From the
Ornithine decarboxylase (ODC) of African trypanosomes is an
important target for anti-trypanosomal chemotherapy because of its
remarkable stability in vivo. The in vivo activity
and stability of mammalian ODC are regulated by polyamines. Polyamines
induce antizyme, which inactivates ODC by tight association and
promotes degradation of ODC by the mammalian 26 S proteasome. Here we
found, in contrast to mammalian cells, that polyamines caused no
reduction of ODC activity in Trypanosoma brucei. Mouse ODC
expressed in T. brucei was also unaffected by exogenous
polyamines, suggesting that a mammalian antizyme equivalent may be
absent in T. brucei. The rat antizyme expressed in T.
brucei was found capable of inhibiting mouse ODC activity by the
formation of rat antizyme-mouse ODC complex. However, complex formation
did not lead to degradation of mouse ODC in T. brucei. Further
in vitro experiments suggested the presence of an inhibitory
factor(s) in trypanosome, which interferes with the degradation of
mouse ODC. We also demonstrated the presence of proteasomes in T.
brucei. But the mobility of the trypanosomal proteasome on native
gel is different from that of the mammalian proteasome. Thus, the
absence of antizyme, the presence of inhibitory factor(s), and the
differences between trypanosomal and mammalian proteasome may account
for the stability of mouse ODC in T. brucei cells.
Ornithine decarboxylase (ODC
In mammalian cells, a noncompetitive
inhibitory protein named antizyme is involved in the degradation of ODC
both in vivo and in vitro (14, 15, 16) . The expression of antizyme
in mammalian cells can be induced by polyamines
(9) . Antizyme
inhibits ODC activity by forming an ODC-antizyme complex
(9, 14, 17) . The complex thus formed causes the
exposure of the carboxyl terminus of ODC
(18) , which
accelerates the degradation of ODC by the 26 S proteasome in an
ATP-dependent but ubiquitin-independent manner
(19, 20, 21) . The 26 S proteasome is a large
complex consisting of two functionally distinct units: a central
cylindrical 20 S proteasome with at least three distinct protease
activities, namely trypsin-like, chymotrypsin-like, and
peptidylglutamyl-peptide hydrolase activities, respectively; and
regulatory parts that attach to the central core
(22, 23, 24) . The mammalian 20 S proteasome
contains 20-25 different proteins with molecular sizes of
22-34 kDa resolved on a two-dimensional gel
(24) . The
manifestation of multiple catalytic activities at independent active
sites within a single enzyme complex may be advantageous for the rapid
and selective degradation and inactivation of various types of cellular
proteins. In addition to the selective ubiquitin-independent
degradation of ODC, the 26 S proteasome performs selective degradation
of regulatory proteins related to cell cycle progression through a
pathway that requires substrate ubiquitination
(25, 26) . The 26 S proteasome is also thought to play
other roles in intracellular proteolysis such as antigen presentation
(23) .
In this study we first examined whether an antizyme is
present in T. brucei and found it to be absent. We then
expressed both mouse ODC and rat antizyme in T. brucei by DNA
transformation and noted the anticipated rat antizyme-mediated
inactivation of mouse ODC enzymatic activity through antizyme-ODC
complex formation. However, formation of the complex does not lead to
rapid degradation in T. brucei, which could be attributed to
the presence in T. brucei of an inhibitor(s) of proteasome
function. We have also, for the first time, identified the proteasomes
in T. brucei, which turn out to be distinct from mammalian
proteasomes.
The activity and stability of ODC in many living organisms,
like many other enzymes of biosynthetic pathways, are regulated by the
end products of its associated metabolic pathway. ODC activity varies
inversely with the supply of polyamines among all the eukaryotes so far
tested, including mammals
(9) , Xenopus laevis (36) , Neurospora crassa (38) ,
Saccharomyces cerevisiae (37) , and Pharsarum
polycepharum (43) . The mechanism of inactivation of ODC
varies in different organisms. In Physarum, polyamines induce
the transition from active (A-form) to inactive (B-form) states
(43) . Studies of S. cerevisiae (37) suggest
that polyamines regulate ODC expression at a post-translational step
prior to assembly of the active form of the enzyme. In the fungus
N. crassa, polyamines appear to repress synthesis of ODC as
well as stimulate its degradation
(44) . In mammalian
(9) and Xenopus cells
(36) , inactivation of ODC
is associated with the presence of the ODC antizyme. ODC antizyme is
defined as the inhibitory protein whose synthesis is induced by the
products of ODC, the polyamines
(9) . Antizyme inhibits ODC
activity and mediates the degradation of ODC protein in the vertebrates
(18, 19) . We have examined the effects of exogenous
polyamines on the ODC activity in T. brucei. Our results
indicated that the trypanosomal ODC activity in the procyclic form is
unaffected by the presence of putrescine and spermidine in the culture
medium, contrary to the results in eukaryotic cells examined
previously. In view of the fact that polyamines can effectively enter
T. brucei cells
(7, 39) , our results suggest
that the machinery for polyamine-dictated regulation of ODC activity
and stability may not exist in T. brucei.
The stable
activity and protein level of trypanosomal ODC indicate that an
antizyme homolog is absent in T. brucei, a conclusion
consistent with the negative outcome of several tests applied in our
present studies. In the vertebrates, antizyme inhibits ODC activity by
forming an antizyme-ODC complex. Li and Coffino
(14, 30) have demonstrated previously that a region near the amino
terminus of mouse ODC as well as the carboxyl-terminal portion of rat
antizyme are required for the in vitro association between the
two proteins. Our immunoprecipitation experiments demonstrated that
mouse ODC and rat antizyme are indeed associated in T. brucei cells. However, only 20-25% of mouse ODC was associated with
antizyme in Tb/MOD-AZ. This percentage corresponds well with
the extent of inhibition of mouse ODC activity by antizyme (also
20-25%) from the same T. brucei transformant. We
estimated that both mouse ODC and rat antizyme are expressed at equal
molar ratio in Tb/MOD-AZ. However, the antizyme expressed in
trypanosome is much less reactive than that from in vitro translation. One simple explanation is that there may be some
factor(s) in T. brucei cells partially blocking the
ODC-antizyme association. However, further evidence on the presence of
putative inhibitory factor(s) is currently unavailable.
Li and
Coffino
(18) demonstrated that antizyme binding to mouse ODC
causes the exposure of the carboxyl terminus of the latter and subjects
the enzyme protein to degradation in reticulocyte lysates. Our current
results demonstrated that in T. brucei cells antizyme-mouse
ODC association leads to inactivation of the ODC activity but not to
the degradation of mouse ODC. The results from Fig. 8indicated
that some soluble and heat-labile factor(s) are present in T.
brucei which inhibits the degradation of mouse ODC in the
reticulocyte lysates. This inhibitory factor(s) may also contribute to
the inhibition of mouse ODC degradation mediated by the antizyme in
Tb/MOD-AZ cells. However, the working mechanism of this
inhibitory factor(s) is unclear. It is possible this inhibitory
factor(s) is also the one(s) partially blocking the association between
mouse ODC and antizyme in Tb/MOD-AZ cells.
It has been
shown that degradation of mammalian ODC is mediated by antizyme and 26
S proteasome in reticulocyte lysates
(41) . Here we also
identified the presence of proteasome in T. brucei. However,
the proteasome identified from T. brucei is probably different
from the mammalian counterparts. Their mobility on native gels is
faster than rabbit 26 S and 20 S proteasomes; presumably the complexes
are smaller than 20 S. The substrate specificity analysis revealed that
trypanosomal proteasome preparations demonstrated much higher
trypsin-like activity than chymotrypsin-like activity, whereas the
proteasome preparations from rabbit reticulocyte lysate have the
opposite specificity.
In addition to the antizyme-dependent
degradation of ODC by 26 S proteasome in mammalian systems, it is
generally believed that the 26 S proteasome performs selective
degradation of eukaryotic cellular proteins through a ubiquitination
pathway
(23, 46) . Among the few physiological
substrates identified in the ubiquitin proteolytic pathway thus far
(46) , one of them is the mitotic cyclin
(25) . The
cyclins associate with protein kinases of the p34
-We thank Dr. Jürg Sommer for providing
pTOD, pMOD, and the T. brucei transformation
vector, pTSA-Hyg2. We are grateful to Dr. Martin Rechsteiner
for the antibody against the human 20 S proteasome, and to Dr. O.
Jänne for the antiserum against mouse ODC.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
;
L-ornithine carboxylase, EC 4.1.1.17) is the key enzyme in
de novo synthesis of polyamines, which are ubiquitous in and
essential for cells
(1, 2) . The inhibition of ODC has
been investigated as a potential way to control rapid cell
proliferation and to cure certain infectious deceases
(3) .
Indeed,
-DL-difluoromethylornithine (DFMO), a catalytic,
irreversible inhibitor of ODC, has been found to cure infections of
Trypanosoma brucei in mice and Trypanosoma gambiense in man
(4, 5) . DFMO depletes the pool of
putrescine and spermidine in African trypanosomes, resulting in
cessation of cell growth and eventual elimination of the parasites by
the host immune response
(5, 6) . The anti-trypanosomal
action of DFMO can be reversed by intraperitoneal injections of
polyamines in the murine model
(7) . The selective DFMO toxicity
toward the trypanosomes may be attributable to a difference in the
in vivo stabilities between trypanosomal ODC and its mammalian
counterpart
(8) . Mammalian ODC is one of the most short lived
cellular proteins in vivo with a half-life of 10-50 min
(1, 9) . On the other hand, we found that the
trypanosomal ODC is very stable in T. brucei, presumably
because a PEST sequence present at the carboxyl terminus of mouse ODC
is absent from T. brucei ODC
(10) . This
carboxyl-terminal PEST sequence could be implicated in the rapid
intracellular turnover of mammalian ODC. Truncation of the 37 residues
at the carboxyl terminus of mouse ODC stabilized the protein in
vivo (11) , and addition of the carboxyl terminus of mouse
ODC to trypanosomal ODC led to the rapid degradation of the chimeric
protein in Chinese hamster ovary cells
(12) , indicating that
the sequence at the mouse ODC carboxyl terminus plays an important role
in bringing about its rapid degradation in vivo. However, when
full-length mouse ODC was expressed in the procyclic form of T.
brucei, we found that the mouse enzyme was stable
(13) .
This observation suggests the absence and/or the inhibition of
necessary machineries in T. brucei cells required for rapid
degradation of mammalian ODC. We decided therefore to look into the
mechanisms behind the unusual stability of mouse ODC in the procyclic
form of T. brucei.
Cell Cultures and Ornithine Decarboxylase Activity
Assay
Procyclic cells of a cloned T. brucei brucei strain TREU667 were continuously cultivated at 26 °C by serial
passages in Cunningham's medium
(27) supplemented with
10% fetal bovine serum (Gemini Bioproducts, Inc.). The hepatoma cells
(HTC) were cultured as described previously
(14) . The
trypanosomal cell suspension was diluted with fresh Cunningham's
medium containing fetal bovine serum to a density of approximately 1
10
cells/ml and incubated at 26 °C overnight
before the ODC activity assay. Cells were washed with cold
phosphate-buffered saline, lysed, and assayed for ODC activity
essentially as described previously
(14) . One unit of ODC
activity is defined as 1 nmol of CO
released from
L-ornithine/h. One unit of antizyme activity is defined as the
amount of antizyme required to inhibit 1 unit of ODC activity.
Plasmid Construction and Transformation of T.
brucei
The rat antizyme partial cDNA clone, Z1
(28, 29) , was obtained from Dr. S. Hayashi. Due to the
absence of a suitably placed methionine initiation codon in the clone,
an in-frame ATG codon was added at the immediate 5`-end of the clone.
The antizyme thus expressed from this construct in Chinese hamster
ovary cells is known to execute all of the expected cellular and
biochemical functions
(30) . An XhoI site was generated
upstream of the ATG codon in the primer sequence
(5`-AAAACTCGAGCATATGCCAGAGAGAAAGAAGGGGA) for polymerase chain reaction.
At the 3`-end of the clone, a BamHI site was added upstream of
the poly(A) tail (primer sequence: 5`-TTTTGGATCCAGATCACTTTATTTATTAG).
The XhoI/ BamHI fragment of the polymerase chain
reaction product was inserted into the XhoI/ BamHI
sites of the vector pTSA-Hyg2
(31) , which contains a
hyg gene whose expression in T. brucei is under the
control of a procyclic acidic repetitive protein promoter. The
nucleotide sequence of the insert was confirmed by sequencing. The
plasmid thus constructed was named pAZ. Plasmids
pMOD300 and pTOD300 containing the inserts of
full-length cDNA encoding mouse ODC and T. brucei ODC,
respectively, were constructed previously from a similar vector
pTSA-Neo2, which has a neo gene controlled by the
procyclic acidic repetitive protein promoter
(13) . The DNAs of
pAZ, pMOD300, and pTOD300 were each
linearized at the MluI site at the center of T. brucei tubulin intergenic region and electroporated into the T.
brucei cells
(31) either individually or in combination.
The stable transformants were selected in the Cunningham's medium
containing 10% fetal bovine serum in the presence of G418 (geneticin,
Life Technologies, Inc.) (50 µg/ml), or hygromycin
(Calbiochem-Novabiochem Corporation) (50 µg/ml), or both.
Southern, Northern, and Slot Blot
Hybridizations
Cellular DNA was isolated from the procyclic
forms of T. brucei according to Hua and Wang
(32) .
Total cellular RNA was isolated as described
(33) , and the
poly(A) RNA was prepared by using the Fast-track mRNA
isolation system (Invitrogen). The Southern, Northern, and slot blot
hybridizations were carried out as described previously
(32) .
Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) and Immunostaining
Cells were
lysed in the lysis solution (2% (w/v) SDS; 125 mM Tris-HCl, pH
6.8; 5% (v/v) -mercaptoethanol; 20% (v/v) glycerol). The lysate
was centrifuged at 16,000
g at 4 °C for 10 min.
The supernatant was fractionated on a 12.5% SDS-PAGE. The separated
proteins were electroblotted onto polyvinylidene difluoride Immobilon
membrane (Millipore Inc.), immunostained, and visualized with the
enhanced chemiluminescence system (Amersham Corp.) as described
previously
(32) .
In Vivo Pulse-Chase Labeling of Protein and
Immunoprecipitation
The trypanosome cells were washed twice
with cysteine/cystine-free Cunningham's medium, then suspended in
the same medium containing
L-[S]cysteine (250 µCi/ml, 600
Ci/mmol) (DuPont NEN) to a density of 5-6
10
cells/ml. After incubation at 26 °C for 1 h, the radioactive
medium was replaced with the regular Cunningham's medium and
incubation continued. Cells were collected at time points of 0, 2, 4
and 6 h. Alternatively, cells were collected immediately after the
L-[
S]cysteine labeling without chasing.
The cells were lysed in RIPA buffer (150 mM NaCl; 0.1% SDS;
0.5% sodium deoxycholate; 1% (v/v) Nonidet P-40; 50 mM
Tris-HCl, pH 8.0)
(34) , and pretreated with a rabbit preimmune
serum. Mouse ODC, trypanosomal ODC, and rat antizyme were
immunoprecipitated with their respective antisera (see below) and
Pansorbin (formalin-fixed Staphylococcus aureus cells)
(Calbiochem-Novabiochem) following a standard protocol
(34) .
HTC cells were labeled in culture medium containing
L-[
S]cysteine as described previously
(30) , and ODC was immunoprecipitated as above. The
immunoprecipitated proteins were subjected to 12.5% SDS-PAGE and
autoradiography.
In Vitro ODC Inhibition Assay
Antizyme
was produced by the in vitro transcription/translation systems
described by Li and Coffino
(18) . Five microliters of the
antizyme thus generated (containing approximately 0.25 unit of
antizyme) was mixed with 0.5 unit of purified native mouse ODC and
incubated on ice for 10 min. Alternatively, lysates from 1.5
10
pAZ-transformed T. brucei cells were
mixed with 0.5 unit of purified native mouse ODC and kept on ice for 10
min. These mixtures were then assayed for ODC activity as described
above.
In Vitro ODC Degradation Assay
Mouse ODC
and antizyme proteins were prepared by the in vitro transcription/translation system as described previously
(30) . Mouse ODC was labeled with
L-[S]methionine during the in vitro translation and mixed with in vitro translated but
unlabeled antizyme followed by the addition of an ATP-regenerating
system as described previously by Li and Coffino
(18) .
Alternatively, the in vitro translated, unlabeled antizyme
sample was replaced with various trypanosomal cell lysates (1.5
10
cells) for the degradation assay in an attempt to search
for activity capable of degrading mouse ODC in the lysates. Treated
mouse ODC was separated by SDS-PAGE and visualized by autoradiography.
Purification and Analysis of
Proteasomes
Proteasomes were isolated from trypanosome and
rabbit blood cells according to Hoffman et al. (35) with some modifications. Briefly, cells were lysed in 10
mM Tris-HCl, pH 7, 1 mM dithiothreitol, 1 mM
ATP, 0.1 mM EDTA by sonication in an ice-ethanol bath. Cell
breakage was monitored under a microscope. After centrifuging the
lysate at 80,000 g for 60 min, glycerol was added to
the supernatant to 20% (v/v). The lysate was then loaded onto a
DEAE-cellulose (Sigma) column equilibrated in the TSDG buffer (10
mM Tris-HCl, pH 7; 25 mM KCl; 10 mM NaCl;
1.1 mM MgCl
; 0.1 mM EDTA; 1 mM
dithiothreitol; 20% glycerol). The column was washed with 5 bed volumes
of TSDG and 5 bed volumes of TSDG containing 100 mM KCl and
eluted finally with TSDG containing 250 mM KCl. The eluate was
then centrifuged at 100,000
g for 15 h at 4 °C.
The pellet was resuspended in TSDG and subjected to electrophoresis in
a nondenaturing gel for 500 V-h as described
(35) . The protease
activity in the gel was detected with a mixture of two fluorogenic
peptides in 30 mM Tris-HCl, pH 7.8; 5 mM
MgCl
; 10 mM KCl; 0.5 mM dithiothreitol; 2
mM ATP for 30 min at 37 °C. The fluorogenic peptides used
were 20 µM Suc-Leu-Leu-Val-Tyr-MCA
(4-methyl-coumaryl-7-amide) and 200 µM Pro-Phe-Arg-MCA
(Sigma). The fluorescent gel was photographed and then stained in 0.2%
Coomassie Brilliant Blue. Alternatively, the fluorescent bands were
excised and soaked in 125 mM Tris-HCl, pH 6.8; 2% (w/v) SDS;
5% (v/v)
-mercaptoethanol; 20% (v/v) glycerol for 15 h at 37
°C. The samples were then subjected to SDS-PAGE and immunostaining
with rabbit anti-human 20 S proteasome antibodies as described above
(35) .
Other Methods
Autoradiographic and
immunostaining results were analyzed with a laser densitometer (LKB
model 2202 Ultrascan). Protein concentrations were determined with
protein assay reagent from Bio-Rad using bovine immunoglobulin (IgG) as
a standard.
Materials
Putrescine and all of the
chemicals for making Cunningham's medium were tissue culture
reagents from Sigma. Spermidine was from Calbiochem. The rabbit
antiserum against antizyme was raised using the glutathione
S-transferase-antizyme fusion protein
(30) . Rabbit
antiserum against trypanosomal ODC was prepared from purified
recombinant T. brucei ODC
(8) . Rabbit anti-mouse ODC
antiserum was a gift from Dr. O. Jänne of the Rockefeller
University. Rabbit antibody against human 20 S proteasome was a
generous gift from Dr. M. Rechsteiner of the University of Utah
(35) . Donkey anti-rabbit IgG-horseradish peroxidase conjugate
was a product from Amersham.
Effect of Polyamines on T. brucei ODC
Activity
Polyamines such as putrescine and spermidine
regulate ODC activities in many living organisms including mammals
(9) , Xenopus (36) , yeast
(37) , and
Neurospora (38) . The presence of excessive polyamines
results in dramatic reductions of ODC activities. We thus investigated
the potential effects of polyamines on the ODC activity of procyclic
T. brucei grown in culture medium. As shown in
Fig. 1A, supplementation of the growth medium with
either putrescine or spermidine up to 5 mM resulted in no
apparent decrease in ODC activity in T. brucei over a period
of 6 h. This is in sharp contrast to the results with rat HTC cell
cultures, in which the ODC activity was markedly reduced upon the
addition of putrescine (Fig. 1 B). It has been shown that
putrescine and spermidine can effectively enter T. brucei cells
(39) . These results thus indicate that there is no
induced inhibition of trypanosomal ODC activity by putrescine or
spermidine, in contrast to all eukaryotic cells examined previously.
Figure 1:
Effect of polyamines on the activity of
ODC. Cells of panel A, T. brucei ( Tb),
panel B, HTC cultures, and panel C, T. brucei transformed with mouse ODC cDNA ( Tb/MOD) were incubated
in the absence or presence of either 5 mM putrescine
( putr.) or 5 mM spermidine ( sper.) for the
indicated time and lysed for ODC activity
assays.
Trypanosomal ODC Is Stable in Polyamine-treated T.
brucei
In mammalian cells and Xenopus, the
synthesis of an inhibitory protein antizyme can be induced by the
presence of polyamines
(9, 17, 36) . Antizyme
reduces ODC activity dramatically and destabilizes ODC protein in
mammalian cells
(15, 16) . Antizyme was also shown to
interact with mouse ODC protein in vitro, inhibit ODC
activity, and accelerate its degradation
(14, 18, 30) . In the above section, we observed
no ODC activity change in the polyamine-treated T. brucei.
Stable activity may result from the balance of both increased
degradation and increased synthesis of trypanosomal ODC in the treated
T. brucei. We therefore examined directly the stability of ODC
protein in the treated trypanosomal cells. T. brucei cells
were pulse labeled with L-[S]cysteine
and then chased in medium supplemented with 5 mM putrescine.
The radiolabeled trypanosomal ODC was immunoprecipitated and analyzed
by SDS-PAGE and autoradiography. The 45-kDa T. brucei ODC
remained stable during the 6-h chase period (Fig. 2 A).
There was no obvious change in migration in SDS-PAGE of the ODC from
T. brucei samples treated with putrescine. On the other hand,
the radiolabeled mouse ODC protein was degraded very rapidly in HTC
cells upon incubation in the medium containing putrescine
(Fig. 2 B), as observed previously
(16) . Slot
blot and Northern hybridizations of T. brucei poly(A
) RNA with a rat antizyme cDNA probe
provided no indication of the presence of T. brucei transcripts homologous to the probe (see Fig. 4). These
results suggest that antizyme is absent in T. brucei.
Figure 2:
L-[S]Cysteine
pulse-chase and immunoprecipitation. T. brucei cells
( panel A) or mouse HTC cells ( panel B) were pulse
labeled with [
S]cysteine as described under
``Experimental Procedures'' and chased for the indicated time
period with nonradioactive cysteine and in the presence of 5
mM putrescine. The cells were lysed, and T. brucei ODC ( panel A) and mouse ODC ( panel B) were
immunoprecipitated with their respective antisera. The proteins were
then analyzed by SDS-PAGE and autoradiography. Lane m in
panel B is the in vitro translated product of mouse
ODC. The arrowhead in panel A indicates the band of
T. brucei ODC; the arrow in panel B points
the mouse ODC.
Figure 4:
Northern analysis of rat antizyme
expressed in T. brucei. Panel A, poly(A)RNA (0.5 µg) isolated from T. brucei stable
transformants was separated on a denaturing agarose gel, transferred
onto a nitrocellulose membrane, and hybridized with the rat antizyme
cDNA probe. Lane 1, Tb/TSA-Hyg2; lane 2,
Tb/AZ; lane 3, Tb/TOD-AZ; lane 4,
Tb/MOD-AZ. The probe was washed off the blot, and the same
blot was hybridized with T. brucei
-tubulin probe
afterward ( panel B).
Expression of Rat Antizyme in T.
brucei
We have previously expressed mouse ODC in its native
form in T. brucei by DNA transformation
(13) (the
transformant is herein designated Tb/MOD). Mouse ODC isolated
from Tb/MOD possesses all of the in vitro biochemical
properties identified in native mouse ODC, but it has little turnover
in the trypanosomal cells
(13) . In the present investigation,
the Tb/MOD cells were incubated with polyamines (putrescine
and spermidine) and assayed for ODC activity. Polyamine treatment
caused no detectable change of the mouse ODC activity
(Fig. 1 C), thus providing further evidence that T.
brucei lacks an antizyme responsive to polyamines. To investigate
whether this stability of mouse ODC in T. brucei cells was
indeed solely due to the absence of antizyme in T. brucei, we
transformed the T. brucei procyclic cells with rat antizyme
cDNA in vector pTSA-Hyg2 (resulting in transformant
Tb/AZ) and selected for stable transformants in the presence
of hygromycin B. Similarly, each of the two stable transformants
Tb/MOD and Tb/TOD which overexpress mouse ODC and
T. brucei ODC, respectively, under the control of a different
vector, pTSA-Neo2
(13) , was also transformed with rat
antizyme cDNA in the pTSA-Hyg2 vector and selected under the
double pressure of G418 and hygromycin B. The two stable double
transformants were designated Tb/MOD-AZ and
Tb/TOD-AZ, respectively. As controls, wild type T.
brucei, Tb/MOD and Tb/TOD were each transformed
with the pTSA-Hyg2 vector without any inserts, generating
Tb/TSA, Tb/MOD-TSA, and Tb/TOD-TSA,
respectively. All of the transformants were then assayed for ODC
activity in the cell lysates. As shown in Fig. 3, the rat
antizyme caused a reduction of total mouse ODC activity by 20-25%
in Tb/MOD-AZ compared with the activities from Tb/MOD
and Tb/MOD-TSA. However, the antizyme had no apparent effects
on the trypanosomal ODC activities (compare Tb/TOD,
Tb/TOD-TSA, and Tb/TOD-AZ in Fig. 3). Southern
analyses (data not shown) indicated that the antizyme cDNA has been
successfully integrated into the T. brucei genome in all of
the stable transformants. Northern hybridizations (Fig. 4)
demonstrated that in each case the integrated antizyme DNA was
transcribed in the poly(A) RNA form at the expected
size, presumably correctly processed. Furthermore, Western blot
analysis confirmed that the antizyme protein was successfully expressed
in the transformants and migrated at expected for a protein of its
size, 26.5 kDa (Fig. 5). To quantitate the level of protein
accumulation of mouse ODC expressed in Tb/MOD-AZ, we analyzed
Tb/MOD-AZ cell extracts by immunoblot, using purified native
mouse ODC as a standard. The signals on the immunoblots were
quantitated by densitometric analysis. The accumulation level of rat
antizyme expressed in Tb/MOD-AZ was similarly examined, using
the purified Escherichia coli-expressed rat antizyme protein
as a standard. The results (not shown) revealed that mouse ODC protein
accumulates at approximately 1.7 pmol/10
cells, whereas
antizyme is present at approximately 1.5 pmol/10
cells.
Thus, rat antizyme was successfully expressed in T. brucei,
and it partially reduced mouse ODC activity in the trypanosomal cells.
Figure 3:
ODC activities of T. brucei and
its stable transformants. Tb, T. brucei;
TSA, Tb/TSA-Hyg2 (vector) transformant; AZ,
Tb/AZ transformant (overexpressing rat antizyme);
TOD, Tb/TOD transformant (overexpressing trypanosome
ODC); TOD-TSA, Tb/TOD-TSA double transformant;
TOD-AZ, Tb/TOD-AZ double transformant (overexpressing
both trypanosome ODC and rat antizyme); MOD, Tb/MOD
transformant (overexpressing mouse ODC); MOD-TSA,
Tb/MOD-TSA double transformant; MOD-AZ,
Tb/MOD-AZ double transformant (overexpressing both mouse ODC
and rat antizyme).
Figure 5:
Immunostaining of rat antizyme expressed
in T. brucei. Panel A, cell lysates from 1
10
cells each were separated by SDS-PAGE and immunostained
with the rabbit antiserum against glutathione
S-transferase-rat antizyme fusion protein. From left to right, first lane, T. brucei ( Tb); second lane, Tb/TSA-Hyg2
( Tb/TSA); third lane, Tb/AZ; fourth
lane, Tb/TOD; fifth lane, Tb/TOD-TSA;
sixth lane, Tb/TOD-AZ; seventh lane,
Tb/MOD; eighth lane, Tb/MOD-TSA; ninth
lane, Tb/MOD-AZ. Panel B, as a control, cell
lysates from 1
10
cells each were immunostained
with mouse monoclonal antibody against chicken
-tubulin. The
upper arrowhead in panel A indicates the nonspecific
signals, and the lower arrowhead indicates the antizyme
band.
Rat Antizyme Is Associated with Mouse ODC but Does
Not Promote Its Degradation in T. brucei
To distinguish
whether inhibition or degradation accounts for capacity of antizyme to
reduce mouse ODC activity, we first determined whether the two proteins
are associated in trypanosomal cells. We labeled cells expressing these
proteins with [S]cysteine and immunoprecipitated
with antibodies directed against antizyme or against ODC. As shown in
Fig. 6A, antiserum against rat antizyme
coimmunoprecipitated mouse ODC along with antizyme from the lysate of
Tb/MOD-AZ. Similarly, antiserum against mouse ODC precipitated
rat antizyme as well as mouse ODC. Densitometric analysis revealed that
approximately 20-25% of the total mouse ODC was coprecipitated
from the Tb/MOD-AZ lysate by the antiserum against rat
antizyme, and about 20-25% of the total rat antizyme was
coprecipitated by the antiserum against mouse ODC, in agreement with
the previously observed extent of inhibition of mouse ODC activity in
the same lysate (Fig. 3). Furthermore, densitometry also
indicated that the autoradiographic signal of MOD is approximately
3-fold stronger than that of AZ. Because mouse ODC contains 12 cysteine
residues
(40) , whereas AZ contains only 4 cysteine residues
(29) , this implies that both MOD and AZ are expressed at an
equal molar ratio in Tb/MOD-AZ. However, the antiserum against
trypanosomal ODC did not coprecipitate any detectable antizyme, nor did
the antiserum against antizyme coprecipitate any trypanosomal ODC from
the lysate of Tb/TOD-AZ (Fig. 6 A). These
results suggest that rat antizyme could bind to mouse ODC but not to
trypanosomal ODC in T. brucei cells, and this association
probably led to the inhibition of mouse ODC activity.
Figure 6:
Rat
antizyme is associated with mouse ODC but does not promote its
degradation in T. brucei. Panel A, T. brucei cells (2 10
) were labeled with
L-[
S]cysteine, lysed, and
immunoprecipitated with antisera. The immunoprecipitants were separated
by SDS-PAGE and visualized by autoradiography. The cell lysates of
different transformants were immunoprecipitated with either the
antiserum against glutathione S-transferase-rat antizyme
fusion protein (
-A) or the antiserum against mouse ODC
(
-M), or the antiserum against T. brucei ODC
(
-T). The arrowhead on the left indicates the nonspecific cross-reaction. Panel B,
pulse-chase experiment. Tb/MOD-AZ cells were pulse labeled
with [
S]cysteine, chased with nonradioactive
cysteine for the indicated time period, and lysed. The lysates were
then immunoprecipitated with antiserum against mouse ODC and analyzed
by SDS-PAGE and autoradiography. The arrowhead on the
right indicates the mouse ODC band.
On the other
hand, when the cells of the trypanosomal transformant containing both
mouse ODC and antizyme ( Tb/MOD-AZ) were subjected to
[S]cysteine pulse-chase experiment followed by
immunoprecipitation with antiserum against mouse ODC, mouse ODC was
seen to be very stable (Fig. 6 B). No sign of degradation
of mouse ODC was observed during an extended 6-h chase period. These
results indicate that although the rat antizyme is associated with
mouse ODC and causes the reduction of its activity in T.
brucei, this association does not promote degradation of mouse ODC
in the trypanosomal cells, in contrast to the results observed in
mammalian cells
(18) .
In Vitro Inhibition And Degradation of Mouse ODC
Initiated by the Rat Antizyme Expressed in
Trypanosomes
Since in vitro incubation of rat
antizyme with mouse ODC can inhibit ODC activity and direct its
degradation
(14, 18) , we determined whether or not
antizyme expressed in T. brucei could do so as well. Purified
mouse ODC (0.5 unit) was mixed either with the antizyme from
Tb/AZ (1.5 10
cell lysate of
Tb/AZ) or with the in vitro translated antizyme
(approximately 0.25 unit of antizyme). The mixtures were incubated on
ice for 30 min and assayed for ODC activity. As shown in Fig. 7,
the antizyme from Tb/AZ was able to inhibit mouse ODC activity
in vitro (compare the columns 4 and 5). The
cell lysate from 1.5
10
cells of Tb/AZ
inhibited about 20% of the mouse ODC activity, i.e. it
contained about 0.1 unit of antizyme activity. Immunostaining of the
antizyme fractionated by SDS-PAGE and densitometric analysis of the
antizyme-specific bands (data not shown) indicated that approximately
30-fold more immunoreactive antizyme from Tb/AZ was needed to
achieve equal inhibitory effect compared with antizyme generated by
in vitro translation. This suggests that antizyme from the
trypanosomal cell lysate is much less active than that produced by
in vitro translation.
Figure 7:In vitro inhibitory effect of
rat antizyme expressed in T. brucei on mouse ODC. The purified
mouse ODC ( mODC) (approximately 0.5 unit) was mixed with
either rabbit reticulocyte lysate ( RRL) or about 0.25 unit of
the in vitro translated antizyme (18) ( iv AZ), or
lysates from 1.5 10
cells of either
pTSA-Hyg2 ( Tb/TSA) or pAZ ( Tb/AZ)
transformants. The mixtures were then assayed for ODC activity. The
endogenous trypanosomal ODC activities (0.5 unit) from Tb/TSA
and Tb/AZ have already been
subtracted.
We further examined the in vitro degradation of native mouse ODC catalyzed by various samples of
rat antizyme. A rabbit reticulocyte extract system was used as the
in vitro degradation system as before
(18) . Mouse ODC
was labeled with L-[S]methionine by
in vitro translation. The labeled mouse ODC was mixed with (i)
the in vitro translated antizyme; (ii) the trypanosomal cell
lysates from Tb/AZ; (iii) the lysates from untransformed
T. brucei; (iv) a combination of (i) and (ii); and (v) a
combination of (i) and (iii). The mixtures were then each added to the
rabbit reticulocyte lysate for the in vitro mouse ODC
degradation. Fig. 8 A demonstrates that mouse ODC is
degraded in the presence of the in vitro translated antizyme,
as observed previously
(18, 19) . However, the antizyme
expressed in Tb/AZ did not promote in vitro degradation of mouse ODC. Furthermore, the mouse ODC degradation
promoted by the in vitro translated antizyme was inhibited by
the cell lysate of untransformed T. brucei or that of
Tb/AZ (Fig. 8 A). These results suggest the
presence of some inhibitory factor(s) in the trypanosomal cell lysates,
which may interfere with the degradation of mouse ODC mediated by
antizyme.
Figure 8:In vitro degradation of mouse
ODC. Mouse ODC was labeled with [S]methionine
( *m) by in vitro translation (18), mixed with the
in vitro translated antizyme ( ivAZ) and/or T.
brucei cell lysates (1.5
10
cells), followed
by the addition of rabbit reticulocyte extract and an ATP-regenerating
system (18). After incubation at 37 °C for 1 h, the mixtures were
analyzed by SDS-PAGE and autoradiography. Panel A, from
left to right: first lane,
S-labeled mouse ODC ( *m); second lane,
labeled mouse ODC plus in vitro translated antizyme
( ivAZ); third lane, mouse ODC plus the lysate of
T. brucei; fourth lane, mouse ODC plus the lysate of
Tb/AZ; fifth lane, mouse ODC plus in vitro translated antizyme and the lysate of T. brucei;
sixth lane, mouse ODC plus in vitro translated
antizyme and the lysate of Tb/AZ. Panel B, T.
brucei cell lysates were centrifuged (16,000
g),
and the unboiled and boiled supernatant ( s) and pellet
( p) fractions were added to the in vitro degradation
system and analyzed by SDS-PAGE and
autoradiography.
To characterize further the property of the degradation
inhibitory factor(s), sonicated trypanosome cell lysate was centrifuged
at 16,000 g for 30 min. The supernatant and the pellet
after resuspension were added to the above mentioned in vitro degradation system, or both fractions were boiled for 5 min before
being added to the in vitro degradation system. The results in
Fig. 8B indicated that this inhibitory factor(s) is in
the soluble fraction and heat-labile.
Identification of Proteasomes in T.
brucei
Mouse ODC is degraded by the 26 S proteasome in the
presence of antizyme in vitro (19, 41, 42) . We therefore determined
whether proteasomes are present in trypanosomal cells. Trypanosomal
cells and rabbit blood cells were each fractionated by a method used
previously for mammalian proteasome purification, essentially as
described
(35) . The resulting preparations were each subjected
to electrophoresis on a native gel, and the protease activity was
detected by overlaying the gel with a mixture of the fluorogenic
peptides Pro-Phe-Arg-MCA and Suc-Leu-Leu-Val-Tyr-MCA. The expected two
bands were present in the mammalian derived preparation, corresponding
to the 26 S and 20 S proteasomes (Fig. 9 A). The
corresponding preparation from T. brucei also showed two
distinct bands of activity, but both bands migrated faster than the
rabbit 26 S and 20 S proteasomes. One of these two bands (see band
a in Fig. 9 A) migrated slightly faster than the
rabbit 20 S proteasome, and there is a protein band observed on the
Coomassie Blue-stained gel corresponding to this activity
(Fig. 9 B). However, the major proteolytic activity
( band b in Fig. 9 A) from T. brucei migrated much faster than the others. We then excised the 20 S
proteasome band of rabbit and activity bands a and b of trypanosome from the nondenaturing gel. They were subjected to
SDS-PAGE and immunostained with the antibodies against the human 20 S
proteasome
(35) . As shown in Fig. 9 C, the
antibodies cross-reacted with the proteins of activity band a from T. brucei by reacting strongly with a protein band
at 48 kDa and somewhat weakly with two other protein bands at 51 and
106 kDa, respectively. These sizes are much larger than those from the
rabbit 20 S proteasome (24-32 kDa) recognized by the same
antibodies
(35) . It is yet to be determined whether these
T. brucei proteins on the immunostaining blot represent the
genuine subunits of the proteasome in trypanosome. On the other hand,
no significant cross-reacting signal was observed from the activity
band b on the same immunoblot, suggesting that the proteins in
this activity band are sufficiently distinct from those in the
mammalian 20 S proteasome. It is presently unclear whether the activity
band b represent a distinctive form of T. brucei proteasome or a contaminating protease activity cosedimented at
100,000 g with the proteasome. We thus conclude that a
proteasome is present in T. brucei. It has properties
distinctive from those of the mammalian proteasomes, which may
constitute part of the reasons why mouse ODC cannot be degraded inside
T. brucei even in the presence of rat antizyme.
Figure 9:
Identification of proteasome in T.
brucei. Proteasomes were isolated from rabbit blood cells
( Rb) and T. brucei cells ( Tb) as described
under ``Experimental Procedures.'' The proteasome
preparations were separated on a native gel, and the protease
activities were detected with a mixture of fluorogenic peptides
Suc-Leu-Leu-Val-Tyr-MCA and Pro-Phe-Arg-MCA ( panel A). The
same gel was stained with Coomassie Brilliant Blue ( panel B).
Or, the activity bands (20 S of rabbit, and a and b of T. brucei) were excised and subjected to SDS-PAGE and
immunostaining with antibodies against the human 20 S proteasome
( panel C).
(
)
Two lines of recent
evidence indicated that the chymotryptic activities of proteasomes are
primarily responsible for the major proteolysis of ODC. Mamroud-Kidron
et al. (45) showed that in a mutant yeast cell line
defective in the chymotryptic activity of proteasome, degradation of
mouse ODC and yeast ODC was severely inhibited, whereas in wild type
yeast cells, both proteins were rapidly degraded. Sequence analysis of
peptides cleaved by the mammalian 26 S proteasome in vitro (42) revealed that chymotryptic-like activity of 26 S proteasome
is predominant in the endoproteolysis of ODC, although other type of
activities are also present. These differences between T. brucei and mammalian proteasomes may thus contribute to the stability of
mouse ODC in T. brucei, in addition to the inhibitory
factor(s) discussed above.
family
to form important regulator of the cell cycle. The proteasomes may be
involved in the degradation of mitotic cyclins. For example, mutations
in a proteasome-related gene in yeast cause defects in nuclear
division. These defects can be suppressed by deletion of a mitotic
cyclin gene
(47) . Recently, a mitotic cyclin gene has also been
identified and isolated from T. brucei (48) . Its
protein abundance increases during the mitotic phase and decreases
during the S phase of the cell cycle in the procyclic form of T.
brucei (48) , presumably because of degradation of this
cyclin protein by the cell cycle-regulated proteolysis, namely, the
proteasomes. Furthermore, certain proteins in T. brucei are
known to turn over at different rates during individual phases of its
life cycle. For example, the half-life of cytochrome c is
approximately 1 h in the bloodstream form, but no detectable turnover
of this protein occurs in the procyclic form
(49) . Whether the
proteasomes in the bloodstream forms differ from those in the procyclic
forms by their specific modes of protein degradation could constitute
an interesting subject for further exploration into possible
regulations of the life cycle of T. brucei by alterations of
the proteasome functions.
-DL-difluoromethylornithine; HTC,
hepatoma cells; PAGE, polyacrylamide gel electrophoresis; Suc,
succinyl; MCA, 4-methyl-coumaryl-7-amide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.