(Received for publication, September 4, 1996, and in revised form, October 29, 1996)
From the Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
There is growing evidence that abnormal protein
folding or trafficking (protein kinesis) leads to diseases. We have
used P-glycoprotein as a model protein to develop strategies to
overcome defects in protein kinesis. Misprocessed mutants of the human
P-glycoprotein are retained in the endoplasmic reticulum as
core-glycosylated biosynthetic intermediates and rapidly degraded.
Synthesis of the mutant proteins in the presence of drug substrates or
modulators such as capsaicin, cyclosporin, vinblastine, or verapamil,
however, resulted in the appearance of a fully glycosylated and
functional protein at the cell surface. These effects were
dose-dependent and occurred within a few hours after the
addition of substrate. The ability to facilitate processing of the
misfolded mutants appeared to be independent of the cell lines used and
location of the mutation. P-glycoproteins with mutations in
transmembrane segments, extracellular or cytoplasmic loops, the
nucleotide-binding domains, or the linker region were processed to the
fully mature form in the presence of these substrates. These drug
substrates or modulators acted as specific chemical chaperones for
P-glycoprotein because they were ineffective on the F508 mutant of
cystic fibrosis transmembrane conductance regulator. Therefore, one
possible strategy to prevent protein misfolding is to carry out
synthesis in the presence of specific substrates or modulators of the
protein.
Abnormal protein folding or trafficking is associated with a
growing number of diseases (1, 2, 3). Diseases such as Alzheimer's and
prion-related diseases are characterized by the presence of high levels
of insoluble protein aggregates in brain tissue. These plaques appear
to be aggregates of misfolded -amyloid protein in Alzheimer's
disease or aggregates of misfolded prion protein in the
prion-associated diseases such as Creutzfeldt-Jacob disease or Scrapie
(mad cow) (4, 5). In cystic fibrosis, the major defect is due to
deletion of a single amino acid (
F508) in the cystic fibrosis
transmembrane conductance regulator (CFTR)1
resulting in abnormal trafficking to the plasma membrane. The mutant
CFTR protein is misfolded, retained in the endoplasmic reticulum, and
rapidly degraded (6). Potential therapy for diseases involving folding
and/or trafficking defects in the target protein is to prevent
misfolding during protein biogenesis.
We have used the human multidrug transporter (P-glycoprotein) as a model system for studying ways to prevent protein misfolding. P-glycoprotein appears to be an excellent model system, because we have identified many misprocessed P-glycoprotein mutants. These temperature-sensitive or -insensitive mutant proteins are misfolded, retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates in association with molecular chaperones such as calnexin and Hsc70, and rapidly degraded (7, 8, 9, 10).
P-glycoprotein, the product of the human MDR1 gene, is an energy-dependent pump located at the plasma membrane that interacts with a wide variety of structurally diverse cytotoxic agents that do not have a common intracellular target (11). This protein has clinical importance because it may be one of several mechanisms whereby cancer cells become resistant to chemotherapy.
The protein consists of 1280 amino acids organized in two tandem repeats of 610 amino acids, joined by a linker region of 60 amino acids. Each repeat consists of an NH2-terminal hydrophobic domain containing six potential transmembrane sequences followed by a hydrophilic domain containing a nucleotide-binding site. The organization of the domains is characteristic of members of the ABC superfamily of (ATP-binding cassette) transporters, the best known member being CFTR.
Our goal was to develop a strategy to specifically rescue the misfolded mutants of P-glycoprotein so that they could exit the endoplasmic reticulum and reach the plasma membrane in a functional form. Nonspecific low molecular weight compounds such as glycerol (12, 13) have been shown to nonspecifically affect protein kinesis. Therefore, we wished to determine whether substrates or modulators of P-glycoprotein could act as specific chemical chaperones and have a more rapid effect on processing of misfolded proteins. We show that biosynthesis of the processing mutants in the presence of substrates or modulators of P-glycoprotein results in the relatively rapid appearance of a fully mature and functional transporter at the cell surface.
Wild-type and mutant MDR1 cDNAs, modified to encode the epitope for monoclonal antibody A52 at the COOH-terminal ends of the proteins, were inserted into the mammalian expression vector pMT21 as described previously (7). Oligonucleotide-directed mutagenesis was carried out as described previously (7). For purification purposes wild-type and mutant MDR1 cDNAs were modified to encode for 10 histidine residues at the COOH ends of the proteins (14). The sequence at the COOH terminus of P-glycoprotein that would normally end as TKRQ became TKRAH10LDPRQ.
Expression, Purification, and Measurement of Mg2+-ATPase Activity of P-glycoprotein MutantsHEK
293 cells were transfected with the mutant cDNA constructs. After
24 h, the medium was replaced with fresh medium containing the
desired drug concentration. For purification of P-glycoprotein mutants,
HEK 293 cells transfected with the cDNA coding for the histidine-tagged P-glycoproteins were solubilized with 1% (w/v) n-dodecyl--D-maltoside, and the mutant
P-glycoproteins were purified by nickel-chelate chromatography.
Drug-stimulated ATPase activity was determined as described previously
(14).
Pulse-chase experiments were done as described previously (8).
Immunological ProceduresWhole cell extracts or purified P-glycoprotein samples were subjected to SDS-PAGE, electroblotted onto nitrocellulose, and developed with monoclonal antibody A52 (15) or with a rabbit polyclonal antibody against P-glycoprotein followed by enhanced chemiluminescence (Amersham Corp.) as described previously (9).
The effect of substrates and modulators of P-glycoprotein
on the biosynthesis of two processing mutants were initially studied; G268V in the NH2-terminal transmembrane domain (16) and
Y490 in the NH2-terminal nucleotide-binding domain (17).
Mutant G268V is a temperature-insensitive processing mutant, whereas
mutant
Y490 contains a deletion at an equivalent position to the
F508 mutation in CFTR. The cDNAs coding for these mutant
P-glycoproteins were modified to encode for the epitope of monoclonal
antibody A52. The presence of the epitope provided us with an important tool for following the synthesis of the misfolded mutants. It allowed
us to distinguish the mutant protein from any endogenous P-glycoprotein
that may be induced by the presence of drug substrates. These mutants,
as well as wild-type enzyme, were expressed transiently in HEK 293 cells and then treated for 24 h with various concentrations of
drug substrates. Four structurally different drug substrates or
modulators (capsaicin, cyclosporin, verapamil, and vinblastine) of
P-glycoprotein were tested. Vinblastine is an antitumor agent that is a
substrate of P-glycoprotein, whereas verapamil and cyclosporin A are
inhibitors of drug transport. Capsaicin, the pungent ingredient in
peppers of the Capsicum family, is a substrate of
P-glycoprotein, based on its ability to stimulate ATPase activity (data
not shown). All four compounds are hydrophobic and can readily diffuse
through the plasma membrane to the site of protein synthesis in the
endoplasmic reticulum.
In the absence of drug substrates (Fig. 1A),
the major product of the mutant P-glycoproteins had an apparent mass of
150 kDa compared with 170 kDa for the wild-type enzyme. The 170- and
150-kDa forms of the enzyme represent mature and core-glycosylated
biosynthetic intermediate, respectively. In the presence of capsaicin,
cyclosporin A, verapamil, or vinblastine, however, the amount of mature
enzyme (170 kDa) for both mutants increased in a
dose-dependent manner. The ability to "rescue"
misprocessed mutants appeared to be independent of the location of the
mutation. In addition to the mutants G268V and Y490, we were able to
facilitate processing of P-glycoproteins with mutations in the
predicted transmembrane segments (TM1, G54V; TM5, G300V; TM7, A718L;
and TM9, A841L), in the extracellular loops between transmembrane
segments (G854V), in the cytoplasmic loops (G251V and W803A), in the
nucleotide-binding domains (G427C and S434C), and in the linker region
connecting the two halves of the molecule (E707A) (data not shown).
The most potent of the four compounds was cyclosporin A. More than 50%
of the mutant protein was present as the mature form of the enzyme in
the presence of 2-10 µM of cyclosporin A, 5-20 µM vinblastine, 12.5-50 µM verapamil, or
75-150 µM capsaicin. The highest concentration of
capsaicin (300 µM) appeared to be quite toxic to the
cells. Except for vinblastine, the cells continued to proliferate in
the presence of the various drug substrates. Vinblastine, which is an
inhibitor of microtubule assembly, did not cause immediate cell death
but resulted in the detachment of the cells from the dish. Other
hydrophobic compounds that are not substrates of P-glycoprotein, such
as 3-methoxy-tyramine or 3-hydroxy-4-methoxyphenethyl amine, had no
effect on the processing of the misfolded mutants (data not shown). The
effects of drug substrates on folding appear to be specific to
P-glycoprotein because the drug substrates of P-glycoprotein could not
rescue the temperature-sensitive CFTR F508 processing mutant (Fig.
1B).
Restoration of processing of the misfolded mutants appeared to be quite
rapid and occurred within a few hours after the addition of drug
substrates to the medium. Fig. 2A shows that
after 4 h in the presence of 15 µM cyclosporin A,
about 50% of mutant G268V was present as the fully mature (170-kDa)
form of the enzyme and that after 24 h, more than 80% of the
mutant protein was present in the fully mature form. The total amount
of P-glycoprotein also increased dramatically in the presence of
cyclosporin A. These results suggested that the drug stabilized the
mutant protein resulting in decreased turnover. This was confirmed by
pulse-chase studies. Fig. 2B shows that in the absence of
cyclosporin A, the 150-kDa P-glycoprotein of mutant G268V was not
processed to the mature enzyme. The core-glycosylated protein was
rapidly degraded (half-life about 2 h), and there was little
product remaining after 8 h. In the presence of cyclosporin A,
however, the kinetics of maturation of the P-glycoprotein of mutant
G268V was similar to that of wild-type enzyme. By 4 h
post-labeling, the majority of the 150-kDa protein was processed to the
mature enzyme (170 kDa). The processed P-glycoprotein was stable for at
least 24 h. Cyclosporin A had no detectable effect on the
processing of the wild-type enzyme.
Detection of P-glycoprotein at the Cell Surface and Measurement of Drug-stimulated ATPase Activity
Cell surface labeling was
performed (8) to determine whether the mutant proteins reached the
plasma membrane. HEK 293 cells were transfected with the
histidine-tagged mutant cDNAs and then incubated in the presence or
the absence of 15 µM cyclosporin A. The transfected cells
were then treated with periodate to convert the carbohydrate moieties
to aldehydes, followed by addition of biotin-LC-hydrazide (Pierce).
Biotin-LC-hydrazide is a nonpermeable compound that forms covalent
attachments to extracellular glycoproteins after periodate oxidation
(18). The histidine tagged P-glycoprotein mutants were then purified
by nickel-chelate chromatography and immunoblotted with
streptavidin-conjugated horseradish peroxidase. Fig. 3
shows that wild-type but not the mutant P-glycoproteins, was present at
the cell surface when expression was done without drug substrate. When
the transfected cells were incubated in the presence of 15 µM cyclosporin A, however, both mutant proteins were
detected at the cell surface.
To test if the mutant P-glycoproteins were active when expressed in the
presence of drug substrates, we attempted to purify the mutant
P-glycoproteins by nickel chelate chromatography for measurement of
drug-stimulated ATPase activity. We have previously found that
wild-type but not misprocessed P-glycoprotein containing a histidine
tag can readily be recovered by nickel-chelate chromatography (10).
Apparently the processing mutants are misfolded, resulting in masking
of the histidine tag. We modified the cDNAs of the mutant
P-glycoproteins to code for 10 tandem histidine residues at the COOH
end of the molecule to facilitate purification by nickel-chelate
chromatography (14). The cDNAs of the mutant P-glycoproteins were
transiently expressed in HEK 293 cells and then incubated for 24 h
in the presence or the absence of 15 µM cyclosporin A. Histidine-tagged P-glycoprotein was isolated by nickel-chelate
chromatography. The majority of the wild-type P-glycoprotein was bound
to the nickel column and was eluted with 0.3 M imidazole regardless of whether expression was carried out in the presence or the
absence of cyclosporin A. This was determined by loading equivalent
amounts of the flow-through and eluted fractions on SDS-PAGE followed
by immunoblotting with a polyclonal antibody against P-glycoprotein
(data not shown). By contrast, most of the P-glycoprotein of mutants
G268V and Y490 grown without drug substrate were recovered in the
flow-through fractions during nickel-chelate chromatography. In the
presence of 15 µM cyclosporin A, however, the majority of
the mutant P-glycoproteins were recovered by nickel-chelate
chromatography and had an apparent mass of 170 kDa. Similar results
were obtained when the transfected cells were incubated in the presence
of capsaicin, verapamil, or vinblastine (data not shown).
Drug-stimulated ATPase activity of the wild-type and mutant
P-glycoproteins was measured in the presence of verapamil, vinblastine, or colchicine after the addition of lipid. Fig. 4 shows
that the purified P-glycoprotein of mutant Y490 after expression in
the presence of cyclosporin resulted in a functional molecule that exhibited a similar pattern of drug-stimulated ATPase activity as the
wild-type enzyme. Similarly, drug-stimulated ATPase activity was
detected in the mutant G268V after expression in the presence of
cyclosporin A. The observation that mutant G268V exhibits reduced activity is consistent with previous observations that several glycine
to valine mutations in the cytoplasmic loops of P-glycoprotein also
alter the substrate specificity of the enzyme (16). These results show
that the presence of drug substrates during biosynthesis of the
misfolded proteins results in the appearance of a functional transporter at the cell surface.
Drug substrates or modulators of P-glycoprotein appear to be acting as powerful "chaperones" for processing misfolded P-glycoproteins. All misprocessed mutants of P-glycoprotein that we have tested could be converted to the fully mature form of the enzyme, even when the mutations were located in different domains of the molecule. The effects of these substrates or modulators were also independent of the cell lines because the misfolded mutants could be rescued when expressed either transiently in HEK 293 cells (this study) or stably in NIH 3T3 cells (data not shown).
The ability of drug substrates to facilitate folding appears to be
specific for P-glycoprotein because these substrates were ineffective
on the CFTR mutant (F508). Reversal of the misfolding phenotype of
the
F508 CFTR mutant can be accomplished by incubation at lower
temperatures (19) or by exposure to nonspecific low molecular weight
compounds such as glycerol (12, 13), trimethylamine N-oxide,
or deuterated water (13). In our hands, the effectiveness of glycerol
treatment or lowering the growth temperature to facilitate processing
was cell line-dependent. For example, some misfolded P-glycoprotein mutants such as G714A are temperature- and
glycerol-sensitive only when stably expressed in NIH 3T3 cells but not
when expressed in HEK 293 cells (data not shown). In addition,
maturation of the misfolded P-glycoproteins in the presence of glycerol
or by lowering the incubation temperatures is a slow process that
usually requires 24-72 h. By contrast, the appearance of the mature
form of any of the misprocessed mutants of P-glycoprotein occurs within 2-4 h after addition of any drug substrate (Fig. 2). Another
interesting observation is that misfolded mutants that are temperature-
and glycerol-insensitive, such as G251V, G268V, and E707A could also be
rescued by these drug substrates when expressed in either HEK 293 or
NIH 3T3 cells (data not shown).
The exact mechanism of how these specific drug substrates or modulators
facilitate processing of misfolded P-glycoproteins mutants is not
known. A possible explanation is that the drug-binding sites(s) in
P-glycoprotein are formed early in the folding intermediates during
biosynthesis. Occupation of the drug-binding site(s) stabilized the
folding intermediates in a "near native" conformation, thus escaping the cell's quality control mechanism (Fig. 2B).
Recently, Qu and Thomas (20) studied the effects of the CFTR F508 on the thermodynamic stability and folding yield of the nucleotide-binding domain 1 and concluded that the major deleterious effect of the mutation was to allow accumulation of a folding intermediate that was
prone to self-association. The
F508 mutation had little effect on
the thermodynamic stability of the folded nucleotide-binding domain 1. The mutation also did not appear to enhance in vivo proteolysis because inhibition of proteasomes (21, 22) did not enhance
the efficiency of maturation of the full-length
F508 CFTR. These
results suggest that mutations that cause misprocessing slow one or
more folding steps, resulting in an increased concentration of the
intermediate that is prone to self-aggregation. Therefore, in
P-glycoprotein, it is possible that the occupation of the drug-binding site(s) in the early stages of folding may reduce the concentration of
the intermediate that is prone to self-aggregation.
In summary, the results of this study demonstrate that a potential strategy in the treatment of diseases involving trafficking/misfolding of proteins would be to identify specific synthetic and natural substrates or modulators and to include these during biosynthesis.
We are grateful to Dr. David H. MacLennan for the use of the A52 epitope and monoclonal antibody used in this study. We thank Dr. J.R. Riordan (Mayo Clinic, Scottsdale, Arizona) for monoclonal antibody M3A7 and Dr. Randal Kaufman (Boston, MA) for pMT21.