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J Biol Chem, Vol. 275, Issue 17, 13109-13117, April 28, 2000
Degradation of DNA Topoisomerase I by a Novel Trypsin-like Serine
Protease in Proliferating Human T Lymphocytes*
Hui-Jye
Chen §,
Ching-Long
Hwong§,
Cheng-Hsu
Wang¶, and
Jaulang
Hwang§
From the Institute of Biochemistry, School of Life
Science, National Yang-Ming University, Taipei 112, Taiwan, the
§ Institute of Molecular Biology, Academia Sinica, Taipei
115, Taiwan, and the ¶ Division of Hematology-Oncology, Department
of Internal Medicine, Chang Gung Memorial Hospital,
Taipei 105, Taiwan
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ABSTRACT |
DNA topoisomerase I (Topo I) contributes to
various important biological functions, and its activity is therefore
likely regulated in response to different physiological conditions.
Increases in both the synthesis and degradation of Topo I were
previously shown to accompany phytohemagglutinin stimulation of
proliferation in human peripheral T lymphocytes. The mechanism of this
degradation of Topo I has now been investigated with both in
vivo and in vitro assays. The activity of a nuclear
protease that specifically degrades Topo I was induced in proliferating
T lymphocytes. The full-length Topo I protein (100 kDa) was
sequentially degraded to 97- and 82-kDa fragments both in
vivo and in vitro. The initial site of proteolytic
cleavage was mapped to the NH2-terminal region of the
enzyme. The degradation of Topo I in vitro was inhibited by aprotinin or soybean trypsin inhibitor, suggesting that the enzyme responsible is a trypsin-like serine protease. Furthermore, Topo I
degradation by this protease was
Mg2+-dependent. The Topo I-specific protease
activity induced during T lymphocytes proliferation was not detected in
Jurkat (human T cell leukemia) cells and various other tested human
cancer cell lines, possibly explaining why the abundance of Topo I is
increased in tumor cells.
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INTRODUCTION |
The superhelical state of DNA is an important determinant of DNA
structure and function. The enzyme DNA topoisomerase I (Topo I)1 regulates DNA topology
and is evolutionarily conserved in both mode of action and amino acid
sequence from yeast to mammals. Topo I relaxes DNA supercoils by
cleaving a single strand of duplex DNA and passing the complementary
DNA strand through the cleaved strand before religation (1).
Biochemical and genetic studies have shown that Topo I plays important
roles in DNA replication (2, 3), RNA transcription (4-6), DNA
recombination (7-10), chromosome condensation (11-13), and the
maintenance of genomic stability (1, 14). During DNA replication, Topo
I removes the positive supercoils that accumulate in the unreplicated
portion of topologically restrained DNA as a result of the unwinding of
the double helix. Topo I also acts as a swivel to release the torsional
strain in DNA during RNA transcription. The enzyme contributes to
illegitimate recombination between a cleavage complex and an exogenous
DNA strand bearing a 5'-hydroxyl end. Moreover, Topo I regulates the
initiation of transcription through direct interaction with
transcription factors (15-17), and it catalyzes the phosphorylation of
SR proteins, which are essential for RNA splicing (18).
Because Topo I participates in multiple biological functions, its
expression is likely strictly regulated under various physiological conditions. Indeed, deregulation of Topo I results in fatal defects in
cells. Thus, overexpression of yeast Topo I in Saccharomyces cerevisiae resulted in a 6-12-fold increase in the frequency of illegitimate recombination (19). Overexpression of human or yeast Topo
I in Escherichia coli or of mammalian Topo I in mammalian cells resulted in cell death (20-22). The abundance of human Topo I
protein is increased in tumors of the colon (23, 24), ovary (25),
prostate (24), lung (26), esophagus (27), or acute myelogenous and
acute lymphocytic leukemia (28), compared with that in the
corresponding normal tissues. These observations also suggest that it
is important for cells to maintain a normal level of Topo I protein.
The cellular concentration of a protein is determined by both its rate
of synthesis and its rate of degradation. Control of protein stability
is becoming a critical point for modulating gene expression, and the
degradation of various proteins is known to be regulated. Thus, in
eukaryotes, mitotic cyclins, G1 cyclin, and Cdk inhibitors
are degraded at specific times during the cell cycle (29); proteolysis
of p53 and c-Jun contributes to the control of cell growth and
proliferation (29); a plant phytochrome is degraded after exposure to
red light (30); and the rate of degradation of several important
biosynthetic enzymes is increased in the presence of their end products
(31, 32). In prokaryotes, the heat shock sigma factor, HtpR, is
transiently stabilized in response to an increase in temperature (33).
However, little is known about regulation of the stability of Topo I protein.
We have previously shown that PHA-induced T cell proliferation is
accompanied by increases in both the abundance of Topo I mRNA and
the synthesis of Topo I protein. However, we also detected a
concomitant rapid degradation of Topo I under these same conditions (34). In the present study, we examined the mechanism of this rapid
degradation of Topo I protein in PHA-stimulated proliferating T cells.
We demonstrate that the degradation of Topo I is mediated by a nuclear
trypsin-like serine protease and that the initial site of cleavage by
this protease is located in the NH2-terminal region of Topo
I. Our results also suggest that the abundance of Topo I is increased
in tumor cells as a result of lacking the trypsin-like serine protease
activity in the nucleus of these cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Lactacystin was kindly provided by Dr. E. J. Corey (Department of Chemistry, Harvard University). Aprotinin,
leupeptin, pepstatin, calpain I inhibitor
(N-acetyl-Leu-Leu-norleucinal), calpain II inhibitor
(N-acetyl-Leu-Leu-methioninal), and E64
(N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine) were from Roche Molecular Biochemicals. All cell culture reagents were
from Life Technologies, Inc. Reagents and apparatus for gel electrophoresis were from Bio-Rad. Nitroblue tetrazolium,
5-bromo-4-chloro-3-indolyl phosphate, and alkaline
phosphatase-conjugated goat antibodies to rabbit IgG for immunoblot
analysis were from Promega. Rabbit antibodies to glutathione
S-transferase (GST) were from Santa Cruz Biotechnology, and
horseradish peroxidase-conjugated goat antibodies to rabbit IgG were
from Southern Biotechnology Associates. All other chemicals were of
analytical grade and from Sigma.
Cells and Culture Conditions--
Human blood was obtained from
healthy donors and mixed with heparin sulfate (20 units/ml).
Lymphocytes were isolated from the heparinized blood by density
gradient centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech)
and were cultured at a density of 2 × 106 cells/ml in
RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin
(50 IU/ml), streptomycin (50 µg/ml), and 1 mM glutamine.
The peripheral resting T lymphocytes were stimulated into proliferation
by incubation in the presence of PHA (100 µg/ml) (Wellcome
Diagnostics) at 37 °C. Jurkat cells (human T cell leukemia cell
line), A2780 cells (human ovarian cell line), U937 cells (human
monoblastic cell line), HL60 cells (human leukemia cell line), K562
cells (human erythroleukemia cell line), and Epstein-Barr virus-transformed B lymphocytes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. H460 cells (human non-small cell lung carcinoma cell line), A431 cells (human epidermoid cell line), Saos-2 cells (human osteosarcoma cell line), KB cells (human epidermoid carcinoma cell line), HepG2 cells (human hepatoblastoma cell
line), and HeLa cells (human cervical carcinoma cell line) were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. All cells were maintained under an atmosphere of
5% CO2 and 95% air at 37 °C.
Expression Constructs and in Vitro Translation--
Human Topo I
cDNA was subcloned into the pET-22b plasmid (Novagen), and the
resulting construct was subjected to in vitro transcription
and translation with TNT wheat germ extract (Promega) in the presence
of [35S]methionine (Amersham Pharmacia Biotech). The
translation products were used directly in the in vitro
assay of Topo I degradation (see below). Various regions of human Topo
I cDNA, generated either by the polymerase chain reaction or by
restriction enzyme digestion, were also subcloned into the plasmid
pGEX-KG (P-L Biochemicals) for expression of the encoded Topo I
fragments as GST fusion proteins. Some of the resulting constructs
(GST, GTOPI-4, GTOPI-6, and GTOPI-7) were subjected to in
vitro transcription and translation with an E. coli T7
S30 extract (Promega) in the presence of [35S]methionine.
The other GST-Topo I constructs were introduced into E. coli
BL21 (DE3), and expression of the fusion proteins was induced by
isopropyl- -D-thiogalactopyranoside. The fusion proteins
were purified with the use of glutathione-agarose affinity columns.
Both the fusion proteins produced by in vitro translation, and those purified with the affinity columns were used for the in
vitro assay of Topo I degradation.
Preparation of Cytosolic and Nuclear Extracts--
Cells were
fractionated according to a modified version of the method of Dyer and
Herzog (35). Cells (1~5 × 107) were collected by
centrifugation at 200 × g for 5 min at 4 °C, washed
with phosphate-buffer saline, and lysed by suspension in 100 µl of
sucrose buffer I (0.32 M sucrose, 3 mM
CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT, 0.5% (v/v) Nonidet P-40). The lysate was
centrifuged at 500 × g for 5 min at 4 °C to pellet
nuclei, and the resulting supernatant was transferred to a fresh tube,
mixed with 0.22 volume of 5× cytoplasmic extraction buffer (150 mM Hepes-NaOH, pH 7.9, 0.7 M KCl, 15 mM MgCl2), and centrifuged at 12,000 × g for 15 min at 4 °C. The resulting supernatant (cytosolic extract) was transferred to a fresh tube, into which glycerol was added to a final concentration of 25% (v/v), and the
mixture was divided into portions and stored at  70 °C. The nuclear
pellet was washed with sucrose buffer I lacking Nonidet P-40, with
recentrifugation at 500 × g for 5 min at 4 °C. The resulting supernatant was removed, and the nuclei were resuspended in
20 µl of low salt buffer (20 mM Hepes-NaOH, pH 7.9, 25%
glycerol, 1.5 mM MgCl2, 20 mM KCl,
0.5 mM DTT). After the addition of 20 µl of high salt
buffer (20 mM Hepes-NaOH, pH 7.9, 1.5 mM
MgCl2, 0.8 M KCl, 0.2 mM EDTA, 1%
Nonidet P-40, 0.5 mM DTT) to lyse the nuclei, the mixture
was incubated on a rotary platform at 4 °C for 20 min, diluted 1:2.5
with diluent (25 mM Hepes-NaOH, pH 7.6, 25% glycerol, 0.1 mM EDTA, 0.5 mM DTT), and centrifuged at
12,000 × g for 15 min at 4 °C. The resulting
supernatant (nuclear extract) was divided into portions and stored at
70 °C.
In Vitro Assay of Topo I Degradation--
5 µl of the
[35S]methionine-labeled in vitro translated
proteins or 250 ng of the purified GST-Topo I fusion proteins were
incubated for various times at 37 °C in a final volume of 30 µl
with nuclear or cytosolic extracts (10 µg of protein) isolated from
proliferating (resting T lymphocytes stimulated with PHA for 72 h
at 37 °C) or resting T lymphocytes in the incubation buffer (20 mM Hepes-NaOH, pH 8.0). After the addition of Laemmli
sample buffer, the mixture was boiled for 5 min and then fractionated
by SDS-polyacrylamide gel electrophoresis on a 10 or 12.5% gel. The
gel was subjected to fluorography with 2,5-diphenyloxazol to detect the
in vitro translated 35S-labeled proteins. The
degradation of the purified GST fusion proteins was detected by
immunoblot analysis.
Immunoblot Analysis--
At various times after stimulation with
PHA, lymphocytes were harvested and lysed in an ice-cold solution
containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 8.3 µM aprotinin, 50 µM leupeptin, 30 mM sodium pyrophosphate, 50 mM NaF, 100 mM Na3VO3, 150 mM NaCl,
1% Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM EDTA, and 50 mM Tris-HCl, pH 8.0. The
mixture was incubated on a rotary platform at 4 °C for 20 min and
centrifuged at 26,000 × g for 30 min at 4 °C. A
portion (100 µg of protein) of each cell lysate was mixed with
Laemmli sample buffer, boiled for 5 min, and fractionated by
SDS-polyacrylamide gel electrophoresis on a 7.5% gel. The separated
proteins were transferred to a polyvinylidene difluoride membrane
(Millipore), which was then incubated for 2 h at room temperature
in TBS buffer (125 mM NaCl, 25 mM Tris-HCl, pH
8.0) containing 1.5% (w/v) nonfat dried milk. The membrane was then
incubated for 90 min with rabbit antiserum to human DNA Topo I (1:5000
dilution) (34) in TBST buffer (125 mM NaCl, 0.05% (v/v)
Tween 20, 25 mM Tris-HCl, pH 8.0), washed three times with TBST buffer, and incubated for an additional 30 min with alkaline phosphatase-conjugated goat antibodies to rabbit IgG. After another three washes in TBS buffer, the membrane was incubated for ~5 min
with a solution containing nitroblue tetrazolium (0.33 mg/ml) and
5-bromo-4-chloro-3-indolyl phosphate (0.165 mg/ml) to allow visualization of immune complexes.
The in vitro cleavage of GST-Topo I fusion proteins
(GTOPI-1, -2, -3, -5, -8, and -9) was detected by immunoblot analysis with rabbit antibodies to GST and horseradish peroxidase-conjugated goat antibodies to rabbit IgG; immune complexes were visualized by
enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
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RESULTS |
Induction of Nuclear Protease Activity Specific for Topo I by PHA
in Peripheral T Lymphocytes--
Our previous observation that Topo I
is degraded rapidly during PHA stimulation of T lymphocyte
proliferation (34) led us to propose that PHA may induce a protease
activity that is responsible for this degradation. To investigate this
proposal, we first harvested human T lymphocytes at various times after
PHA stimulation and examined the degradation of Topo I by immunoblot
analysis. The amount of Topo I gradually increased with time of
exposure to PHA, and a partial degradation of Topo I into two
smaller immunoreactive fragments was first detected 12 h after PHA
stimulation and increased thereafter (Fig.
1), indicating that a protease activity
was indeed induced in the proliferating T cells.
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Fig. 1.
Proteolysis of Topo I in PHA-stimulated human
T lymphocytes. T cells were incubated with PHA for the indicated
times, harvested, and resuspended in lysis buffer. Cell lysates (100 µg of protein) were subjected to immunoblot analysis with antiserum
to human Topo I. The positions of Topo I and of the two predominant
degradation products are indicated.
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To characterize the proteolytic activity responsible for the
degradation of Topo I, we developed an in vitro degradation
assay. We produced [35S]methionine-labeled human Topo I
by in vitro transcription and translation and incubated the
labeled protein with nuclear or cytosolic extracts isolated from either
resting or proliferating T cells. The 35S-labeled Topo I
(100 kDa) was degraded in a time-dependent manner in the
presence of cytosolic or nuclear extracts from either resting (Fig.
2A) or proliferating (Fig.
3A) T cells. However, only
nuclear extract from proliferating T cells generated a pattern of Topo I degradation similar to that observed in intact cells (Figs. 1 and
3A), characterized by two predominant proteolytic
intermediates of 97 and 82 kDa that appeared to be produced
sequentially. Whereas a mixture of protease inhibitors (aprotinin (5.8 µM), leupeptin (42 µM), pepstatin (1.46 µM), and PMSF (574 µM)) inhibited the degradation of Topo I in vitro by nuclear or cytosolic
extracts from resting T cells (Fig. 2B) or by cytosolic
extract of proliferating T cells (Fig. 3B), these inhibitors
had no effect on Topo I degradation by nuclear extract of proliferating
T cells (Fig. 3B), suggesting that this latter proteolysis
is specific.
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Fig. 2.
Degradation of Topo I by cytosolic or nuclear
extracts of resting T lymphocytes in vitro.
[35S]Methionine-labeled Topo I was incubated for the
indicated times at 37 °C with cytosolic (RCE) or nuclear
(RNE) extracts of resting T cells either in the absence
(A) or presence (B) of a mixture of protease
inhibitors (aprotinin (5.8 µM), leupeptin (42 µM), pepstatin (1.46 µM), and PMSF (574 µM)). The reaction mixture was then analyzed by
electrophoresis and fluorography. The uppermost labeled band
corresponds to full-length Topo I.
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Fig. 3.
Degradation of Topo I by cytosolic or nuclear
extracts of proliferating T lymphocytes.
[35S]Methionine-labeled Topo I was incubated for the
indicated times at 37 °C with cytosolic (PCE) or nuclear
(PNE) extracts of proliferating T lymphocytes either in the
absence (A) or presence (B) of protease
inhibitors as described in the legend to Fig. 2. The positions of Topo
I and of the predominant 97- and 82-kDa fragments of Topo I are
indicated.
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The 82-kDa processing intermediate was more prominent in the in
vitro assay of Topo I degradation by nuclear extract of
proliferating T cells (Fig. 3) than in the in vivo assay of
Topo I degradation (Fig. 1). To investigate whether this difference
might be due to an effect of temperature, given that the in
vivo assay was performed at 4 °C and the in vitro
assay was performed at 37 °C, we compared the patterns of in
vitro degradation of Topo I at 37, 25, 15, and 4 °C. Although
Topo I was degraded at all tested temperatures, the rate of proteolysis
increased with increasing temperature, and the 82-kDa product was less
evident at 4 °C than at 37 °C (Fig.
4). The in vitro degradation
pattern at 4 °C was thus most similar to the in vivo
pattern, suggesting that the degradation of Topo I into 97- and 82-kDa
fragments is mediated by the same proteolytic activity both in
vivo and in vitro.
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Fig. 4.
Temperature dependence of Topo I degradation
in vitro. [35S]Methionine-labeled
Topo I was incubated for 5 min at the indicated temperatures in the
presence of protease inhibitors (aprotinin, leupeptin, pepstatin, and
PMSF) and in the absence or presence of nuclear extract from
proliferating T cells. Topo I degradation was then analyzed as
described in the legend to Fig. 2. PNE, nuclear extracts of
proliferating T lymphocytes.
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To determine whether this proteolytic activity degrades other proteins,
we incubated two nuclear proteins, the Cdk inhibitor p27 and the tumor
suppressor p53 (Fig. 5), as well as
bacterial GST (see Fig. 11A) with nuclear extract of
proliferating T lymphocytes. None of these three proteins was degraded
under conditions similar to those of the Topo I degradation assay. The
protease activity thus appears to be relatively specific for Topo
I.
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Fig. 5.
Resistance of p53 and p27 to degradation by
nuclear extract of proliferating T lymphocytes.
[35S]Methionine-labeled p53 and p27 proteins were
incubated for 5 min at 37 °C in the presence of protease inhibitors
(aprotinin, leupeptin, pepstatin, and PMSF) and in the absence or
presence of nuclear extract of proliferating T cells. The integrity of
the two proteins was then analyzed as described in the legend to Fig.
2. The leftmost lane contains molecular size standards.
PNE, nuclear extracts of proliferating T lymphocytes.
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Characterization of the Proteolytic Activity Responsible for the
Degradation of Topo I--
Protein turnover in mammalian cells has
been shown to be mediated by three distinct pathways (36): the
ubiquitin-26 S proteasome pathway, which requires ATP, and the
lysosomal and Ca2+-dependent pathways, which
are ATP-independent. To determine whether Topo I is degraded by the
ubiquitin-26 S proteasome pathway, we therefore examined the effects of
the ATP analog ATP- -S, of depletion of ATP by apyrase, and of the 26 S proteasome inhibitors hemin and lactacystin on the degradation of
Topo I by nuclear extract of proliferating T cells. The degradation of
the 97-kDa Topo I fragment to the 82-kDa fragment was partially
inhibited by 1 mM ATP--S (Fig.
6A), and that of the
full-length protein to the 97-kDa fragment was partially blocked at 10 mM ATP--S and was completely inhibited at 40 mM ATP--S (Fig. 6B). These data thus suggested that the degradation of Topo I may be
ATP-dependent. However, depletion of ATP by apyrase failed
to block Topo I degradation (Fig.
7A). To resolve these
apparently inconsistent results, we examined the effect of ATP on Topo
I proteolysis. The degradation of Topo I was inhibited by ATP in a
concentration-dependent manner (Fig. 6C),
similar to the effect of ATP--S (Fig. 6B). As a control, we examined the effect of cAMP; this nucleotide had no effect on the
degradation of Topo I (data not shown).
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Fig. 6.
Effects of ATP--S
and ATP on the degradation of Topo I in vitro.
[35S]Methionine-labeled Topo I was incubated for 5 min at
37 °C in the presence of the indicated concentrations of ATP--S
(A and B) or ATP (C) and in the
absence or presence of nuclear extract of proliferating T cells. The
degradation of Topo I was then analyzed as described in the legend to
Fig. 2. The leftmost lane in A contains molecular
size standards. PNE, nuclear extracts of proliferating T
lymphocytes.
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Fig. 7.
Effects of apyrase and lactacystin on the
degradation of Topo I in vitro. A,
[35S]methionine-labeled Topo I and nuclear extract of
proliferating T lymphocytes, each of which had been pretreated or not
with apyrase, were incubated in the indicated combinations with a
mixture of protease inhibitors (aprotinin, leupeptin, pepstatin, and
PMSF) for 5 min at 37 °C. B,
[35S]methionine-labeled Topo I was incubated for 5 min at
37 °C in the presence of the indicated concentrations of lactacystin
and in the absence or presence of nuclear extract of proliferating T
cells. Topo I degradation was analyzed as described in the legend to
Fig. 2. PNE, nuclear extracts of proliferating T
lymphocytes.
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We have previously shown that the binding of ATP to Topo I results in a
conformational change in the enzyme, whereas cAMP has no such effect
(37). Thus, the inhibition of Topo I degradation by ATP or ATP--S
may be attributable to such a conformational change that renders the
enzyme resistant to protease digestion. The conformation of Topo I may
thus be a determining factor of the stability of the protein. However,
we also cannot rule out the possibility that the inhibitory effect of
ATP or ATP--S on the Topo I proteolysis is due to a titration of
Mg2+, because Mg2+ is required for Topo I
proteolysis (see Fig. 9B). Our data indicate that ATP is not
required for the proteolysis of Topo I. Neither lactacystin (Fig.
7B) nor hemin (data not shown) inhibited the degradation of
Topo I, further indicating that Topo I proteolysis is not mediated by
the ATP-dependent 26 S proteasome pathway in proliferating
T lymphocytes.
The effects of E64 as well as of calpain I inhibitor and calpain II
inhibitor on Topo I degradation were examined to determine the roles of
the lysosomal and Ca2+-dependent proteolysis
systems, respectively, in this phenomenon. None of these three
inhibitors blocked the proteolysis of Topo I (data not shown).
Together, our data suggest that the degradation of Topo I is not
mediated by the ubiquitin-dependent 26 S proteasome, lysosomal, or Ca2+-dependent proteolytic pathways.
Role of a Trypsin-like Serine Protease in the Degradation of Topo
I--
Proteases can be classified into four types on the basis of
their active sites: serine proteases, cysteine proteases,
metalloproteases, and aspartic proteases. Proteases of each type are
inhibited by corresponding specific blockers. To characterize the type
of protease responsible for the degradation of Topo I, we examined the
effects of various protease inhibitors, including aprotinin, PMSF,
pepstatin, leupeptin, soybean trypsin inhibitor, EDTA (Fig.
8), N-ethylmaleimide, N- -tosyl-L-lysine chloromethyl ketone,
tosyl-L-phenylalanine chloromethyl ketone, chymostatin, and
caspase inhibitors (data not shown), on Topo I degradation by nuclear
extract of proliferating T cells. Of these various agents, only EDTA,
aprotinin (trypsin inhibitor, pancreas type, from bovine lung), and
soybean trypsin inhibitor exhibited an inhibitory effect on Topo I
degradation. The degradation of the 97-kDa fragment of Topo I to the
82-kDa fragment was inhibited by aprotinin at concentrations of 50 and 167 µM (Fig. 8B); however, aprotinin failed to
block the degradation of the full-length protein to the 97-kDa
intermediate. Soybean trypsin inhibitor blocked the degradation of
full-length Topo I in a concentration-dependent manner,
with complete inhibition of proteolysis apparent at a concentration of
150 µM (Fig. 8C). Taken together, these
results suggest that the protease responsible for Topo I degradation is
an EDTA-sensitive, trypsin-like serine protease.
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Fig. 8.
Effects of various protease inhibitors on the
degradation of Topo I in vitro.
[35S]Methionine-labeled Topo I was incubated for 5 min at
37 °C in the absence or presence of aprotinin, leupeptin, pepstatin,
EDTA, or PMSF at commonly used concentrations (A), aprotinin
or leupeptin at higher concentrations (B), or the indicated
concentrations of soybean trypsin inhibitor (C) and in the
absence or presence of nuclear extract of proliferating T cells, after
which Topo I degradation was analyzed as described in the legend to
Fig. 2. PNE, nuclear extracts of proliferating T
lymphocytes.
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Requirement of Mg2+ for Degradation of Topo I--
The
degradation of the 97-kDa fragment of Topo I to the 82-kDa fragment was
inhibited by 1 mM EDTA (Fig. 8A). Furthermore, degradation of the full-length enzyme to the 97-kDa intermediate was
inhibited in the presence of 2 mM EDTA (Fig.
9A). These observations suggested that Mg2+ or Ca2+ might be required
for Topo I proteolysis. We therefore investigated the effect of the
Ca2+-specific chelator EGTA and addition of
Ca2+ on this process. EGTA had no effect on the degradation
of Topo I at concentrations up to 2 mM (Fig.
9A), and the addition of various concentrations of
Ca2+ also did not stimulate proteolysis of Topo I (data not
shown), indicating that Ca2+ is dispensable for proteolysis
of Topo I. We further investigated the role of Mg2+ in Topo
I degradation by examining the effect of addition of various
concentrations of Mg2+ to the assay mixture in the presence
of 2 mM EDTA. The addition of Mg2+ reversed the
inhibitory effect of EDTA on the degradation of Topo I in a
concentration-dependent manner (Fig. 9B),
indicating that Mg2+ is required for Topo I
proteolysis.
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Fig. 9.
Effect of Mg2+ on the degradation
of Topo I in vitro. A, nuclear extract
of proliferating T lymphocytes was preincubated for 5 min at room
temperature with the indicated concentrations of EDTA or EGTA and was
then incubated for an additional 5 min at 37 °C with
[35S]methionine-labeled Topo I. B, nuclear
extract pretreated with 2 mM EDTA as in A was
incubated for 5 min at 37 °C with
[35S]methionine-labeled Topo I in the presence of the
indicated concentrations of Mg2+. The degradation of Topo I
was analyzed as described in the legend to Fig. 2. PNE,
nuclear extracts of proliferating T lymphocytes.
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Mapping of the Site of Proteolytic Cleavage in Topo I--
To
localize the site of proteolytic cleavage in Topo I, we constructed
recombinant GST fusion proteins containing various fragments of Topo I
(Fig. 10). The fusion proteins were
either expressed in E. coli and purified by glutathione
affinity chromatography or translated in vitro, and their
degradation was then examined with the in vitro assay. Only
the GTOPI-1 fusion protein (amino acids 1-138 of Topo I) was cleaved
by the nuclear extract of proliferating T lymphocytes (Fig.
11, A-D). To assess whether
the degradation of GTOPI-1 and that of full-length Topo I are likely
mediated by the same protease, we examined the effect of soybean
trypsin inhibitor on the proteolytic cleavage of GTOPI-1. The
degradation of GTOPI-1 was inhibited by soybean trypsin inhibitor in a
dose-dependent manner (Fig. 11E), suggesting
that the proteolysis of GTOPI-1 and that of full-length Topo I are
mediated by the same trypsin-like serine protease. The fusion protein
data indicate that the initial site of Topo I cleavage, which reduces
the molecular size of the protein from 100 to 97 kDa, is located within
the NH2-terminal 50 residues of the protein.
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Fig. 10.
Schematic representation of GST-Topo I
fusion proteins and summary of in vitro cleavage
data. The human Topo I cDNA, including the positions of the
initiation (ATG) and termination (TAG) codons as well as of restriction
enzyme sites, is shown above the representations of the various GTOPI
fusion proteins (with the solid bars corresponding to the
Topo I sequence in each). The results of the in vitro
cleavage assays for each of the fusion proteins (Fig. 11) are
summarized on the left, with a minus sign
representing no cleavage and a plus sign indicating
cleavage.
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Fig. 11.
Localization of the initial site of
proteolytic cleavage in Topo I. GST (A) and the
indicated GST-Topo I fusion proteins (B-D) were incubated
for 10 min at 37 °C in the absence or presence of nuclear extract
from proliferating T lymphocytes and in the presence of a mixture of
protease inhibitors (aprotinin, leupeptin, pepstatin, and PMSF).
E, GTOPI-1 was incubated as in A but in the
additional presence of the indicated concentrations of soybean trypsin
inhibitor. The degradation of the various fusion proteins was then
analyzed either by electrophoresis and fluorography or by immunoblot
analysis. PNE, nuclear extracts of proliferating T
lymphocytes.
|
|
Lack of the Trypsin-like Serine Protease Activity in Human Tumor
Cell Lines and Epstein-Barr virus-transformed B Cells--
We next
investigated whether the protease activity responsible for the
degradation of Topo I in normal proliferating T lymphocytes is also
expressed in human transformed lymphocyte and other cell lines. We
prepared cytosolic and nuclear extracts of A2780, Jurkat, HeLa, H460,
KB, Saos-2, A431, HepG2, K562, U937, HL60, and phorbol 12-myristate
13-acetate-treated HL60 cells as well as of Epstein-Barr virus-transformed B lymphocytes. These extracts were then examined for
their ability to cleave full-length Topo I in vitro.
Full-length Topo I was sequentially processed to the 97- and 82-kDa
fragments by nuclear extract of proliferating T lymphocytes (Fig.
12A), whereas Topo I
remained intact during incubation with either nuclear extract (Fig.
12A) or cytosolic extract (data not shown) of Jurkat cells. The trypsin-like serine protease activity was also not detectable in
nuclear extract of the A2780 ovarian cancer cell line (Fig. 12B) or in the extracts of Epstein-Barr virus-transformed B
lymphocytes or any of the other tested cancer cell lines (data not
shown). The activity of this protease thus appears to be induced when resting T lymphocytes enter proliferation rather than being expressed in actively cycling cells.
View larger version (48K):
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|
Fig. 12.
Effects of nuclear extracts of Jurkat cells
and A2780 cells on the stability of Topo I in
vitro. [35S]Methionine-labeled Topo I was
incubated for the indicated times at 37 °C in the presence of
protease inhibitors (aprotinin, leupeptin, pepstatin, and PMSF) and in
the absence or presence of nuclear extracts of proliferating T
lymphocytes, Jurkat cells (JNE) (A) or A2780
cells (B). The degradation of Topo I was then analyzed as
described in the legend to Fig. 2. PNE, nuclear extracts of
proliferating T lymphocytes.
|
|
| |
DISCUSSION |
Regulation of protein stability has been revealed to be important
in cell cycle progression, signal transduction, transcription factor
activity, tumor suppression, and oncogenesis. Abnormalities in protein
degradation often result in disruption of normal physiological status
and underlie various diseases (38-40). However, little has been known
of the stability or turnover of human Topo I (41).
We previously showed that the abundance of Topo I is regulated when
human peripheral T lymphocytes stimulated with PHA. Topo I protein was
thus shown to be synthesized in large amounts but also rapidly degraded
when resting T lymphocytes are induced to proliferate (34). We have now
established an in vitro assay of Topo I proteolysis that
appears to mimic the degradation of the protein in intact cells. We
have also characterized a novel trypsin-like serine protease that
appears to mediate the degradation of human Topo I in PHA-stimulated T
lymphocytes. This protease is localized to the nucleus and degrades
Topo I sequentially into 97- and 82-kDa fragments; the latter undergoes
further degradation in vitro with prolonged incubation times
(Fig. 12A).
Topo I exists in cells as a phosphorylated protein under various
physiological conditions (42-45). To rule out the possibility that the
97-kDa intermediate is actually a less phosphorylated or
unphosphorylated form of full-length Topo I, we investigated the effect
of addition of a phosphatase inhibitor (okadaic acid (1 and 100 nM) and sodium vanadate (20~500 µM)) to the
degradation assay mixture; the inhibitor had no effect on the pattern
of Topo I degradation (data not shown). Furthermore, the generation of the 97-kDa product in vitro was inhibited in a
concentration-dependent manner by soybean trypsin
inhibitor. The 97-kDa intermediate thus indeed appears to be a product
of Topo I proteolysis. We also showed that Mg2+ is required
for the degradation of Topo I in vitro. However, it remains
unclear whether Mg2+ acts as a cofactor for the protease or
is required for Topo I itself. Although a requirement of
Mg2+ for proteolysis is rare, several precedents exist. For
example, Mg2+ is required both for the proteolytic function
of amidopeptidase (46-48) and for the ubiquitin-dependent
26 S proteasome degradative pathway (29).
Desai et al. (41) showed that Topo I is degraded by the
ubiquitin-dependent 26 S proteasome pathway in Chinese
hamster ovary cells and that Topo I degradation by this pathway is
stimulated by camptothecin. Fu et al. (49) also showed that
the camptothecin-induced degradation of Topo I in KB cells is mediated
by the ubiquitin-dependent 26 S proteasome. However, in our
study, apyrase, lactacystin, and hemin did not affect the degradation
of Topo I in vitro. The possibility that both the
trypsin-like serine protease and the ubiquitin-dependent 26 S proteasome pathway are responsible for the degradation of Topo I in
our system appears to be excluded by the observation that the abundance
of Topo I did not decrease with incubation time (from 5 to 30 min)
after inhibition of the trypsin-like serine protease with 200 µM soybean trypsin inhibitor (data not shown). Thus, the
degradation of Topo I may be mediated predominantly by the trypsin-like
serine protease in normal proliferating T cells, whereas Topo I
proteolysis is mediated by the ubiquitin-dependent 26 S
proteasome in cells with camptothecin-induced DNA damage.
No intracellular protease with the characteristics (protease inhibitor
sensitivity and subcellular localization) of the trypsin-like protease
detected in the present study has been previously described. Hirschhorn
et al. (50) suggested that a trypsin-like protease activity
is required for the increase in RNA transcription in the nucleus when
resting T lymphocytes stimulated with PHA. It remains to be determined
whether this protease is identical to that described in the present
study. Our results also appear to differ from those of Grayzel et
al. (51), who did not detect an increase in broad spectrum
proteolytic activity (measured with casein and hemoglobin as
substrates) in response to exposure of resting T lymphocytes to PHA.
This apparent discrepancy may be due to the difference in substrates
used in the respective assays.
The susceptibility of a protein to proteolysis can be regulated by
phosphorylation (29), conformation (52), and the existence of intrinsic
destabilizing sequences (29). In the present study, the addition of
various types of kinase inhibitors or of calf intestinal phosphatase to
the nuclear extract of proliferating T cells had no effect on the
degradation of Topo I by this extract (data not shown), suggesting that
the phosphorylation state of Topo I does not affect its susceptibility
to proteolysis. In addition, recombinant Topo I produced with the use
of a baculovirus expression system is a phosphoprotein, and the
purified phosphoprotein was also degraded by the trypsin-like serine
protease in our in vitro assay (data not shown). We
previously showed that Topo I exists in at least two distinct
conformations; one conformation binds ATP and catalyzes the
phosphorylation of SR proteins, whereas the other form binds to and
relaxes superhelical DNA (37). Our present observation that Topo I
degradation was inhibited by ATP and ATP--S thus suggests that the
conformation of ATP-bound Topo I is resistant to digestion by the
trypsin-like serine protease, whereas the conformation of DNA-bound
Topo I is sensitive to the action of this protease. However, we cannot
rule out the possibility that this ATP inhibition is through the
titration of the Mg2+ ions.
The site of initial cleavage in Topo I was mapped to the
NH2-terminal 50 amino acids of the protein. Cleavage at
this site is likely responsible for initiating proteolysis, given that
the 97-kDa product appeared to be an essential intermediate for further degradation. We were not able to map the second cleavage site, possibly
because cleavage at the first site by the trypsin-like serine protease
results in a structural change in the 97-kDa intermediate that renders
the second site susceptible to attack by the protease. On the basis of
this hypothesis, given that, of the various GST-Topo I fusion proteins
studied, only GTOPI-1 possesses the first cutting site, these
constructs would not have been informative with regard to the
localization of the second cleavage site.
Studies with human (53) and yeast (54) Topo I have revealed that the
NH2-terminal domains of these enzymes are dispensable for
catalytic activity but that they may play an important role in the
in vivo function of Topo I. Shaiu and Hsieh (55) recently showed that the hydrophilic NH2-terminal domain of
Drosophila Topo I targets to transcriptionally active loci.
It is therefore possible that human Topo I has a similar function.
Interestingly, all of the proteolytic cleavage sites of human Topo I
described to date are located in the NH2-terminal domain of
the protein. Thus, in addition to the results of the present study, the
NH2-terminal domain of Topo I has previously been shown to
be susceptible to proteolysis (56), and Topo I degradation induced by
apoptosis was shown to occur by cleavage at the NH2
terminus (57). Some cues were provided to explain why all the
proteolytic cleavage sites occurred on the NH2-terminal
domain of Topo I. The NH2-terminal portion of Topo I forms
an extensive structure (56) that would appear inherently susceptible to
protease attack. Furthermore, removal of the NH2-terminal
domain may facilitate proteolytic attack on the remaining portion of
Topo I. Functionally, removal of the NH2-terminal domain
may inactivate the biological function of Topo I. An example is
Drosophila Topo I that deletion of NH2 terminus
impairs its targeting to actively transcribed gene and blocks the
involvement of Topo I in RNA transcription (55). Therefore, the
activity of Topo I may be finely tuned by scissoring off its
NH2 terminus immediately to inactivate its biological functions and to facilitate the proteolysis of its residual portion.
The rapid increase in the abundance of Topo I apparent on stimulation
of proliferation in peripheral T cells likely ensures the effective
execution of the associated DNA replication and gene transcription.
However, such a rapid increase in Topo I abundance also confers a
potential risk of genomic instability. Thus, overexpression of Topo I
in yeast promotes illegitimate recombination (19). Illegitimate
recombination events are usually associated with chromosomal
aberrations, which, in turn, are implicated in carcinogenesis and many
heritable diseases. In addition, the transcriptional activity of other
genes may be affected by changing in Topo I abundance (58). This change
could be involved in the activation of oncogenes, lost expression of
tumor suppressor genes, or deregulation of genes whose products
contribute to recombination. Therefore, it is likely that Topo I is
removed by proteolysis immediately after its abundance has increased to
meet the physiological requirements of the cell.
The abundance of Topo I in cancer cells has been shown to be an average
of 14-fold greater than that in the corresponding mucosal cells of
individuals with colon cancer (23, 24). A similar phenomenon has also
been detected for other cancers (24-28). However, the mechanism
responsible for this difference remains unclear. In the present study,
we have shown that a novel trypsin-like serine protease activity that
mediates the degradation of Topo I is induced in normal proliferating T
cells, but this activity is not detectable in Jurkat cells and other
various cancer cell lines. The lack of activity of this trypsin-like
serine protease in nuclear extracts of these cells may thus explain why
the abundance of Topo I is increased in cancer cells.
| |
ACKNOWLEDGEMENTS |
We thank all those individuals who donated
blood for this study.
| |
FOOTNOTES |
*
This work was supported by Grant NSC 88-2318-B-001-007-M15
from the National Science Council of Taiwan and by Academia Sinica.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Inst. of Molecular
Biology, Academia Sinica, Taipei 115, Taiwan. Tel.: 886-2-2789-9217; Fax: 886-2-2782-6085; E-mail: jh@ccvax.sinica.edu.tw.
| |
ABBREVIATIONS |
The abbreviations used are:
Topo I, DNA
topoisomerase I;
PHA, phytohemagglutinin;
GST, glutathione
S-transferase;
DTT, dithiothreitol;
PMSF, phenylmethylsulfonyl fluoride;
ATP--S, adenosine-5'-(-thio)triphosphate.
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H.-J. Chen, C.-M. Lin, C.-S. Lin, R. Perez-Olle, C. L. Leung, and R. K.H. Liem
The role of microtubule actin cross-linking factor 1 (MACF1) in the Wnt signaling pathway
Genes & Dev.,
July 15, 2006;
20(14):
1933 - 1945.
[Abstract]
[Full Text]
[PDF]
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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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