated enzymes of processing mutants are nonfunctional. ATPase activity of P-gp that is prevented from undergoing maturation Figure 1. Isolation of mature and core-glycosylated P-gp. HEK293 cells expressing histidine-tagged P-gp mutants G268V, G269V or Y710A were incubated with (1) or without (2) 10 mM cyclosporin A (cyclo) for 24 h. The cells were harvested and solubilized with n-dodecyl-b-D-maltoside and the cell extracts were subjected to nickel-chelate chromatography with 20 mM imidazole in the wash buffers. P-gp was eluted with 300 mM imidazole; samples were subjected to immunoblot analysis with rabbit anti-P-gp polyclonal antibody and enhanced chemiluminescence. The positions of the core-glycosylated (150) and mature (170) P-gp are indicated.
stimulated ATPase activity. In contrast, the histidinetagged core-glycosylated 150 kDa protein of the processing mutants could not be purified unless the imidazole concentration during the washes was lowered from 80 mM to 20 mM. Under these modified conditions, the core-glycosylated protein could be isolated in sufficient quantities for measuring drugstimulated ATPase activity. Accordingly, the histidine-tagged 150 kDa protein of mutants G268V, G269V, and Y710A were isolated (Fig. 1, lanes 1, 3, and 5). These mutants were analyzed because the major P-gp product was the 150 kDa core-glycosylated biosynthetic intermediate (18, 21). To convert the processing mutants G268V, G269V, and Y710A to the mature enzyme, the mutants were synthesized in the presence of 10 mM cyclosporin A to induce maturation (19). Cyclosporin A is a particularly useful P-gp substrate for inducing maturation of the processing mutants since it is relatively nontoxic to the cells, and its very hydrophobic nature allows the compound to readily diffuse into the cell. Isolation of the mutants by nickel-chelate chromatography after synthesis with cyclosporin A resulted in the recovery of the mature 170 kDa mutant P-gp (Fig. 1, lanes 2, 4, and 6). The isolated mature 170 kDa and core-glycosylated 150 kDa P-gps were assayed for verapamil and vinblastine-stimulated ATPase activity. Verapamil causes the greatest stimulation of the ATPase activity of P-gp (29), whereas vinblastine-stimulated ATPase activity correlates well the turnover number of vinblastine transport (30). Figure 2 shows that the mature form of all three processing mutants exhibited verapamil and vinblastine-stimulated ATPase activities. The mature enzyme of mutant Y710A had activities that were similar to wild-type P-gp. As reported previously (19), the mature enzyme of mutants G268V and G269V had ;50 to 75% of the activity of wild-type P-gp. By 1726
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It was possible that the 150 kDa core-glycosylated P-gp of the processing mutants G268V, G269V, and Y710A were inactive because the point mutations inherently caused misfolding of the protein and subsequent retention in the endoplasmic reticulum. Therefore, another approach for assaying the activity of the core-glycosylated P-gp is to prevent maturation of P-gp by inhibitors. The proteasome inhibitors lactacystin and MG-132 have been reported to inhibit maturation of the cystic fibrosis transmembrane conductance regulator (31) and human P-gp (23). A histidine-tagged Cys-less P-gp containing an R113A mutation and grown in the presence of 2 mM MG-132 was assayed for drug-stimulated ATPase activity. The Cys-less P-gp was used because it was active, but its rate of maturation was relatively slower than the wild-type enzyme (16). This relatively slower rate of maturation was used to advantage since it was possible to ‘trap’ the Cys-less P-gp as a core-glycosylated intermediate. By contrast, the wild-type P-gp matured quite rapidly, such that a very small amount
Figure 2. Drug-stimulated ATPase activities of mature and core-glycosylated P-gp of processing mutants. Equivalent amounts of histidine-tagged mature and core-glycosylated P-gp of wild-type and mutants G268V, G269V, and Y710A, isolated by nickel-chelate chromatography, were reconstituted with lipid and assayed for verapamil- (1 mM) and vinblastine- (0.05 mM) stimulated ATPase activities. Fold stimulation is the ratio of activity with drug substrate to that without drug substrate.
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Figure 3. Effect of MG-132 on expression of P-gp. HEK293 cells were transfected with Cys-less P-gp cDNA containing an R113A mutation. After 16 h, the media was replaced with fresh media containing various amounts (0 –20 mM) of MG132. After another 24 h, the cells were solubilized with SDS sample buffer and subjected to immunoblot analysis with rabbit anti-P-gp polyclonal antibody.
of the core-glycosylated P-gp still matured (leakage) into the mature enzyme even in the presence of MG-132 (data not shown). The R113A mutation was included since it stabilizes P-gp against proteolytic digestion during biosynthesis, and it resulted in increased yield of enzyme (23). Figure 3 shows that the major product of the Cys-less(R113A) mutant in the presence of increasing concentrations of MG-132 was the 150 kDa core-glycosylated intermediate. At MG-132 concentrations greater than 0.63 mM, expression of the 170 kDa mature P-gp was inhibited (Fig. 3, lane 6). In the presence of 1.25 mM MG-132, the 150 kDa core-glycosylated P-gp was the major product (Fig. 3, lane 5). Accordingly, the histidinetagged Cys-less(R113A) P-gp grown with or without 2 mM MG-132 was isolated by nickel-chelate chromatography. As shown in Fig. 4A (lane 4), the 150 kDa
core glycosylated Cys-less(R113A) P-gp could be recovered by nickel-chelate chromatography when the mutant was grown with MG-132. By contrast, in the absence of MG-132, there was approximately equal amounts of 150 kDa core-glycosylated and 170 kDa mature P-gps (Fig. 4A, lane 3). Equivalent amounts of the isolated Cys-less(R113A) P-gps were reconstituted with lipid and assayed for drug-stimulated ATPase activity. Figure 4B shows that P-gp expressed in the presence of MG-132 showed no drug-stimulated ATPase activity. In the absence of MG-132, however, P-gp exhibited 10.3- and 4.7-fold stimulation of ATPase activity in the presence of verapamil and vinblastine, respectively. This suggested that MG-132 likely ‘trapped’ the 150 kDa core-glycosylated P-gp in a conformation that was unable to couple drug binding to ATP hydrolysis. ATP-induced conformational changes in the transmembrane domain The absence of drug-stimulated ATPase activity in the 150 kDa core-glycosylated P-gp suggested that the protein may not undergo ATP-induced conformational changes. It has been shown that ATP hydrolysis induces conformational changes in P-gp (32, 33) through the transmembrane (TM) domains (27, 28). The ATPinduced conformational changes in the TM domains can be detected by disulfide bond formation between Cys332 in TM6 and Cys975 in TM12. Cross-linking
Figure 4. Drug-stimulated ATPase activity of P-gp after expression in the presence or absence of MG-132. HEK293 cells were transfected with pMT21 vector (control) or histidine-tagged Cys-less(R113A) P-gp cDNA (P-gp). After 16 h, the media was replaced with fresh media (2) or media containing 2 mM MG-132 (1). The cells were harvested after another 24 h and P-gp was isolated by nickel-chelate chromatography using 20 mM imidazole in the wash buffers. The eluted samples were subjected to immunoblot analysis using a rabbit anti-P-gp polyclonal antibody (A). Equivalent amounts of each sample were reconstituted with lipid and assayed for drug-stimulated ATPase activity with 1 mM verapamil or 0.05 mM vinblastine (B). BLOCKING THE MATURATION OF P-GLYCOPROTEIN
1727
Figure 5. Disulfide cross-linking of P-gp after expression with MG-132. HEK 293 cells were transfected with Cys-less P-gp cDNA containing mutations L332C/L975C and then incubated with (1MG-132) or without (2MG-132) MG-132 for 24 h. Membranes were then prepared and treated with (1) or without (2) copper phenanthrolene (CuPhen) for 5 min at 37°C in the presence (1ATP) or absence (2ATP) of 10 mM ATP. The samples were then subjected to immunoblot analysis with a rabbit anti-P-gp polyclonal antibody and enhanced chemiluminescence. The positions of the cross-linked (Xlink), mature (170), core-glycosylated (150), and P-gp proteolytic digestion product (130) are indicated.
sible explanation was that the processing of the carbohydrate moieties of P-gp as it traversed the Golgi apparatus was essential for function. To test this hypothesis, residues in the consensus glycosylation sites (N91, N94, and N99) were mutated to alanine (35), and a histidine tag was attached at the COOH terminus of the protein (26) to facilitate purification of the mutant P-gp by nickel-chelate chromatography (Fig. 6A). Equivalent amounts of 170 kDa mature and 140 kDa glycosylation-deficient P-gp were reconstituted with lipid and assayed for drug-stimulated ATPase activity. Figure 6B shows that the 140 kDa glycosylation-deficient P-gp exhibited verapamil- and vinblastine-stimulated (15.9- and 7.8fold, respectively) ATPase activities that were similar to the wild-type P-gp. Therefore, the presence of carbohydrate moieties on P-gp is not essential for coupling drug binding to ATPase activity. These results support the findings of Schinkel et al. (35), who showed that the glycosylation-deficient P-gp was able to confer drug resistance on cells expressing this mutant P-gp.
between these two cysteine residues occurs only in the presence of an oxidant while P-gp is hydrolyzing MgzATP. No cross-linking was observed in the presence of MgzADP, nonhydrolyzable ATP analog (AMP-PNP) or with MgzATP and vanadate. Vanadate inhibits ATP hydrolysis by P-gp (34). Cys-less P-gp containing the mutations L332C and L975C was expressed with or without MG-132 and cross-linked in the presence or absence of MgzATP. Figure 5 shows that ATP induces cross-linking in mutant L332C/L975C when expressed in the absence of MG-132. P-gp containing a disulfide bond between TM6 and TM12 migrates with lower mobility on SDS-PAGE (Fig. 5, lane 4) (27, 28). In the presence of MG-132, however, the 150 kDa coreglycosylated mutant L332C/L975C was not crosslinked by oxidant in the absence or presence of ATP (Fig. 5, lanes 6 and 8). These results show that the 150 kDa core-glycosylated mutant did not exhibit the same conformational changes during ATP hydrolysis as observed in the 170 kDa mature enzyme. Carbohydrate moieties of P-gp are not required for P-gp function The results indicate that the 150 kDa core-glycosylated intermediates isolated from processing mutants or by preventing maturation of the core-glycosylated P-gp with MG-132 were not capable of coupling drug binding to ATP hydrolysis. One pos1728
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Figure 6. Comparison of drug-stimulated ATPase activities of glycosylated and unglycosylated P-gp. Histidine-tagged wildtype and unglycosylated (minus CHO) P-gp were isolated by nickel-chelate chromatography and subjected to immunoblot analysis with rabbit anti-P-gp polyclonal antibody and enhanced chemiluminescence (A). The positions of mature (170) and unglycosylated (140) P-gp are indicated. Equivalent amounts of each sample were also reconstituted with lipid and assayed for verapamil- and vinblastine-stimulated ATPase activity (B).
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mature enzyme and that MG-132 prevents P-gp from folding into the mature trypsin-resistant P-gp.
DISCUSSION
Figure 7. Protease sensitivity of P-gp after expression in the presence or absence of MG-132. HEK293 cells expressing A52-epitope-tagged Cys-less(R113A) P-gp were treated with (1) or without (2) 2 mM MG-132 for 24 h. Membranes were then prepared and treated with various concentrations of TPCK-trypsin. The reactions were stopped by addition of trypsin inhibitor; the samples were subjected to immunoblot analysis with monoclonal antibody A52. The positions of mature (170) and core-glycosylated (150) P-gp are indicated.
TPCK-trypsin sensitivity of P-gp expressed with or without MG-132 Another plausible explanation for the lack of activity in the 150 kDa core-glycosylated intermediates of P-gp was that the presence of point mutations or synthesis in the presence of MG-132 may ‘trap’ the transporter in an inactive conformation. We previously demonstrated that P-gp processing mutants are present in the cell as partially unfolded proteins (20) and are ;100-fold more sensitive to trypsin digestion compared to the 170 kDa mature enzyme. To test whether the proteasome inhibitor MG-132 also affected the proper folding of P-gp, we assayed for sensitivity of P-gp to trypsin digestion. Membranes were prepared from HEK293 cells expressing Cys-less(R113A) P-gp that were grown with or without MG-132. The membranes were then treated with various concentration of trypsin. Figure 7 shows that P-gp expressed in the presence of MG-132 was very sensitive to trypsin digestion. The 150 kDa coreglycosylated intermediate was not detected after treatment with 10 mg/ml trypsin. By contrast, the 170 kDa mature enzyme when expressed in the absence of MG-132 was relatively resistant to trypsin. It required the use of 100-fold more trypsin (1000 mg/ ml) before it became undetectable by immunoblot analysis. The trypsin sensitivity of the 150 kDa coreglycosylated P-gp in the absence of MG-132 was also similar to that seen in the presence of MG-132. These results are consistent with the explanation that the 150 kDa core-glycosylated P-gps are inactive because they are structurally different from the 170 kDa BLOCKING THE MATURATION OF P-GLYCOPROTEIN
The results in this study suggest that human P-gp does not form a functional transporter immediately after synthesis, but must undergo further folding to be active. The presence of point mutations (processing mutants) or expression in the presence of proteasome inhibitor MG-132 prevented P-gp from folding into the mature and functional enzyme. It appears that the structure of mature P-gp is quite different from the early biosynthetic intermediates, because mature P-gp is relatively resistant to trypsin. In this respect, the 150 kDa P-gp that accumulates in MG-132-treated cells resembles the biosynthetic intermediates found in P-gp processing mutations. It was previously shown that the 150 kDa protein of processing mutants were also ;100-fold more sensitive to digestion with trypsin (20). Expression of P-gp processing mutants in the presence of drug substrate, however, induced the transporter to adopt a trypsin-resistant structure that was trafficked to the cell surface and exhibited drug-stimulated ATPase activity. It appeared that drug substrates affected biosynthesis of P-gp by inducing superfolding of the transmembrane domains (20). Structural differences between mature and coreglycosylated proteins have also been reported for the cystic fibrosis conductance regulator (CFTR), another member of the ABC superfamily of transporters. The most common mutation in cystic fibrosis is a deletion of F508 (DF508) (36). The mutation causes the CFTR protein to be retained inside the cell instead of being at its proper location on the cell surface (37). Recently, it was shown by protease digestion studies that the structure of DF508 CFTR was different from the mature wild-type CFTR (38). It was interesting that the proteolytic digestion pattern of DF508 CFTR resembled that of the immature core-glycosylated wild-type CFTR. This suggests that, as with point mutations in P-gp, the DF508 processing mutation in CFTR does not significantly alter the structure of CFTR, but interferes with the folding steps that occur after synthesis of the protein. This is supported by the finding that DF508 CFTR can be induced to fold properly and be correctly targeted to the plasma membrane if synthesis is carried out at reduced temperatures (39) or in the presence of chemical chaperones such as glycerol, trimethylamine N-oxide, or deuterated water (40, 41). Maturation of CFTR and P-gp can be inhibited by proteasome inhibitors. Expression of wild-type CFTR in the presence of MG-132 completely blocks maturation of the protein (31). This study also reported 1729
Figure 8. Schematic representation of processes that block maturation of P-gp. P-gp synthesis begins in the endoplasmic reticulum, where quality control mechanisms such as molecular chaperones calnexin and Hsc70 may ensure proper folding of the protein (21). The presence of mutations such as G341C (1); processing mutations (2); or the presence of proteasome inhibitors, such as MG-132 (3), prevent maturation of P-gp and the P-gps are rapidly degraded. Maturation of these mutant P-gps can be promoted by either removing the protease sensitive site (A), expressing the processing mutants in the presence of drug substrates (B), or carrying out expression in the absence of proteasome inhibitors (C).
that MG-132 slowed maturation of P-gp. The authors also did not observe any accumulation of a 130 kDa P-gp breakdown product in the presence of MG-132. This is contrary to our findings (20, 23), and likely reflects differences in the cell lines used. There is, however, a difference between CFTR and P-gp in that the DF508 CFTR core-glycosylated and immature wild-type CFTR proteins are functional. Patch-clamp studies of the endoplasmic reticulum of cells expressing DF508 and wild-type CFTR suggest that core-glycosylated proteins form chloride channels that are normally regulated (24). This suggests that the structural requirements to form a regulated channel may not be as stringent as those required to form a drug transporter. It is not clear how proteasome inhibitors interfere with maturation of CFTR and P-gp. One possibility is that inhibition of a proteasome by inhibitors such as MG-132 could have pleiotropic effects on the cell metabolism as a result of accumulation of undegraded proteins. In CFTR, it has been shown that maturation is an ATP-dependent process (42). Another possibility is that the proteasome directly plays a role in the folding process by acting as a potential chaperone. Since most of the P-gp and CFTR molecules are accessible from the cytoplasm during synthesis in the endoplasmic reticulum, they could be in direct contact with proteasomes. Recent evidence from studies of yeast Ste6p processing mutants supports the notion that the proteasome may have such a role (43). The Ste6p protein is a yeast ABC transporter that transports a mating factor. Loayza et al. (43) reported that overexpression of the Hrd2p proteasome subunit in yeast promoted the appearance of functional Ste6p processing mutants at the cell surface. Thus, the proteasome could play a dual role in the folding of ABC transporter; they could promote folding of the proteins as well as mediate degradation of incorrectly folded proteins (44, 45). 1730
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Studies of human P-gp have identified several steps that prevent maturation of the enzyme; the steps are summarized in Fig. 8. Some mutations such as G341C and Q347C (23, 46) expose a proteolytic site in the first extracellular loop that result in degradation of the protein during or immediately after synthesis. These mutants can be rescued, however, by mutating the protease-sensitive site (23). Another type of folding defect is that caused by point mutations that result in misprocessing of P-gp. In these mutations, the P-gp accumulates as inactive, partially folded intermediates that do not seem to be able to overcome a barrier to further folding. In the presence of drug substrates, however, these barriers are removed and the protein folds correctly and is delivered to the cell surface in an active form (19). By contrast, expression in the presence of proteasome inhibitors blocks proper folding and maturation of P-gp. These findings suggest there are several steps during P-gp synthesis and maturation that could be potential targets for inhibition during chemotherapy. Potential strategies would be to identify a pharmacological agent(s) that causes exposure of hypersensitive-protease sites such as R113 (Fig. 8, step 1), that can induce the same alterations as in the processing mutants (e.g., G268V, Fig. 8, step 2), or that blocks protein folding steps as seen with protease inhibitors (Fig. 8, step 3). Learning how to manipulate such steps in the endoplasmic reticulum may also contribute to strategies to treat other diseases that involve defective protein folding and/or trafficking (47– 49). We thank Dr. David H. MacLennan for the A52 epitope and antibody and Dr. Randal Kaufman (Boston, Mass.) for pMT21. This work was supported by grants from the National Institutes of Health (RO1-CA80900), the Medical Research Council of Canada, and the Canadian Cystic Fibrosis Foundation. D.M.C. is a Scientist of the Medical Research Council of Canada and the Canadian Cystic Fibrosis Foundation Zellers Senior Scientist.
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The FASEB Journal
Received for publication February 8, 1999. Accepted for publication July 6, 1999.
LOO AND CLARKE
