(Received for publication, July 1, 1996, and in revised form, September 15, 1996)
From ProScript, Inc., Cambridge, Massachusetts 02139
The natural product lactacystin exerts its
cellular antiproliferative effects through a mechanism involving
acylation and inhibition of the proteasome, a cytosolic proteinase
complex that is an essential component of the ubiquitin-proteasome
pathway for intracellular protein degradation. In vitro,
lactacystin does not react with the proteasome; rather, it undergoes a
spontaneous conversion (lactonization) to the active proteasome
inhibitor, clasto-lactacystin -lactone. We show here
that when the
-lactone is added to mammalian cells in culture, it
rapidly enters the cells, where it can react with the sulfhydryl of
glutathione to form a thioester adduct that is both structurally and
functionally analogous to lactacystin. We call this adduct lactathione,
and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active
-lactone. We have studied the kinetics of this reaction under appropriate in
vitro conditions as well as the kinetics of lactathione
accumulation and proteasome inhibition in cells treated with
lactacystin or
-lactone. The results indicate that only the
-lactone (not lactacystin) can enter cells and suggest that the
formation of lactathione serves to concentrate the inhibitor inside
cells, providing a reservoir for prolonged release of the active
-lactone.
Lactacystin is a natural product that was originally discovered for its ability to induce neurite outgrowth and differentiation in a mouse neuroblastoma cell line, Neuro 2A (1). Subsequent work (2, 3) demonstrated that the biological effects of lactacystin result from its ability to acylate and inhibit the proteasome, a ubiquitous intracellular protein-degrading machine (for reviews, see Refs. 4, 5, 6, 7, 8, 9, 10). Because the proteasome is involved both in the normal turnover of cellular proteins (11) and in the processing and degradation of regulatory proteins that control cell growth and metabolism (12), proteasome inhibitors can have profound biological consequences.
As part of a program to develop proteasome inhibitors into novel
therapeutic agents, we have undertaken detailed studies of the
mechanism of lactacystin. In a recent report (13), we demonstrated that
lactacystin per se is not a proteasome inhibitor. Rather, lactacystin in aqueous solution can spontaneously undergo an
intramolecular reaction (lactonization) to form the active proteasome
inhibitory species, clasto-lactacystin -lactone. Herein
we have extended these studies to examine the mechanism of proteasome
inhibition by lactacystin in cultured cells. We show that cells are
relatively impermeable to lactacystin, but highly permeable to
-lactone. Thus, the efficacy of lactacystin as a proteasome
inhibitor in cells will depend on the lactonization of lactacystin in
the medium to generate
-lactone. Once inside the cell,
-lactone
has multiple fates. It can hydrolyze to the inactive dihydroxy acid, it
can react with the proteasome, or it can react with GSH to form a GSH
conjugate with properties that are analogous to lactacystin. This GSH
conjugate, which we call lactathione, is inactive as a proteasome
inhibitor. Nevertheless, like lactacystin, it can spontaneously
regenerate the active
-lactone via intramolecular lactonization.
These findings bring forth interesting questions pertaining to the
relationship between the metabolism of
-lactone and its efficacy as
a proteasome inhibitor in vivo. In addition, the data point
to several practical considerations for the ever increasing use of
these compounds to study the biological function of proteasomes.
Lactacystin and clasto-lactacystin
-lactone were prepared by the method of Corey et al. (14,
15). Lactathione was prepared by mixing
-lactone (49 mg, 0.229 mmol)
in 10 ml of acetonitrile with glutathione (360 mg, 1.17 mmol) and 1.35 ml of 1.00 N NaOH (1.35 mmol). The mixture was stirred for
4 h at room temperature, and 1 N HCl was added to
obtain pH 4. The solution was concentrated on a rotovap and then
lyophilized. The dry material was resuspended in a small volume of
water and subjected to reverse-phase HPLC1
on a 19 × 300-mm Delta-Pak C18 column (Waters).
Lactathione and the lactathione isomer (see Fig. 1) were collected in
separate fractions eluting from the column. Lyophilization of the
fractions afforded 48 mg of lactathione and 7.8 mg of the lactathione
isomer. The structure of the synthetic materials was confirmed by
proton magnetic resonance spectrometry and electrospray ionization mass spectrometry.
-[3H]Lactone at a specific activity of
3.4 Ci/mmol was prepared from
-lactone by the method described
(3).
HPLC Analyses of Lactathione and Lactacystin
The HPLC methodology has been described in detail (13). Briefly, the stationary phase was a C18 column (Vydac, 218TP54), and the mobile phase consisted of a gradient of methanol (0.05% trifluoroacetic acid) in water (0.06% trifluoroacetic acid).
Analysis of Lactathione HydrolysisThe values for the rate
constants given in Scheme 1 were estimated based on measurements of the
GSH dependence of the steady-state rate constant for lactathione
hydrolysis in phosphate-buffered saline at pH 7.4 and 37 °C and on
separate measurements of -lactone hydrolysis under the same
conditions. The spectrophotometric assays and data analysis were the
same as those used in a previous work to study lactacystin hydrolysis
(13).
Lactathione Inactivation of Proteasome Activity
The 20 S proteasome and PA28 activator were purified from rabbit reticulocytes as described (13). Hydrolysis of the substrate succinyl-Leu-Leu-Val-Tyr 7-amino-4-methylcoumarin by the 20 S proteasome-PA28 complex was assayed by continuously monitoring the fluorescence of the liberated 7-amino-4-methylcoumarin (13). kinact values for lactathione were estimated by analysis of progress curves obtained upon addition of lactathione (13) at a final concentration of 2 µM.
General Cell CultureCell lines were obtained from American Type Culture Collection. Jurkat cells (human T cell leukemia, clone E6-1) were grown in RPMI 1640 medium (JRH Biosciences) supplemented with 10% heat-inactivated FBS (Sigma) and antibiotics (1000 units/ml penicillin and 100 µg/ml streptomycin) (Sigma). HeLa cells (human erythroid carcinoma) were grown in Dulbecco's modified Eagle's medium (DME/HIGH modified, JRH Biosciences) supplemented with 10% FBS and antibiotics as described above. C2C12 cells (mouse myoblast) were grown in Dulbecco's modified Eagle's medium (DME/HIGH modified) supplemented with 10% heat-inactivated FBS and antibiotics described as above. All cells were grown at 37 °C in an atmosphere of 5% CO2. Cells were passaged biweekly and used for experimental purposes in exponential growth phase.
GSH Depletion StudiesFor GSH depletion, Jurkat cells were
kept at low density (5 × 106 cells/ml) for 24 h in the presence of 1 mM buthionine sulfoxime (16).
Following this treatment, the cells were harvested, resuspended in
fresh medium, and aliquoted into 24-well plates at a density of 5 × 107 cells/ml for measurement of lactathione accumulation
or 7 × 106 cells/ml for measurement of IL-2
production. Aliquots of 2 × 106 cells were harvested
and washed, and total intracellular glutathione was measured
enzymatically using the GSH-GSSG recycling assay (17).
Following exposure to
-lactone or lactacystin at the concentrations and times indicated in
the figure legends, cells were harvested and washed twice with cold
PBS. To each cell pellet (5 × 107 cells) in a
microcentrifuge tube was added 200 µl of cold methanol with 0.1%
trifluoroacetic acid, and the pellet was homogenized with a motorized
pestle. The tubes were centrifuged at 16,000 × g for
10 min at 4 °C. The supernatant was collected and dried in a vacuum
centrifuge. The dried samples were resuspended in water with 0.06%
trifluoroacetic acid and subjected to reverse-phase HPLC (see
above).
Jurkat cells at 7 × 106 cells/ml in
medium were treated for 30 min with different concentrations of
-lactone. The cells were harvested and washed twice with cold PBS.
The resulting pellets were ground with a motorized pestle in 50 µl of
cold 5 mM HEPES, pH 7.5, with 1 mM
dithiothreitol. The samples were centrifuged for 10 min at 16,000 × g in the cold, and the supernatants were collected. The
total soluble protein was measured using the Coomassie Plus Protein
Assay (Pierce). For measurement of 20 S proteasome peptidase activity,
10 µl of each sample (40-80 µg of total protein) was diluted into
a cuvette containing 2 ml of 20 mM HEPES, 0.5 mM EDTA, pH 8, at 37 °C with 0.035% (w/v) SDS and 10 µM succinyl-Leu-Leu-Val-Tyr 7-amino-4-methylcoumarin. The
substrate hydrolysis was measured by continuously monitoring the
fluorescence of the liberated 7-amino-4-methylcoumarin. After 3-4 min,
lactacystin was added to a final concentration of 10 µM,
and the substrate hydrolysis was monitored for another 6-8 min. This
lactacystin treatment is sufficient to inhibit the peptidase activity
of purified 20 S proteasome by
99% under the same assay conditions
(data not shown). Thus, the residual activity seen in crude extracts
following lactacystin treatment presumably represents background from
some other peptidase(s). This background was typically
5% of the
total activity and was subtracted out.
C2C12 cells were
aliquoted into 12-well plates at 2 × 104 cells/well
and grown overnight. The medium was exchanged with methionine-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 10% dialyzed FBS and 1 µCi of
[35S]methionine. The cells were grown for 48 h in
the labeling medium and then washed three times with cold PBS
containing 2 mM methionine. Fresh medium containing 10%
FBS and 2 mM methionine was then added, and the cells were
incubated for 2 h. The cells were washed once with PBS containing
2 mM methionine and cultured for an additional 4 h
with medium containing 50% FBS, 2 mM methionine, and
various concentrations of -lactone or lactacystin as indicated in
Fig. 5. The media were harvested into microcentrifuge tubes,
trichloroacetic acid was added to a final concentration of 10% (v/v),
and the tubes were placed on ice for 15 min. The tubes were centrifuged at 16,000 × g for 5 min in the cold, and 100-µl
aliquots of the resulting supernatants were subjected to liquid
scintillation counting.
Assay of Stimulated IL-2 Production in Jurkat Cells
Jurkat cells in 24-well plates at 7 × 106 cells in 1 ml of medium were treated with 0.1 µg of phorbol 12-myristate 13-acetate and 3.5 µg of ionomycin. After 4 h, the culture medium was collected and assayed for IL-2 using the Human IL-2 Quantikine kit (R&D Systems) according to the manufacturer's instructions.
Pulse-Chase ofFour 15-cm plates of HeLa cells were grown to confluence
and harvested to obtain 2.9 × 107 cells. The cells
were suspended in 5 ml of medium, and 5 µl of 1.5 mM
-[3H]lactone in acetonitrile was added (25 µCi of
total 3H). The cells were incubated for 30 min, and 40 ml
of ice-cold PBS was added. The cells were pelleted by centrifugation,
the supernatant was removed, and the cells were resuspended in 6 ml of
fresh medium. The cell suspension was aliquoted into microcentrifuge tubes at 4.8 × 106 cells/tube and pelleted by
centrifugation. The supernatants were removed, and the cell pellets
were resuspended in 500 µl of fresh medium. The tubes were incubated
for the times indicated in Fig. 7A. At the indicated times,
the cells were pelleted, and the supernatant was collected and
acidified with 1 µl of concentrated trifluoroacetic acid. Tritium in
the medium samples was determined by subjecting 50-µl aliquots to
liquid scintillation counting. For HPLC analysis (e.g. Fig.
7B), aliquots of the medium were diluted 4:1 with water (0.06% trifluoroacetic acid) and filtered to remove particulate matter, and 180-µl aliquots of the filtered material were injected onto the column. Liquid scintillation counting of the samples before
and after filtration showed that all of the tritium was recovered in
the filtrate.
-In aqueous solution,
clasto-lactacystin -lactone readily reacts with
N-acetyl-L-cysteine (NAC) to form the thioester
adduct that is lactacystin (13). This prompted us to speculate that an
analogous thioester adduct would form upon mixing
-lactone with GSH.
Fig. 1 shows the UV chromatograms of a reaction mixture containing
-lactone and GSH along with controls for the two
components incubated separately. Two new peaks were observed in the
chromatogram of the reaction mixture. In addition, the peak
corresponding to the
-lactone had disappeared, but no peak
corresponding to clasto-lactacystin dihydroxy acid was
detected, indicating that the lactone had not hydrolyzed. The two new
products were subsequently prepared in quantities sufficient for proton
NMR and mass spectral analysis (data not shown). The larger of the two
peaks (M + H = 521.8) corresponds to the predicted thioester
adduct (Scheme 1), for which we suggest the trivial name
lactathione. The smaller peak (M + H = 521.8) represents an isomer
of lactathione, whose exact structure and origin are currently under
investigation.
In aqueous solution,
lactacystin undergoes spontaneous hydrolysis to yield NAC and
clasto-lactacystin dihydroxy acid. As shown in our previous
work (13), this process proceeds exclusively through the intermediacy
of the -lactone, and this latter species is solely responsible for
proteasome inactivation. The hydrolysis of lactathione was studied
employing the same methodology, and we obtained similar results as
summarized in Scheme 1. Also, the inactivation of 20 S proteasome
chymotrypsin-like activity by lactathione was examined as a function of
GSH concentration as shown in Fig. 2. Increasing the GSH
concentration, which will lower the steady-state level of
-lactone
(Scheme 1), also slows proteasome inactivation (Fig. 2). Likewise, at
pH 6.5, where lactathione is stable because lactonization is suppressed
(data not shown), the rate of proteasome inactivation by lactathione is
greatly reduced (kinact/[I]
0.04 mM
1 s
1). Thus, the properties
of lactathione in vitro are entirely analogous to
lactacystin. Like lactacystin, lactathione does not function as a
proteasome inhibitor, but it can serve as a source of the active
species, clasto-lactacystin
-lactone.
Lactathione Formation in Cells
Of particular interest in
Scheme 1 is the second-order rate constant for combination of
-lactone with glutathione to form lactathione, 3.6 M
1 s
1, measured in PBS at
37 °C. This result suggested that in the physiological range of
intracellular GSH ([GSH]
1 mM), intracellular
-lactone would react rapidly (t1/2
3 min) to
form lactathione. Fig. 3 shows chromatographic analyses
of extracts from Jurkat cells that had been exposed to
-lactone,
lactacystin, or vehicle (dimethyl sulfoxide). In the extracts from both
-lactone- and lactacystin-treated cells, a peak with the same
retention time as lactathione (21.4 min) was observed. This component
is absent in the extract from vehicle-treated cells. The identity of
this component as lactathione was confirmed in separate experiments by
collecting it and subjecting it to mass spectral analysis. The major
component that was detected had a mass identical to synthetic
lactathione (see above). Interestingly, only lactathione, and not
lactacystin, could be detected in the extracts from lactacystin-treated cells. Although the absence of lactacystin in extracts from
lactacystin-treated cells is a negative result, control experiments
suggest that it is significant. The retention time of lactacystin in
this chromatographic system is 24 min, which is well resolved from any
of the background peaks. Also, as a control for recovery, we
supplemented extracts from naive cells with lactathione and lactacystin
and then prepared the extracts for chromatography. The peaks
corresponding to both lactathione and lactacystin were observed (data
not shown), and both were obtained with 79% overall recovery through
the procedure.
In light of these control experiments, we feel that our inability to
detect lactacystin in extracts from lactacystin-treated cells suggests
that either lactacystin does not enter these cells or that it enters
the cells and is very rapidly converted to lactathione (e.g.
the conversion might be catalyzed by a GSH transferase). To test these
possibilities, we examined the effect of glutathione depletion on
accumulation of lactathione inside the cells. A treatment of Jurkat
cells with the -glutamylcysteine synthase inhibitor, buthionine
sulfoxime, that was sufficient to lower intracellular GSH
concentrations by >95% had the expected effect of lowering lactathione accumulation in both
-lactone- and lactacystin-treated cells (Table I). Nevertheless, no lactacystin could be
detected by chromatographic analysis of the GSH-depleted,
lactacystin-treated cells. Additionally, we tested the ability of a
commercially available rat liver GSH transferase preparation to
catalyze lactathione formation. The integrity of this enzyme
preparation was confirmed by monitoring the conjugation of
1-chloro-2,4-dinitrobenzene in a standard assay (18). Nevertheless, no
lactathione formation could be detected when lactacystin was tested as
a substrate (data not shown). Also, lactacystin showed no ability to
inhibit GSH transferase activity in the standard assay (data not
shown).
|
We next examined the kinetics of lactathione accumulation in Jurkat
cells treated with -lactone or lactacystin using the HPLC assay.
Representative data are shown in Fig. 4A.
Lactathione accumulation in
-lactone-treated cells could be detected
at the earliest time point that we were capable of obtaining. This time point is ~2 min, which is the time it takes to harvest and wash the
cells after adding the drug. The intracellular lactathione concentration reaches a maximum at 15-30 min and then decays with a
t1/2 of ~30 min. As a rough approximation, we can
estimate the intracellular volume from the mass of the cell pellet
assuming 1 mg/µl. 5 × 107 Jurkat cells have a mass
of ~50 mg (i.e. 50-µl cell volume). Thus, the
intracellular concentration of lactathione at the peak is ~140
µM, which is higher than the starting concentration (50 µM) of
-lactone in the medium. We have also performed
this experiment with HeLa cells, and the shape of the time course is
nearly identical (data not shown). Nevertheless, the peak
concentrations of lactathione observed in HeLa cells is lower by about
two-thirds compared with Jurkat cells for the same dose of
-lactone.
This could reflect differences in the permeability of the drug in the
different cell types; however, we suspect that this observation most
likely reflects the condition that the HeLa cells are adherent cells
and the Jurkat cells grow in suspension. On one occasion, we first
harvested HeLa cells and then treated them with
-lactone in
suspension, and the lactathione accumulation was three times higher
than when the cells were treated on the plates.
The time course for lactathione accumulation in cells treated with
lactacystin differed substantially from that with -lactone (Fig.
4A). The peak level was lower, and it was obtained at a longer time (45-60 min). Nevertheless, the decay was slower, so that
at 60 min, the concentrations of lactathione in the lactacystin- and
-lactone-treated cells are equal, and at longer times, the lactathione concentration is higher in the lactacystin-treated cells.
The same observations were made in experiments with HeLa cells (data
not shown). These results, together with consideration of the mechanism
of lactacystin hydrolysis as elucidated in our previous work (13),
suggested a model whereby only
-lactone, but not lactacystin, can
enter cells. To illustrate this point, we have performed a calculation
of the time dependence of
-lactone concentration in aqueous medium
starting at zero time with equal concentrations of either
-lactone
or lactacystin (Fig. 4B). Starting with
-lactone, the
-lactone concentration decays in a first-order process due to
hydrolysis. Stating with lactacystin, the
-lactone concentration is
initially zero and with time shows a transient accumulation. This
represents the balance between production of
-lactone via the
lactonization of lactacystin and
-lactone destruction via
hydrolysis. The concentrations of lactacystin relevant to the cell
culture experiments are relatively low (
50 µM), so that the NAC produced in the lactonization step never accumulates to a level
that is high enough to drive the back-reaction (i.e.
re-formation of lactacystin from
-lactone and NAC) to an appreciable
extent. Thus, one need only consider a simple model of two sequential first-order reactions for the calculation of
-lactone concentration when starting with lactacystin. There are two crucial features of this
system (Fig. 4B) that are relevant for consideration of the
data (Fig. 4A). First, the concentration of
-lactone will eventually be higher when starting with lactacystin. The timing of this
(i.e. the time point at the intersection of the two curves) will depend on the relative magnitudes of the rate constants for lactonization and hydrolysis. Second, because the hydrolysis of lactacystin proceeds exclusively through the intermediacy of
-lactone, the areas under the curves are equal. Both of these
features parallel the lactathione accumulation data. First, we observed
that at longer times (
60 min), lactathione concentration was higher
in the lactacystin-treated cells. Second, the areas under the curves for the data in Fig. 4A (calculated by a
model-independent approach (linear trapezoidal rule)) are 470 (nmol/105 cells) × min for the
-lactone-treated cells
and 410 (nmol/105 cells) × min for the lactacystin-treated
cells. Thus, these kinetics suggest that a relationship of
proportionality exists between intracellular lactathione
accumulation and the extracellular concentration of
-lactone. These results led us to propose the model of Scheme 2, where we hypothesize that lactonization of
lactacystin occurs extracellularly and that
-lactone is the only
species that can enter the cells. Once inside,
-lactone can
reversibly react with GSH to form lactathione, or it can inactivate the
proteasome.
Proteasome Inhibition in Cells Treated with Lactacystin and
The rapid hydrolysis of -lactone in aqueous
solution (Scheme 1) is unavoidable in cell culture experiments.
Nevertheless, this is offset to a large extent because of its rapid
entry into cells (Fig. 4B). As a result, low micromolar
concentrations of
-lactone in medium can effect inhibition of the
proteasome in cells. This is illustrated in Fig.
5A, which shows a dose-response curve for the
inactivation of 20 S proteasome peptidase activity in Jurkat cells
treated with
-lactone. A 30-min incubation of the cells with
1
µM
-lactone was sufficient to inactivate the proteasome peptidase activity by
90%. Inhibition of proteasome peptidase activity in cells can substantially suppress overall intracellular protein turnover (11). Fig. 5B shows
dose-response curves for
-lactone and lactacystin inhibiting the
degradation of metabolically labeled "long-lived" proteins in C2C12
cells. Either compound was able to inhibit this turnover to a level of ~60%, and both showed similar potencies (IC50
1 µM). Given the 5-10-fold lower activity that has been
observed for lactacystin compared with
-lactone in vitro
(3, 13), these results were initially surprising. However, considering
the different time scales for the cell culture experiments
versus the in vitro assays of enzyme activity,
the observation of similar potencies for the two compounds makes good
sense. For example, the acid-soluble radioactivity that is used to
monitor protein degradation in C2C12 cells (Fig. 5B) is
accumulated over 4 h. In contrast, the assays with purified
enzyme, as a matter of convenience, are designed so that the full
extent of inactivation is obtained within a much shorter interval (
30
min). As illustrated in Fig. 4, at early times, the concentration of
-lactone generated by lactonization of lactacystin is relatively
low; however, given enough time, the cumulative exposure of cells to
-lactone will be the same for both compounds.
The activation of the transcription factor NF-B and, subsequently,
the expression of the genes that NF-
B controls are dependent on the
proteasome (12). The proteasome functions in this pathway by degrading
I
B
, an inhibitor of NF-
B that, under basal conditions, binds
to NF-
B and sequesters it in the cytoplasm. The mechanism involves a
signal-induced phosphorylation and ubiquitination of I
B
, which
targets this protein for degradation via the ubiquitin-proteasome pathway (19). Expression of the human IL-2 gene depends on NF-
B activation (20) and thus provides a useful system for studying a
specific biological role of the proteasome and a specific consequence of proteasome inhibition (i.e. inhibition of IL-2
production). Fig. 6A shows the ability of
lactacystin to suppress IL-2 production by stimulated Jurkat cells as a
function of NAC concentration. The rationale for this experiment can be
understood by reference to Scheme 2. Extracellular NAC will lower the
extracellular
-lactone concentration by driving the back-reaction to
form lactacystin. Thus, unless lactacystin can enter the cells, the
extracellular NAC should prevent proteasome inactivation. In the
absence of NAC, 10 µM lactacystin applied 30 min prior to
the stimulus decreased IL-2 production to ~10% of the control value
(Fig. 6A). With increasing concentrations of NAC, IL-2
production was restored in the lactacystin-treated cells, reaching
~80% of the control value at 5 mM NAC. We also measured
lactathione accumulation under these same conditions, and the results
are presented in Fig. 6B. Addition of NAC suppressed intracellular lactathione accumulation in lactacystin-treated cells,
and the dose response mirrors the restoration of IL-2 production (i.e. prevention of proteasome inhibition) in the cells.
Independently of the effect of lactacystin, NAC suppressed IL-2
production by about one-third at 5 mM. This is most likely
due to the well documented ability of NAC to prevent NF-
B activation
(21), presumably reflecting the involvement of reactive oxygen
intermediates in the signaling pathway and the ability of antioxidants
such as NAC to buffer them (22).
Metabolic Fate of Lactathione
Inside cells, glutathione
conjugation to xenobiotic electrophiles such as
clasto-lactacystin -lactone is a common mechanism for
detoxification of these compounds. In this aspect, the formation of
lactathione upon entry of
-lactone into cells may serve as a buffer
against
-lactone-mediated proteasome inactivation. Alternatively, the ability of lactathione to spontaneously regenerate the
-lactone (via lactonization) suggests that lactathione could serve as a reservoir for the prolonged release of the active species. The rapid
accumulation of lactathione in
-lactone-treated cells (Fig. 4A) suggests that the cells are freely permeable to this
molecule, whereas the observation that lactathione accumulates to a
level that exceeds that of extracellular
-lactone suggests that the cells are relatively impermeable to lactathione. Thus, lactathione formation concentrates
-lactone inside the cell, albeit in an inactive form. However, this concentrating effect may be
counterbalanced by subsequent metabolism of the lactathione. In
vitro at physiological GSH concentrations (
1 mM),
lactathione is very stable (t1/2
15 h). In
Jurkat or HeLa cells, lactathione disappears, with a half-time of ~30
min (e.g. Fig. 4A). This loss of lactathione from
the cells may be mediated by a glutathione sulfur conjugate export pump in the cell membrane (23, 24, 25). Irrespective of the mechanism, it is
clearly too fast to be accounted for by spontaneous lactonization of
lactathione and subsequent hydrolysis of the
-lactone. To examine
the fate of intracellular lactathione, we prepared radiolabeled
-lactone and performed pulse-chase experiments in HeLa cells. Fig.
7A shows the time course for the appearance
of tritium in the medium from cells following a 30-min pulse of
-[3H]lactone. These data are superimposed upon the
time course for lactathione accumulation in HeLa cells treated with
-lactone measured in a separate experiment. The appearance of
tritium in the medium mirrored the loss of lactathione from inside the
cells, suggesting that intracellular lactathione was transported out of
the cells. To ascertain the identities of the tritiated species, aliquots of chase medium were subjected to reverse-phase HPLC, fractions were collected, and the column fractions were subjected to
liquid scintillation counting (Fig. 7B). The major peak of radioactive material had a retention time coincident with lactathione (21-22 min). A second broad peak of radioactive material at an earlier
retention time (8-11 min) may correspond to
clasto-lactacystin dihydroxy acid (retention time = 8 min; see Fig. 1), which would be produced from lactonization and
hydrolysis of lactathione during the chase interval. However, the peak
fraction is at 10-11 min, which more closely corresponds to the
cysteine sulfur adduct of the
-lactone (i.e.
desacetyllactacystin), which has a retention time of 10 min in this
chromatographic system (data not shown). This species could potentially
be generated via the sequential actions of
-glutamyl transpeptidase
and dipeptidases upon export of lactathione from the cells (for review,
see Ref. 26). Near the beginning of the chase interval, the major
portion of the tritium was found associated with the lactathione peak
(retention time = 24 min). At longer times, the major potion of
the tritium was found associated with the earlier eluting peaks
(retention time = 8-11 min). These kinetics suggest that
lactathione is the predominant form being exported from the cells, with
subsequent generation of the other species by either spontaneous
chemical conversion (i.e. lactonization and hydrolysis) or
enzyme-catalyzed reactions taking place outside the cells. A final
point worth noting is that in vivo, the pathway for
detoxification that is initiated by GSH conjugation is completed by
uptake of the resulting cysteine sulfur conjugates into kidney tubules,
where they are acted upon by N-acetyltransferases to produce
mercapturic acids that are excreted in urine. Thus, the ultimate fate
of lactacystin in vivo is predicted to be excretion of the
mercapturate derived from lactathione, i.e.
lactacystin.2