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RESEARCH COMMUNICATION |
a Department of Biology, University of Rome `Tor Vergata' Rome, Italy;
b Laboratorio Ultrastrutture, Istituto Superiore di Sanità, Rome; Italy;
c Biochemistry Laboratory, IDI-IRCCS, Rome, Italy;
d Department of Biomedical Sciences, Università `G. D' Annunzio' Chieti, Italy; and
e Cell Biology Laboratory `L. Spallanzani'-IRCCS, Rome, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cell death • human neuroblastoma cells • staurosporine • histone H2B • microtubules • chromatin • glutathione
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-carboxamide
groups of peptide-bound glutamine residues serve as acyl donors and
primary amino groups of several compounds function as acceptor
substrates 1-3)
. The reaction results in the
posttranslational modification of proteins by establishing
(-glutamyl)lysine and N,N-bis(-glutamyl)polyamine isodipeptide
linkages (4)
. The tTG cross-linking activity is highly
regulated at the posttranscriptional level by GTP, polyamines, and
nitric oxide (5
, 6
). tTG is undetectable in
most mammalian cells and its mRNA is specifically transcribed as a
consequence of the induction of apoptosis (1
,
7
). tTG-dependent formation of covalent cross-links in
apoptotic cells leads to the polymerization of substrate proteins that
can be dismantled only by the proteolytic degradation of the protein
chains (8
, 9
). Overexpression of tTG induce apoptosis in various mammalian cells and the few clones resistant to tTG transfections show a marked reduction in their growth capacity not only in vitro 10-12) , but also when xenografted into SCID mice (13) . In keeping with these findings, the transfection of human neuroblastoma cells with an expression vector containing a segment of the human tTG cDNA in antisense orientation resulted in a decrease in both spontaneous and retinoic acid-induced apoptosis (12) . The question arises as to whether in the early stages of apoptosis a regulated tTG-mediated posttranslational modification of specific protein substrate(s) might play a role in the commitment to apoptosis. We have recently shown that the retinoblastoma protein (pRB) gene product, which plays an important role in controlling both cell release from the G1 phase and apoptosis (14) , is posttranslationally modified by a tTG during the early phases of apoptosis (7) . Although the precise position of tTG in the cascade of events leading to apoptosis is not yet established, a large body of evidence indicates that tTG induction parallels or slightly preecedes bcl-2 down-regulation and is not susceptible to its inhibitory effect (12) .
Recent data suggest that cell death by apoptosis and tTG might be involved in degenerative diseases of human central nervous system (15 , 16 ). Huntington's disease and others are caused by genes with expanded CAG triplet repeats in their coding region that result in extended polyglutamine domains within the expressed proteins (15 , 16 ). Affected individuals are characterized by accumulation of intracellular inclusions in the brain, which are composed of insoluble aggregates of proteins containing the reiterated glutamine residues (15 , 16 ). The quantity of these protein aggregates correlates with severity of the disease and the rapid progression to cell death 15-17) . Recent findings have proposed that tTG, by promoting the formation of these insoluble aggregates, might play a major role in the pathogenesis of these neurodegenerative diseases (17) . The outcome of this progressive cell damage is a dramatic cell loss due to the induction of apoptosis, which, as recently reported for the Huntington's disease, can be partially blocked by tTG inhibitors (17) .
To gain further insight into how tTG and apoptosis are regulated in neural cells, we have identified and characterized cellular proteins that specifically interact with and eventually act as substrates of tTG during the neuronal cell's commitment to apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell cultures and transfections
The parental SK-N-BE(2)
line and its transfected
sublines (Neo and TGA) were grown in RPMI with 10% (v/v)
heat-inactivated fetal calf serum, 2 mM L-glutamine, 1.2 g/l
bicarbonate, nonessential amino acids (1%v/v), and 15 mM HEPES in a
humidified atmosphere with 5% (v/v) CO2 at 37°C.
Transfection studies were performed by using the calcium phosphate
precipitation technique as described by Melino et al.
(12)
. The Neo clone was transfected only with pSV2Neo
plasmid containing resistance to neomycin; TGA clone, which
overexpresses tTG, was also cotransfected with the pSG5-tTG construct
that contains the tTG full-length cDNA. Cell growth was evaluated by
plating 50 to 100 x 103 cells per 25 cm2
flask. For STS experiments, cells were treated first with 10 µg/ml
CHX for 30 min, then 2 µM STS was added for various time intervals.
Control cells were treated with 0.2% dimethyl sulfoxide. In the
experiments with the caspase inhibitor, Z-VAD (100 µM) was added
1 h before CHX treatment.
Cell labeling and immunoprecipitation
Prior to metabolite labeling, cells were washed with serum- and
methionine-free Dulbecco modified Eagle's medium (Sigma). Equal
amounts of cells (15 x106 cells) were exposed for 24 h with 300 µCi of [35S]methionine. Cells were lysed at
4°C in isotonyc lysis RIPA buffer [150 mM NaCl, 1% NP-40, 0.5%
DOC, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris HCl pH 7.5/8 with
freshly added protease inhibitor (10 mM benzamidine hydrochloride, 10
mM chymostatin, 1 mM EDTA, 0.7 µg/ml pepstatin A, and 1 µg/ml
leupeptin). The anti-tTG, GST P1-1, and ß-tubulin mAb's were added
overnight at 4°C, and immunoprecipitates were captured with 10%
(v/v) protein A-Sepharose (Pharmacia, Uppsala, Sweden) for 1 h.
Immunoprecipitates were solubilized with SDS-polyacrylamide gel
electrophoresis (PAGE) sample buffer and electrophoresed through 10%
SDS-polyacrylamide gels. Gels containing
[35S]methionine-labeled protein were fixed with 10%
glacial acid acetic and 30% methanol overnight, enhanced by soaking in
a fluorography enhancing solution. Gels were dried and autoradiography
was performed at -70°C.
Sequencing of tTG binding proteins
The proteins that coprecipitated with anti tTG antibodies were
separated by SDS-PAGE and electrotransferred onto a polyvinylidene
difluoride membrane (PVDF) (Immobilon P, Millipore) according to
Matsudaira (19)
. Sequence analysis was performed with an
Applied Biosystems model 473A pulsed liquid sequencer with an on-line
PTH-amino acid analyzer.
`Tissue' transglutaminase activity measurement
Cells were mechanically removed from flasks, washed in
phosphate-buffered saline (PBS) without Ca2+ and
Mg2+, and sonicated at 4°C for 20 s. tTG activity
was measured by detecting the incorporation of
[3H]putrescine into N,N'-dimethylcasein, as described
previously (20)
.
Western and ligand blot analysis
For immunoblot, proteins were electroblotted overnight at 4°C
on nitrocellulose membranes. Filters were blocked for 1 h with PBS
containing 3% gelatin. In GST P1-1 and ß-tubulin immunoblotting,
unconjugated anti-mouse immunoglobulin G (IgG) (5 µg/ml, Sigma) was
added to the blocking solution to prevent the cross reaction between
the mouse IgG chains and the secondary antibodies. All additional
immunostaining steps were performed in PBS with 0.05% Tween 20
(PBS-Tween) at room temperature. Filters were incubated with first mAb
for 2 h. The secondary antibody horseradish peroxidase-conjugated
goat anti-mouse (Bio-Rad) was reacted for 1 h. The reaction was
developed by using an enhanced ECL chemiluminescence detection system
(Amersham).
In ligand blot analysis, the membranes were incubated with 3% bovine serum albumin (BSA) in buffer A (50 mM tris-HCl, pH 7.5, and 150 mM NaCl) and then with 10 µg/ml of the purified tTG in buffer A containing 10 mM CaCl2 and 2 mM DTT. After washing with buffer A containing 10 mM CaCl2 and 0.5% Triton X-100, the membranes were incubated with anti-human tTG antibody. In controls, BSA was used instead of purified tTG. Protein substrates binding tTG were visualized by treatment with ECL reagents (Amersham).
Transmission electron microscopy analysis
For ultrathin sectioning and postembedding tTGase labeling,
cells were fixed with 3% paraformaldehyde in 0.2 M cacodylate buffer
(pH 7.3) at 4°C. After washing, cells were postfixed with 1% osmium
tetroxide in the same buffer for 5 min on ice. Cells were then
dehydrated through graded ethanols and embedded in Lowicryl
(21)
. Ultrathin sections were obtained with an LKB
ultramicrotome (Ultratome Nova), stained with mAb anti-tTG (final
dilution 1:50), and incubated with anti-mouse IgG-gold conjugate (gold
particles 10 nm, final dilution 1:5, Sigma). After the postembedding
procedure, sections were stained for 10 min with uranyl acetate and
lead citrate. Finally, sections were observed with a Zeiss EM10C
electron microscope.
Quantitation of apoptosis
To quantitate apoptosis, cells subjected to the different
treatments were cultured in 25 cm2 flasks, cells floating
in the culture medium for 24 h were collected by centrifugation at
800 x g for 5 min and the resulting pellet was pooled
with the cells recovered from flasks. Cell death by apoptosis was
evaluated by flow cytometry using propidium iodide staining on a
FACScan flow cytometer (Becton-Dickinson, Mass.), as described
previously (12)
.
GST P1-1 production, purification, and measurement
Human native enzyme GST P1-1 was produced in Escherichia
coli cells and purified, as described previously (22
,
23
). The enzymatic activity of GST was assayed
spectrophotometrically at 25°C with 1-chloro-2,4-dinitrobenzene
(CDNB) as a cosubstrate under the conditions reported below.
Spectrophotometric measurements were performed in a double beam Uvicon
940 spectrophotometer (Kontron Instruments) equipped with a
thermostatted cuvette compartment. Initial rates were measured at
0.1 s intervals for a total of 12 s after a lag time of
5 s. Enzymatic rates were corrected for spontaneous reaction. A
suitable amount of GST P1-1 sample (typically 1 µg of protein) was
added to a cuvette containing 0.1 M phosphate buffer pH 6.5, plus EDTA
0.1 mM, in the presence of 1 mM glutathione (GSH) (1 ml final volume).
The reaction was started by adding 1 mM CDNB and monitored at 340 nm
(= 9600 M-1 cm-1) (24)
.
GST P1-1 as an in vitro substrate of tTG
GST P1-1 (1.0 mg/ml) was incubated in 0.15 M Tris-HCl buffer (pH
8.3) containing 5 mM CaCl2, 10 mM DTT, and 90 mM NaCl in
the presence of tTG (0.3 mg/ml) at 37°C (0.25 ml final volume).
Aliquots were withdrawn from the mixture at different times, assayed
for GST activity, and subjected to SDS-PAGE and Western blot analysis
as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Characterization of tTG binding proteins in TGA cells
At the molecular level, tTG participates and is able to induce
apoptosis through mechanisms as yet unclear (27)
. We have
attempted to identify the proteins able to interact with tTG during
apoptosis. In a preliminary set of experiments, we verified that the
antibody used in this study was able to selectively precipitate the tTG
protein, as assessed by Western blotting after immunoprecipitation
(Fig. 2A
).Immunoprecipitations with preimmune sera do not result in any
detectable proteins (data not shown). To characterize the proteins that
interact with tTG, TGA cells were metabolically labeled for 24 h
with [35S]methionine before immunoprecipitation. SDS-PAGE
analysis of the immunoprecipitates showed that at least two markedly
labeled proteins with molecular masses between 22 and 12 kDa
coprecipitated with tTG. The specificity of the interaction between tTG
and the proteins described above was confirmed by 1)
blocking the tTG binding with different amounts of purified tTG (Fig. 2B
); 2) the absence of any precipitable
protein(s) in cells transfected with pSV2-Neo which do not show any
detectable tTG by both Western blotting (Fig. 1A
, lane 1)
and immunoprecipitation (Fig. 2B
); and 3) using
SK-N-BE(2)
derived clones, in which synthesis of the tTG
was blocked by the expression of an anti-sense tTGA construct (data not
shown; 12).
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The presence in the immunoprecipitates of additional proteins that incorporated a lower amount of radioactivity was revealed by prolonging the exposure of autoradiography (data not shown). The analysis of total proteins coprecipitated by tTG in nonradiolabeled TGA cells confirmed that, in addition to the low molecular mass bands detected by the [35S]methionine labeling (Fig. 2B ), two polypeptides with a molecular mass of about 110 and 50 kDa were also coprecipitated by anti-tTG mAb (Fig. 2C ). Furthermore, protein staining of the immunoprecipitates revealed that the broad band of ~20 kDa detected by autoradiography was indeed composed of two bands of 22 and 14 kDa (compare Fig. 2B vs. Fig. 2C ). The difference in the radiolabeling among the proteins coprecipitating with tTG likely reflects, in addition to the relative quantity, a difference in their turnover rate (compare Fig. 2B with Fig. 2C ).
To identify these tTG binding proteins, large-scale tTG immunoprecipitates of human TGA cells were electrophoresed and electroblotted onto PVDF membranes and the bands were microsequenced by Edman degradation. The resulting amino acids sequences were analyzed by computer search, using the SWISS-PROT data bank as the main source (Table 1 ).This approach allowed us to identify the tTG binding polypeptides shown in Fig. 2C as the ß-tubulin (50 kDa; band 2), the histone H2B (14 kDa; band 4), and two GST P1-1 fragments of about 22 and 12 kDa (band 3 and 5 respectively). Due to the small amount of purified protein present in the tTG immunoprecipitates, we were unable to sequence the 110 kDa protein (band 1 in Fig. 2C ). To confirm the specificity of tTG interaction with ß-tubulin and GST P1-1, we immunoprecipitated TGA-derived cell extracts with both anti-ß-tubulin and the anti-GST P1-1 antibodies, and detected the presence of tTG in the immunoprecipitates by Western blotting. Fig. 3A shows that immunoprecipitates obtained by using both the anti-ß-tubulin and the anti-GST P1-1 mAb contain high levels of tTG. By using anti-ß-tubulin and GSTP1-1 mAb, we also demonstrated that these two proteins were able to coprecipitate each other (Fig. 3B, C ), thus indicating the presence in TGA cell cytoplasm of a protein complex including tTG, ß-tubulin, and GSTP1-1.
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To verify whether the cytoskeleton acts as a specific binding site for tTG, we carried out the enzyme immunolocalization on detergent-extracted TGA cells by transmission electron microscopy (TEM) (Fig. 4A, B ).Although most of the immunostaining was detected in the cytoplasm, a notable tTG staining was also observed on the nuclear matrix (Fig. 4B ). The gold particles observed at high magnification in the cytoplasm were present as regularly alligned clusters showing a distance between the single gold particles of about 8 nm (Fig. 4A ), which may be referred to as the estimated space between two molecules of ß-tubulin in the microtubules heterodimer structure (28) .
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To study whether the tTG binding proteins described above act as potential substrates, we performed ligand blot analysis (see Materials and Methods). Protein extracts obtained from untreated TGA were separated by SDS-PAGE, transferred onto membranes, and incubated in the presence or absence of exogenous purified tTG, which was finally revealed by the anti-tTG antibody. Data reported in Fig. 4C show that TGA cell extracts contain three low molecular mass proteins, ranging from 25 and 12 kDa, which specifically bind purified tTG on SDS-PAGE analysis. The molecular mass of these putative tTG substrates coincides with that of histone H2B and the two fragments of the GST P1-1 that coimmunoprecipitate with tTG (compare Fig. 2C and Fig. 4C ). In keeping with these findings, recent in vitro studies have shown that the histone H2B acts as a good tTG substrate (29) . Although previous in vitro studies have shown that tubulin may also act as substrate of tTG (30) , no modification of the molecular mass of ß-tubulin was observed in TGA cells when compared with cells transfected with pSV2-Neo (Fig. 5B ).
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GST P1-1 acts as substrate for tTG
To determine whether GST P1-1 acts as a substrate for
tTG, we investigated whether overexpression of tTG leads the
posttranslational modification of GST P1-1 protein in TGA cells. As
shown in Fig. 5
, overexpression of tTG in these neuroblastoma cells was
paralleled by an accumulation of immunoreactive GST P1-1 high molecular
mass products, whereas the GST P1-1-derived cleavage product with a
molecular mass of lower than 20 kDa was present in both control and TGA
cells (Fig. 5A
).
To confirm that GST P1-1 acts as substrate for tTG, we incubated purified GST P1-1 in the presence of purified tTG. Figure 5C shows that high molecular mass GST P1-1 immunopositive polymers were produced in vitro by incubating purified guinea pig tTG with purified human GST P1-1 (Fig. 5C ), thus confirming that GST P1-1 acts as tTG substrate both in vitro and in cells.
To investigate the effect of the tTG-dependent posttranslational modification on the GST P1-1 protein, we measured GST P1-1 enzyme activity after incubation with tTG both in the presence and absence of GSH (Fig. 6A ).The tTG-dependent polymerization of the GST P1-1 observed after incubation and SDS-PAGE analysis was paralleled by a reduction of transferase activity (compare Fig. 5C and Fig. 6A ). The presence of GSH in the incubation mixture was able to prevent the inhibition of the enzyme activity as well as its tTG-dependent polymerization (Fig. 6A, B ).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The intracellular localization of tTG has been a controversial issue for the past 20 years (5) . Although recent findings suggest that nucleosomes are good candidate glutaminyl substrates for the tTG catalyzed reaction processes (29 , 33 ), very little is known about possible interaction between core histones and tTG in vivo. Here, we demonstrate that tTG interacts with histone H2B in mammalian cell lysates; furthermore, we show that this interaction might take place in vivo, as indicated by the enzyme subcellular localization in the nuclear matrix. These data are in keeping with very recent findings showing that, in another human neuroblastoma (SH-SY-5Y) cell line, 93% of total tTG is localized in the cytosol and 7% in the nucleus (34) . The entry of tTG into the nucleus can be enhanced by the nuclear envelope breakdown occurring in cells undergoing apoptosis (35) ; in fact, this event may expose the chromatin to the tTG localized in the cytoplasm. Consistent with this hypothesis, it has been shown that chromatin can bind tubulin (36) .
Previous studies have indicated that tubulin and microtubule-associated protein may act as good substrates for tTG (15) . Although we demonstrate here a specific interaction between tTG and ß-tubulin, no detectable posttranslational modification of ß-tubulin was observed in tTG transfected cells, suggesting that microtubules in neural cells act as a binding site for the enzyme. This finding is particularly relevant with respect to the recently suggested role for the enzyme in neurodegenerative diseases, such as Huntington's disease and others caused by genes with expanded CAG triplet repeats in their coding region 15-17) . Affected individuals are characterized by accumulation of intracellular inclusions in the brain that are composed of insoluble aggregates of proteins containing reiterated glutamine residues. The quantity of these protein aggregates correlates with severity of the disease and the rapid progression to cell death 15-17) . One established property of a glutamine residue is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction (4) . Huntingtin is a cytosolic protein found primarily in association with microtubules in a protein complex including huntingtin-associated protein and dynactin P150 (Glued), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent transport of membranous organelles (37 , 38 ). Therefore, the colocalization of tTG, huntingtin, and its associated proteins on microtubules might favor the interaction of the enzyme with its substrates and, consequently, the tTG-dependent cross-linking. We show here that, in apoptosis-prone TGA cells, tTG is part of a protein complex including ß-tubulin and GST P1-1. These findings are consistent with previous data suggesting a specific localization of GST P 1-1 on microtubules (39) . The interaction of tTG with GST P1-1 at the level of ß-tubulin might lead to the posttranslational modification of the GST P1-1, as suggested by the presence of enzyme's covalent multimers in TGA cells. It has recently been shown that many genes induced during apoptosis encode proteins that may generate or respond to oxidative stress (40) . Massive GSH depletion characterizes the early phases of apoptosis (41 , 42 ). GST-dependent GSH conjugation is a key detoxication reaction that prevents redox cycling and the accumulation of reactive oxygen species; thus, GSTs have a cytoprotective effect involving the elimination of reactive chemical species originating from oxidative metabolism (43) . The question arises as to the functional implications of the tTG-dependent polymerization of GST P1-1. We show that the tTG-dependent posttranslational modification of GST P1-1 is paralleled by a rapid decrease in its catalytic activity in a GSH-dependent manner. These results indicate that GST P1-1 oligomerization leads to functional inactivation of the enzyme, thus suggesting that the tTG-dependent posttranslational modification might involve one or more glutamine residues located in or nearby the GSH binding site (44) . In particular, the residues Gln 51 and/or Gln 64, both strictly conserved among the Pi class, have been reported to be important determinants for the binding of GSH. Based on the analysis of 67 tTG substrate repeats present in two tTG-substrate proteins, Zeeuwen et al. (45) have recently proposed a consensus sequence for TG substrates. As shown in Fig. 7 ,the Gln 51 of the human GST P1-1 shows significant identity with the tTG consensus [GQ(DL)PVK vs. GQLP-K], thus suggesting this glutamine as a potential acyl donor residue.
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The functional inactivation of GST P1-1 may represent an important means by which tTG participates in the irreversible commitment of cells to apoptosis. Considering the early and dramatic depletion of GSH that occurs in cells undergoing apoptosis (42) , the finding that only the GST P1-1 not bound to GSH is susceptible to tTG-dependent posttranslational modification is particularly intriguing. In fact, the tTG-dependent GST P1-1 inactivation might represent a mechanism that leads to the accumulation of increasing amounts of reactive oxigen species, which in turn may lead to the induction of apoptosis.
Is the GST P1-1 a substrate for cystein protease? Many cellular proteins (actin, hungtintin, histone H2B, pRB, and troponin) that are cleaved by caspases and calpains during the cell death execution phase (26 , 46 ) have also been recently shown to undergo tTG-dependent polymerization in apoptosis (7 , 17 , 47 , 48 ), thus indicating that tTG and thiol proteases share many target proteins. Here we show that tTG binds two GST P1-1-derived products cleaved after the Tyr3 and the Asp94 (Fig. 7) . This latter product is interesting, as the cysteine proteases activated during apoptosis cleave after an aspartate residue (26) . The DMVND-G motif found at the GST P1-1 cleavage site (Fig. 7) represents a good potential caspase consensus; in fact, the Gly residue after the Asp94 is present in many caspase substrates, including poly(ADP-ribose)polymerase (Fig. 7) . Val residue in position P3 has also been found in many caspase substrates, including the pro-interleukin 1ß (26) . Considering that the overexpression of tTG in TGA cells is paralleled by an increase of GST P1-1 polymerization, it is tempting to speculate that the tTG-dependent posttranslational modification and enzyme cleavage might be related events.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 L.P. and M.G.F. contributed equally to this
paper.
3 Abbreviations: mAb, monoclonal antibody; BSA, bovine serum albumin; CDNB, dinitrobenzene; CHX, cycloheximide; GSH, glutathione; GST, glutathione S transferase; IgG, immunoglobulin G; PBS, phosphate-buffered saline; pRB, retinoblastoma protein; PVDF, polyvinylidene difluoride membrane; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel eledtrophoresis; STS, staurosporine; tTG, `tissue' transglutaminase; TEM, transmission electron microscopy; Z-VAD, Z-Val-Ala-DL-Asp-fluoromethylketone.
Received for publication June 23, 1998.
Revision received September 24, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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: Cleavage site of the GST P1-1
truncated form of 22 kDa. 