From the Department of Experimental Radiation Oncology, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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Using differential display, a cDNA fragment
was identified as being overexpressed in a mouse lymphoma cell line
that had gained resistance to cell death after exposure to a variety of
agents used in cancer therapy. The full-length cDNA of 1.1 kb that
was cloned contained an open reading frame coding for a previously unidentified 28-kDa mammalian protein, p28. p28 showed significant homologies to a large family of stress response proteins that contain a
glutathione S-transferase (GST) domain. In correspondence with the sequence homology, p28 was found to bind glutathione; however,
GST or glutathione peroxidase activity could not be demonstrated. Northern analysis of the mRNA of this protein showed abundant expression in mouse heart and liver tissues, whereas anti-p28 antibody
binding identified p28 expression in mouse 3T3 cells and early passage
mouse embryo fibroblasts. Subcellular protein fractionation revealed
p28 localization in the cytoplasm, but with thermal stress p28
relocated to the nuclear fraction of cellular proteins. Based on
sequence homology and protein activity we conclude that p28 acts as a
small stress response protein, likely involved in cellular redox
homeostasis, and belongs to a family of GST-like proteins related to
class To cope with a variety of adverse environmental influences,
including temperature extremes, toxins, nutritional deprivation, and
oxidative damage, cells from bacteria to mammals have developed common
molecular responses, including alterations in gene expression that
up-regulate heat shock or stress response proteins. These stress
response proteins can act as molecular chaperones by binding to
denatured or misfolded proteins, by regulating the correct folding of
proteins, by dissociating protein aggregates, or by facilitating the
transfer of proteins to specific cellular locations (for review, see
Refs. 1-5). At least one heat shock protein regulates glutathione
levels, resulting in shifts in cellular redox status (6). Stress
response mechanisms, like many biological phenomena, are double-edged
swords in that the induction of heat shock proteins or detoxification
enzymes such as glutathione S-transferases (GSTs)1 have beneficial
effects Such an increase in resistance of tumor cells to cytotoxic drugs or
ionizing radiation is observed in the murine lymphoma cell model
described below. The cell line, named LY-as, was derived from a
syngeneic mouse B cell lymphoma designated LY-TH (12). This cell line
proved to be very susceptible to apoptotic cell death induced by
ionizing radiation (13) or chemotherapeutic drugs when maintained
in vitro.2
However, during in vitro culture this cell line reproducibly loses its high susceptibility to apoptosis and becomes much more resistant to the cytotoxic agents mentioned (13).2 This
resistant cell type was designated LY-ar (13). The gain in resistance
is associated with an up-regulation of Bcl-2 oncoprotein and doubling
of the cellular content of reduced glutathione (15). Modulation of
glutathione levels by cysteine deprivation or by treatment with diamide
or diethyl maleate, agents that deplete cellular thiols, after
radiation results in a restoration of radiosensitivity and apoptosis
induction characteristic of the parental LY-as cell line
(15).2 The Bcl-2 protein level does not change as a result
of cysteine deprivation (15), but it has been suggested that it is
nuclear GSH levels that are critical in determining the extent of
apoptosis and that nuclear membrane-associated Bcl-2 is responsible for maintenance of those levels (16).
To determine how the cellular phenotype changes from sensitive to
resistant, we examined differences in the level of gene expression
between the two cell lines by using the differential display technique.
Apart from the expression differences of several known
genes,3 we also found a
cDNA fragment exclusively expressed in the resistant cell line
LY-ar that showed no homology to mRNAs of known mammalian proteins.
Here we describe the cloning of the full-length cDNA of which this
fragment is a part. The cDNA codes for a novel protein with a
calculated molecular mass of 28 kDa. This protein shows homologies to
small stress proteins and GSTs, which all belong to a large ancient
protein superfamily of proteins that are related by their sequence and
tertiary structure to class Cell Culture--
LY-as and LY-ar cells were passaged in RPMI
1640 medium supplemented with 10% fetal bovine serum and antibiotics
(100 units/ml penicillin, 100 µg/ml streptomycin). The cells were
incubated at 37 °C in a humidified atmosphere containing 5%
CO2. For thermal stress the cells, in tissue culture
flasks, were submerged in a water bath (Haake DC1) at the appropriate
temperature (±0.1 °C) for 1 h.
RNA Isolation and Differential Display--
Small scale RNA
isolations for the differential display were performed using RNAzol B
(TEL-TEST Inc.), which is a modification of the method of Chomczynski
and Sacchi (19). Poly(A)+ RNA was isolated from total RNA
using the Poly(A)Tract System III (Promega) according to the
instructions given by the manufacturer. Differential display polymerase
chain reaction (PCR), the cloning of the differentially expressed
fragments, and the sequencing of these fragments was done as described
previously.3
Full-length cDNA Cloning and Sequencing--
The full-length
cDNA was cloned by rapid amplification of cDNA ends (RACE) PCR
using the Marathon cDNA Amplification Kit
(). Briefly, double-stranded cDNA was
synthesized from 1 µg of poly(A)+ RNA isolated from LY-ar
cells. The cDNA was ligated to adapters, and RACE PCR was performed
with the gene-specific primer GGGGAAATCACAGTTTTCAGACATG and the adapter
primer AP1 using the enzyme mix and the buffers of the Expand Long
Template PCR System (Boehringer Mannheim). The cycling conditions were
as follows: 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 3 min. The PCR products were separated by agarose
gel electrophoresis. The major amplification product was recovered from
the gel and cloned into the pCR2.1 vector and transfected into
competent Eschericia coli from the TA-Cloning Kit
(Invitrogen). After colony selection, sequencing was performed with a
cycle sequencing kit (Amersham Pharmacia Biotech) using
33P-labeled primer according to the protocol given by the manufacturer.
Northern Blots--
Tissue-specific expression levels of p28
mRNA were determined by Northern blot analysis using a commercially
available mouse multiple tissue Northern blot membrane
(). The 32P-labeled probe was
generated from the coding sequence of the cDNA using PCR as
described previously.3 Hybridization and washes were
performed exactly as proposed by the membrane manufacturer (Amersham
Pharmacia Biotech).
Protein Expression in E. coli--
The largest cDNA product
produced by RACE PCR and subsequently subcloned into the pCR2.1 plasmid
as described above was cut from pCR2.1 with EspI and
EcoRV and then subcloned in frame into the EcoRV
site of the pET32(+) vector (Novagen). The expression vector was
transformed into BL21(DE3) cells, and protein expression was induced by
incubating the cells with 1 mM
isopropyl- Protein Preparation and Immunoblotting--
p28 was localized
according to the protocol of Dyer and Herzog (20), with the exception
that dithiothreitol was not included, by producing subcellular
fractions of LY-ar cells. A nuclear fraction was produced by lysis of
the cells in isotonic buffer containing 0.5% Nonidet P-40 followed by
centrifugation for 5 min at 700 × g to pellet the
nuclei. A mitochondria-rich fraction was obtained by centrifugation of
the supernatant at 10,000 × g for 30 min, and then the
microsomal fraction was separated from the cytoplasmic fraction
(supernatant) by a 2-h centrifugation at 100,000 × g.
To determine the localization of p28 after heat treatment, the cells
were heated to the appropriate temperature for 1 h, immediately washed once in cold PBS, and then lysed for 3 min on ice in a lysis
buffer containing 10 mM Tris (pH 7.4), 5 mM
MgCl2, 10 mM NaCl, 0.1 mM
phenylmethylsulfonyl fluoride, and 0.5% Triton X-100. The particulate
and detergent-soluble fractions were separated by a 5-min
centrifugation step at 16,000 × g. Further
discrimination of the localization of p28 was carried out as in the
work of Dyer and Herzog (20) on cells heated or not to 44 °C for
1 h before lysis and separation into nuclear, mitochondrial, and
microsomal fractions.
Proteins were separated on a 12% acrylamide gel using the Laemmli (21)
buffer system and then electrotransfered to a nitrocellulose membrane
(Amersham Pharmacia Biotech). After the membrane was blocked with
BLOTTO, it was incubated for 1 h in antiserum diluted 1:200 with
BLOTTO. After three 15-min washes in Tris-buffered saline containing
0.05% Tween 20 (TBST), the membrane was incubated for 1 h in a
1:1500 dilution of peroxidase-conjugated goat anti-rabbit antibody
(Amersham Pharmacia Biotech) in BLOTTO. After three final 15-min washes
in TBST, the blot was developed using a chemiluminescence detection kit
(ECL, Amersham Pharmacia Biotech).
GSH Affinity Precipitation--
For GSH affinity precipitation,
proteins from 5 × 106 LY-as and LY-ar cells were
radiolabeled with 50 µCi [35S]methionine (Amersham
Pharmacia Biotech) over 3 h. The cells were lysed in the same
lysis buffer used as above for p28 localization after thermal stress. A
50-µl volume of lysate representing cytoplasmic proteins and
containing ~1.5 × 106 cpm was diluted with 450 µl
of PBS and mixed with 30 µl of amino-linked GSH-Sepharose (Sigma
G-9761). The mixture was incubated at 4 °C for 1 h, and then
the GSH-Sepharose was washed three times for 15 min in PBS at 4 °C.
The bound proteins were solubilized and released from the GSH-Sepharose
matrix by boiling the GSH-Sepharose in SDS gel loading buffer (21),
separated on a 12% polyacrylamide gel, and detected by
autoradiography. p28 was identified on the same membrane by
chemiluminescence as described above.
GST and Glutathione Peroxidase Activities--
GST activities
were determined by monitoring the production of thioethers, as
1-chloro-2,4-nitrobenzene, trans-4-phenyl-3-buten-2-one, ethacrynic acid, and androstene-3,17-dione were conjugated with GSH, by
UV absorbance spectrophotometry (22). Glutathione peroxidase activity
was determined with the substrate cumene hydroperoxide in a coupled
enzymatic reaction with GSH-reductase and NADPH again by
spectrophotometry as described previously (23).
Molecular Cloning of the Full-length cDNA Sequence--
After
using the random primer TCGATACAGG and the anchored primer
T12VG for PCR and subsequent reverse transcription of LY-as and LY-ar mRNA, differential display resolved two bands with sizes of 135 and 128 bp that appeared only in the gel lane containing the
LY-ar cDNA sample (Fig.
1A). Sequencing revealed that
both fragments were similar to one another and only differed slightly in their polyadenylation site (Fig. 1B). A data base search
did not show any homology of these fragments to mRNAs coding for
known proteins. Because these cDNA fragments are necessarily 3'
because of the anchored primer used, 5' RACE PCR was used to obtain the upstream cDNA sequence. The position of the gene-specific primer used to amplify the cDNA is shown in Fig. 1B. The 5'
RACE yielded a single product of ~1.1 kb, which was homogeneous as
judged directly from the cycle sequencing of this PCR product. Northern
analysis of mRNA expression using the cDNA as probe against RNA
from both cell lines confirmed that p28 RNA was expressed exclusively
in LY-ar cells (Fig. 1C). The full-length cDNA sequence
is given in Fig. 2. The size of the
cDNA, 1159 bp, was in good agreement with the size of the
hybridization signal seen in the Northern blot, 1.3 kb. The position of
the start codon was verified by an in-frame stop codon upstream at
position 11.
Tissue-specific mRNA Expression in Mice--
Fig.
3 shows the expression levels of p28
mRNA in various mouse tissues as determined by Northern blot
analysis. The highest levels of p28 mRNA were found in liver, lung,
and heart tissues. Kidney, skeletal muscle, spleen, and brain showed
low levels of p28 transcript, whereas it was almost undetectable in
testicular tissue. In most of the tissues, a major, larger transcript
with a size of ~1.3 kb and a minor, smaller mRNA of ~1.1 kb
were detected. 3' RACE amplification and sequencing of the smaller
cDNA revealed that the size difference was caused by the
alternative polyadenylation site located at base 891. The reason for
the intermediate size of the transcript in spleen cells was not
determined.
Protein Sequence Analysis--
The full-length cDNA described
above contained one long open reading frame coding for a protein of 240 amino acids with a predicted molecular mass of 27.5 kDa. The
predicted protein had significant sequence homology to a diverse family
of stress response proteins that contain a GST domain (18). Protein
sequence alignments with some important members of this family are
shown in Fig. 4. The closest matching
sequence, 33% identity and 52% similarity, was from a hypothetical
28.5-kDa protein of unknown function found by genomic sequencing of
Caenorabditis elegans (24). p28 also showed 40% identity
and 67% similarity with a 92-amino acid-long protein fragment from
Aplysia californica named protein 9. Its expression was
found to be induced in Aplysia ganglia after depolarization and treatment with serotonin (25). Because protein 9 is a fragment, the
statistical power of the seemingly greater sequence identity and
similarity seen is low, which is why the C. elegans protein was considered a closer match to p28.
p28 also showed significant homologies to a large number of plant
proteins that have an either proven or suspected GST function and are
induced as part of the plant stress response. Of these, the alignments
for the GST GTX1, also called pathogenesis-related protein 1 (26), from
Solanum tuberosum and for the heat shock protein 26A (27)
from soybean are shown in Fig. 4. Both sequences showed an identity of
22 and 24% and a similarity of 42 and 43%, respectively.
Further homologies existed with each domain of the TcAc2 protein (28),
a Trypanosoma cruzi protein that contains a tandemly repeated domain structure that catalyzes the thiol-disulfide exchange between dihydrotypanothione and glutathione disulfide (29). Finally,
p28 showed a significant, albeit lesser, degree of homology to the
sequence of the stringent starvation protein A (30) from E. coli, with 23% identity and 39% similarity. The stringent
starvation protein A is considered a bacterial regulatory protein (31) that is heavily up-regulated after amino acid deprivation (32).
Identification of p28 by Western Blotting--
An antiserum was
raised in rabbits using the recombinant fusion protein described under
"Experimental Procedures" that consists of an N-terminal
thioredoxin linked to amino acids 29-240 of p28. This antiserum
recognized a protein in the Western blot with an apparent molecular
mass of 32 kDa (Fig. 5A) that
was not recognized by preimmune serum (Fig. 5B). The
difference between calculated and apparent molecular mass is likely not
attributable to post-translational modification of the protein, because
recombinant p28 expressed in E. coli also showed the
apparent molecular mass of 32 kDa when separated on an
SDS-polyacrylamide gel (Fig. 5C). The antiserum also
detected high amounts of p28 in mouse fibroblast cell lines. In both
NIH 3T3 and low passage mouse embryonal fibroblasts, the protein could
be detected at precisely the same apparent molecular mass (data not
shown).
GSH Binding and Activity of p28--
Because of the sequence
homologies of p28 to proteins of the aforementioned GST superfamily,
GSH binding by p28 was examined. Radiolabeled cytoplasmic proteins of
LY-ar and LY-as cells were affinity precipitated with GSH linked to
Sepharose, eluted from the GSH-Sepharose matrix, and separated on
SDS-polyacrylamide gels. A number of protein bands of various molecular
masses were apparent in extracts from both LY-ar and LY-as cells, with
the exception of a 32-kDa protein that is apparent in only the LY-ar protein preparations (Fig.
6A). The identification of
this glutathione-binding protein as p28 was confirmed by its reactivity
with p28 antiserum (Fig. 6B).
Using 1-chloro-2,4-nitrobenzene,
trans-4-phenyl-3-buten-2-one, ethacrynic acid, and
androstene-3,17-dione, which are regarded as representative substrates
for many types of GSTs (33), we were unable to demonstrate an enzymatic
function for the recombinantly derived protein. Nor could we
demonstrate glutathione peroxidase activity against the substrate
cumene hydroperoxide.
Subcellular Localization of p28--
Crude nuclear, mitochondrial,
microsomal, and cytoplasmic protein extracts of LY-ar cells were
prepared and analyzed for their p28 content (Fig.
7A). The greatest amount of
p28 was found in the cytoplasmic fraction. Very small amounts of
protein were also detected in the mitochondrial and nuclear fraction;
however, because these two fractions likely contained small amounts of
contaminating cytoplasmic proteins, the primary subcellular
localization of p28 was considered cytoplasmic.
Changes in the Subcellular Localization of p28 during Heat
Shock--
It is well established that small heat shock proteins from
mammalian cells localize into the nucleus or into the
detergent-insoluble fraction of cellular protein preparations after
exposure of cells to elevated temperatures (34). p28 was a cytoplasmic
protein at 37 °C (Figs. 7 and 8). If
LY-ar cells were incubated at elevated temperatures, in this case for
60 min, p28 relocalized from the detergent-soluble fraction,
e.g. cytosol, into the particulate fraction (Fig.
7B). This effect could be observed at temperatures ranging
from 42 to 44 °C. After 60 min at 44 °C, almost all of the p28
signal was found in the particulate fraction. Further fractionation of
cellular proteins revealed that p28 relocalization as a result of heat
exposure was predominantly to the nucleus (Fig. 8A).
Although hyperthermic treatment leads to a rapid induction of some
small heat shock proteins (1), we were unable to observe any heat
inducibility of p28 in LY-as cells.
The sequence homology of p28 suggests that it belongs to a broad
family of proteins evolutionarily related to class The amino acid residues 162-189 in the p28 sequence conform to the
second conserved motif described by Koonin et al. (18). This
domain is not involved in substrate binding, and its function is less
clear. A conserved aspartic acid residue (amino acid 173 in p28) is
considered important (18), and this domain may be a key structural
element in the conserved core of The presence of these conserved domains in p28 justified the assumption
that it would bind glutathione and catalyze the conjugation of
glutathione to other substrates. We were able to demonstrate that p28
binds to glutathione, but regardless of the substrate used, no
enzymatic activity could be associated with the recombinant p28
protein. The substrates used to identify an enzymatic activity for p28
have been shown to be substrates for a number of GST isoenzymes (9).
However, when compared with more common GSTs, recognition of a specific
electrophilic ligand by p28 would be unusual because of the low
specificity for electrophilic ligands by more common GSTs (33).
Furthermore, at least 100 different chemicals are known to activate
GSTs, and many also act as the substrate for the up-regulated GST; so
it is conceivable that a substrate for p28 is among them. Another
reason for the lack of measurable GST activity could be that the
recombinant protein, despite its solubility, is incorrectly folded. We
have, however, observed a similar lack of GST activity by p28 using the
same substrates against mammalian cells that express p28 as part of
their normal growth condition (data not shown).
Induction of many of the plant proteins to which p28 shares homology is
caused by a wide variety of stresses. Fungal infections induce protein
GTX1 in S. tuberosum (26), auxins induce GTXA in
Arabidopsis thaliana (37), and heavy metal exposure or heat shock induces heat shock protein 26A in soybean (38). We therefore tested the hypothesis that p28 might respond to stress, in this case
heat shock. By fractionating cellular proteins based on detergent solubility, we first showed that with thermal stress p28 localized into
the detergent-insoluble fraction of cellular protein isolates. When
examined in more detail, p28 was shown to be localized in the nuclear
fraction as a result of heat shock, an association that is a well known
feature of small heat shock proteins (34). For example, the
constitutively expressed protein hsp27 relocalizes from a
detergent-insoluble fraction to a soluble cytoplasmic fraction after
stress (39, 40), whereas hsp70 relocalizes to the nucleoli when cells
are stressed (41). Concomitant with the p28 localization, the
inducibility of p28 after thermal stress was examined, but no increase
in p28 levels were apparent in LY-ar cells (perhaps p28 is already
expressed maximally) or in LY-as cells, in which p28 expression could
not be measured.
p28 contains a glutathione binding domain and is translocated after
thermal stress. p28 mRNA was up-regulated in mouse tissues associated with higher oxygen tensions such as heart, lung, and kidney
but was less evident in spleen, skeletal muscle, and testicular tissues. LY-ar cells, isolated from LY-as cells after extensive cell
culture, also contain twice the level of cellular reduced glutathione.
Note that although LY-ar cells have twice the glutathione concentration, bithionine sulfoximine, the specific inhibitor of
glutathione synthesis, does not alter the apoptotic propensity of LY-ar
cells (15), and addition of N-acetyl cysteine in millimolar concentrations reduces apoptosis in LY-as cells only slightly (data
not shown). Although generic inhibitors of thiols are effective in
modulating apoptosis as stated in the introduction, it may be the
specific compartmentalization of glutathione that is critical (16).
Because p28 is abundantly expressed in 3T3 and mouse embryo
fibroblasts, it argues against a role for p28 in apoptosis, because neither 3T3 nor embryo fibroblasts undergo radiation-induced apoptosis. In addition, altered gene expression in LY-ar cells3 could
be attributable to LY-ar cells having become somewhat more differentiated. Admittedly, we have no evidence of that having occurred. However, lymphoid cells such as K562 cells are known to
differentiate as a result of environmental stress. In fact, it is
conceivable that although Bcl-2 up-regulation in LY-ar cells is
responsible for blocking apoptosis, it may not be responsible for the
radiation or chemoresistance of LY-ar cells, because sufficient evidence now exists showing that Bcl-2 up-regulation does not predict
resistance (14, 17).
We speculate that p28 may be involved in the cellular defense against
or in the adaptive response to altered cellular redox conditions. LY-as
cells were originally isolated from a mouse B cell lymphoma grown
in situ, and the conversion of LY-as cells to LY-ar cells is
likely a prime example of the redox-adaptive response as these cells
adapt to growth in culture conditions foreign to them, e.g.
high oxygen tension. This may also be the case for 3T3 cells and
especially low passage mouse embryo fibroblasts, in which p28 was
highly expressed. Whether p28 functions directly to alter redox status
by glutathione binding or modulates the activity of other proteins via
binding through its translocation activity is unknown at this time but
is under pursuit. However, the expression and translocation of p28 may
provide cells with a greater diversity of mechanisms for coping with
insults such as thermal stress or altered redox status that may be
responsible for the radiation and chemoresistance of LY-ar cells.
GSTs.
INTRODUCTION
Top
Abstract
Introduction
References
increasing normal tissue stress tolerance, and harmful
effects
enabling tumor cells to resist cytotoxic therapy, especially
by drugs that increase intracellular reactive oxygen intermediates
(6-11).
GSTs (18). This superfamily, which
includes GSTs, small stress proteins, the eukaryotic translation
elongation factor 1
, bacterial dehalogenases, and etherases, as
well as several uncharacterized proteins, is characterized by two
conserved domains, a glutathione binding domain and a second domain of
unknown function. The sequence homology of p28 to the conserved domains
and the similar functional characteristics of p28 to other family
members, in particular relocalization in response to thermal stress and
ability to bind glutathione, argue for the inclusion of p28 as a new
mammalian member of this superfamily.
EXPERIMENTAL PROCEDURES
-thiogalactopyranoside for 3 h. The vector coded for
a fusion protein consisting of a thioredoxin tag, a 6xHis site, and an
enterokinase cleavage site fused to the N terminus of p28. This fusion
protein was purified using a His-Bind Ni2+ affinity column
(Novagen). Antiserum was raised against the purified fusion protein in
rabbits using standard immunization protocols.
RESULTS
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Fig. 1.
Autoradiogram (A) and sequence
(B) of the differential display products obtained from LY-ar
and LY-as RNA. The sequences of the random and anchored primers
(underlined) were TCGATACAGG and T12VG. The 7 nucleotides printed bold distinguish the 135-bp from the
128-bp PCR product; the difference results from slightly different
polyadenylation sites. The italicized nucleotides in the
middle of the sequence are complementary to the
gene-specific primer used for 5' RACE PCR to obtain the
full-length cDNA. C, Northern gel depicting the
differential expression of a 1.1-kb mRNA (upper band)
identified using a probe made from the cDNA product identified by
differential display analysis. 20 µg of total RNA were loaded
per lane. The lower panel depicts the hybridization
signals of a glyceraldehyde-3-phosphate dehydrogenase probe used as a
loading control.
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Fig. 2.
Sequence of the full-length p28
cDNA. This sequence was deposited in GenBank under accession
number U80819. The position of the start codon (base 100) is verified
by an upstream in-frame stop codon (underlined, base 11).
Apart from the major polyadenylation site given in this sequence, a
minor site was identified at base 891. The major site is preceded by a
polyadenylation signal from bases 1129 to 1134 (underlined),
which is an imperfect match to the consensus sequence.
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Fig. 3.
Northern blot showing the abundance of the
p28 mRNA in various mouse tissues. Approximately 2 µg of
poly(A)+ RNA bound on a commercially available membrane
() were hybridized with a probe spanning
the coding sequence of the p28 cDNA. The size of the major
transcript was ~1.3 kb, whereas the minor transcript was ~1.1
kb.
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Fig. 4.
Alignment of the p28 protein sequence with
several homologous proteins. p29, a hypothetical 28.5-kDa
protein from C. elegans (GenBank number P34345); prot.
9, a protein fragment from A. californica, which is
induced by treating ganglia with serotonin along with depolarization
(GenBank number 546766); GTX1, a pathogenesis-induced
protein and probable GST from S. tuberosum (GenBank number
417542); hsp26a, the heat shock protein 26A from soybean
(GenBank number 417148); TcAc2, the second domain of a
trypanosomal thiol-disulfide exchange protein (GenBank number 537611);
SSPA, the stringent starvation protein A from E. coli (GenBank number P05838).
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Fig. 5.
A, immunoblot showing the localization
of a protein at an apparent molecular mass of 32 kDa exclusively in
LY-ar cells by using an antiserum raised against recombinant p28.
B, nonspecific binding of preimmune serum. C,
comparison of molecular masses of native p28 with recombinant p28
produced as described under "Experimental Procedures." Total
cellular protein from 2*105 LY-ar and LY-as cells was
loaded in each lane. Numerical values for protein markers to the
left of A are in kDa.
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Fig. 6.
Binding of p28 to GSH immobilized on a
Sepharose matrix. LY-ar and LY-as cells were radiolabeled with
[35S]methionine. The cytosolic extracts of both cell
lines were precipitated with a GSH-Sepharose matrix. The proteins
binding to this matrix were eluted and then separated on a 12%
acrylamide gel and either detected by autoradiography (A) or
transferred to a nitrocellulose membrane and probed with p28 antiserum
(B).
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Fig. 7.
A, immunoblot showing the original
subcellular fractionation of p28 into cytoplasmic, nuclear,
mitochondrial, and microsomal fractions. B, immunoblot
showing the changes in the intracellular localization of p28
immediately after a 1-h incubation at 37 °C and higher temperatures
(42-44 °C). LY-ar cells were lysed in a buffer containing 0.5%
Triton X-100, and the soluble proteins (s) were separated
from the particulate fraction (p) by a 5-min centrifugation
at 16,000 × g. 20 µg of each protein fraction were
loaded per lane.
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Fig. 8.
A, subcellular localization of p28 in
LY-ar cells after heating, or not, cells for 1 h at 44 °C.
LY-ar cells were lysed and fractionated into nuclear, mitochondrial,
microsomal, and cytoplasmic fractions. 20 µg of protein of each
fraction were loaded per lane. B, the nuclear and
cytoplasmic fractions of proteins isolated after heating at 44 °C
were examined for cross-contamination by Western analysis for histone
H2b, representing nuclear proteins, and actin, representing cytoplasmic
proteins.
DISCUSSION
GSTs (35).
Proteins belonging to this class have two defined motifs, the first of
which contains a glutathione binding domain, whereas the second is
thought to be a core structural domain (18). Amino acids 69-95 of p28
conform to the first motif, which comprises the glutathione binding
domain. By way of comparison of the GST domain of p28 with class
GSTs, analysis of the crystal structure of a GST
class enzyme from
Lucilla cuprina has identified the amino acids within the
first motif that interact with glutathione (36). Amino acids 64-66 in
the Lucilla enzyme, the highly conserved glutamate and
serine and a less conserved arginine, interact with the
-glutamyl
residue of GSH. The corresponding amino acids in p28 are at positions
85 and 86, which are conserved, and position 87, which is a
less-conserved valine. The crystal structure of the Lucilla
enzyme also demonstrated that a serine residue at position 9 is
responsible for activation of the thiol group of GSH. There are four
serine residues in the N-terminal region of p28, at positions 5, 6, 8, and 13, that could fulfill that function. Finally, two other amino
acids interact with the cysteinyl residue of GSH in the
Lucilla enzyme. They are isoleucine and tyrosine at amino
acid positions 52 and 113, respectively, and although valine appears to
be substituted for isoleucine in p28, no amino acid corresponding to
the tyrosine could be identified on the basis of the protein sequence.
The same holds true for the two histidines at positions 38 and 50 of
the Lucilla enzyme, which were found to interact with the
glycine of glutathione.
class GSTs.
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ACKNOWLEDGEMENTS |
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We acknowledge the help and expert advice of Dr. Michael Weil in the field of molecular biology and Marvette L. Hobbs in cell culture. We also acknowledge the help of Dr. Zhaohui Pan and Dr. Jian Kuang for critically reading the manuscript. For the production of the antibody used we acknowledge National Institutes of Health Institutional Core Grant CA16672 and thank the staff of the Department of Veterinary Science of the M. D. Anderson Cancer Center for their skillful support.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA62209 (to M. S., P. C., and R. K.) and National Institutes of Health Training Grant CA77050 (to P. C.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U80819.
Present address: Klinic fur Strahlentherapy und Strahlenbiologie,
Wahringer Gurtel 18-20, A-1090 Vienna, Austria.
§ To whom correspondence should be addressed: Dept. of Experimental Radiation Oncology, Box 66, University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-3424; Fax: 713-794-5369; E-mail: mstory{at}odin.mdacc.tmc.edu.
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ABBREVIATIONS |
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The abbreviations used are: GST, glutathione S-transferase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; PBS, phosphate-buffered saline; TBST, Tris-buffered saline containing 0.05% Tween 20.
2 M. D. Story and R. E. Meyn, submitted for publication.
3 R. Kodym and M. D. Story, submitted for publication.
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