From The Center for Blood Research,
¶ Massachusetts General Hospital and § Department of
Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 and
Manitoba Institute of Cell Biology, University of Manitoba,
Winnipeg, R3E OV9 Canada
Received for publication, June 20, 2000, and in revised form, September 5, 2000
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ABSTRACT |
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The cytotoxic T lymphocyte protease granzyme A
induces caspase-independent cell death in which DNA single-strand
nicking is observed instead of oligonucleosomal fragmentation. Granzyme
A is a specific tryptase that concentrates in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by the
caspase activator granzyme B. Here we show that granzyme A treatment of
isolated nuclei enhances DNA accessibility to exogenous endonucleases.
In vitro and after cell loading with perforin, GrnA
completely degrades histone H1 and cleaves core histones into ~16-kDa
fragments. Histone digestion provides a mechanism for unfolding
compacted chromatin and facilitating endogenous DNase access to DNA
during T cell and natural killer cell granule-mediated apoptosis.
Cytotoxic T lymphocytes
(CTL)1 induce apoptosis by
engagement of cell surface death domain-bearing receptors, such as Fas, or by exocytosis of cytolytic granules containing perforin and serine
proteases called granzymes (Grn) (1-4). Experiments with mice in which
the perforin gene is genetically disrupted suggest that the granule
exocytosis pathway is the primary mechanism for CTL protection from
viral infection and tumors (5). GrnA and GrnB, the most abundant
granzymes, are delivered to the cytosol of targeted cells via perforin,
concentrate in target cell nuclei, and independently and
synergistically induce apoptosis (6-13). Death receptor engagement and
GrnB activate the caspase apoptotic pathway (14). However, CTL induce
target cell death in the presence of caspase blockade and in some cases
in target cells that overexpress Bcl-2 (13, 15-19). This suggests that
CTL also activate caspase-independent cell death pathways.
The most abundant CTL protease, GrnA, induces rapid cell death that has
the features of apoptosis, except for oligonucleosomal DNA degradation
(13). Until recently it was unclear whether GrnA induces any DNA damage
since small fragments of DNA are not released, DNA degradation is not
visualized on agarose gels, and DNA breaks are not generally detected
with terminal deoxynucleotidyltransferase labeling. Induction of cell
death by GrnA was considered slow, based on assays that measure the
release of small radiolabeled DNA fragments into the supernatant.
However, other apoptotic changes occur as rapidly with GrnA loading as
with GrnB loading (Ref. 13, data not shown). DNA damage in the form of
single-strand nicks occurs within 2 h in cells loaded with GrnA.
These breaks can be labeled with Klenow polymerase and visualized on
denaturing alkaline agarose gels. None of the features of GrnA-induced
death are blocked by caspase inhibition or Bcl-2 overexpression.
Moreover, effector caspases are not activated by GrnA loading.
Therefore, the GrnA pathway, including the DNA damage it induces, is
caspase-independent. However, perforin loading of GrnA facilitates the
oligonucleosomal DNA damage of the caspase-dependent DNase
(CAD/DFF40) (20-22) activated by loading GrnB.
Although many of the features of GrnB-induced cell death have been
elucidated with the definition of caspase activation pathways, the
story is incomplete since GrnB induces cell death but not DNA
fragmentation when caspase activation is blocked (13, 19). However,
still less is known about the molecular events triggered by GrnA.
Although GrnA has tryptic activity, it is a highly specific protease.
When comparable concentrations of GrnA and trypsin with equivalent
benzyloxycarbonyl-L-lysine-thiobenzyl esterase
activity are incubated at 37 °C with nuclear lysates, trypsin
completely degrades the sample within 1 h, so that no bands are
visible on SDS-PAGE gels. GrnA, on the other hand, only visibly
degrades 1 of 22 bands after 25 h (23). Moreover, only a few
candidate GrnA substrates, including interleukin-1 While looking for potential nuclear substrates of GrnA, we found that
immobilized histone H1 binds GrnA on far-Western blots and that histone
H1 is cleaved in nuclear lysates by purified CTL granule extracts
(23).3 Recombinant GrnA
degrades purified histone H1 in vitro but requires the
presence of heparan sulfate (HS).3 The HS dependence of the
in vitro cleavage reaction may not be surprising since the
granzymes released from cytolytic granules are complexed to sulfated
proteoglycans and because both GrnA and histone H1 are very basic
(calculated pI values, 9.1 and 11.3, respectively).
Histone H1 is an attractive substrate for a cell death pathway in which
DNA is targeted. The linker histone H1 plays a critical role in
chromatin hypercondensation (35-37), which may protect genomic DNA
from endonuclease digestion. Lys-and Arg-rich histones might be
especially good targets of a tryptase like GrnA. Therefore, we
investigated whether histones might be physiologically relevant targets
of GrnA. Here we show that recombinant GrnA in vitro and in vivo, after cell loading with perforin, degrades not only
histone H1 but also cleaves core histones. The cleavage sites
recognized by GrnA are distinct from those cut by trypsin; in fact
histone H1 is initially cut in a folded core region that is protected from trypsin digestion. We also find that GrnA loading of isolated nuclei disrupts chromatin and facilitates exogenous DNase access to
DNA. Chromatin disruption by histone modification may be essential to
GrnA-induced DNA nicking and may also contribute to the synergy of GrnA
and GrnB in inducing oligonucleosomal DNA fragmentation.
Cell Lines--
K562, Jurkat, COS, and HL60 cells were obtained
from the American Type Culture Collection and grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM HEPES, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 50 µM Granzymes and Perforin--
Recombinant GrnA and enzymatically
inactive Ser Recombinant PHAP II--
PHAP II cDNA (30) was amplified by
polymerase chain reaction from an Epstein-Barr virus-transformed B cell
line library using primers containing BamHI and
Xhol restriction sites (5'-atc gga tcc gat gtc ggc gcc ggc
ggc caa-3' and 5'-atg ctc gag gtc atc ttc tcc ttc atc ctc ctc t-3').
The polymerase chain reaction product was inserted into pET-25b(+)
(Novagen) through the BamHI and Xhol cloning
sites. The PHAP II plasmid was transformed into BL21 cells (Novagen)
and PHAP II expression was induced by adding 1 mM
isopropyl-1-thio- Preparation of Mononucleosomes and Purified
Histones--
Mononucleosomes and histones were purified from HeLa or
HL60 cells as described (40). Briefly, 109 cells were lysed
in Nonidet P-40 lysis buffer (20 mM HEPES (pH 7.5), 0.25 M sucrose, 3 mM MgCl2, 0.2%
Nonidet P-40, 3 mM In Vitro GrnA Cleavage Reaction--
Purified histones or
H1-stripped HeLa cell nucleosomes (0.5 µg of DNA) were incubated with
various amounts of recombinant granzymes in the presence or absence of
heparan sulfate (Sigma), recombinant PHAP II, or random plasmid DNA
(prepared by plasmid DNA purification kit (Qiagen)) in 25-µl reaction
buffer (100 mM Tris HCl (pH 8.0), 50 mM NaCl)
at 37 °C for indicated times. The cleavage reaction was stopped by
adding 5× SDS-loading buffer containing a mixture of protease
inhibitors (50 µg/ml antipain, 2 µg/ml aprotinin, 40 µg/ml
bestatin, 60 µg/ml chymostatin, 10 µg/ml E-64, 1 µg/ml leupeptin,
1 µg/ml pepstatin, and 1 mg/ml 4-(2-aminoethyl)benzenesulfonyl
fluoride (Sigma) to which was added 250 µM GrnA
specific inhibitor Ph-NHCONH-CiEtOIC (a kind gift of J.C. Powers,
Georgia Institute of Technology, Atlanta, GA) (34)). Boiled samples
were electrophoresed on 15% SDS-PAGE gels (for core histones) or 12%
SDS-PAGE gels (for histone H1) and analyzed by immunoblot using
polyclonal antisera against histone H1, H2B, and H3 (Biodesign
International, Inc.) or staining with GelCode® Blue
(Pierce). For N-terminal sequencing of the cleavage products, electrophoresed samples were transferred to a polyvinylidene difluoride membrane (ProBlottTM, Applied Biosystems), and the desired
Coomassie-stained bands were cut and sent for protein sequencing by the
Tufts Core Facility.
Cleavage of Histones in Isolated Nuclei--
Nuclei were
isolated from HL-60 cells by Nonidet P-40 lysis as described above and
washed twice with buffer B (20 mM Tris (pH 7.2), 0.25 M sucrose, 3 mM CaCl2). Nuclei
(1 × 106) in 100 µl of buffer B containing 50 mM NaCl were incubated with the indicated amounts of GrnA
with or without HS at 37 °C for indicated times. The reaction was
stopped by adding 5× SDS-loading buffer and the protease inhibitor
mixture. Boiled samples were analyzed by SDS-PAGE and immunoblot.
Limited Nuclease Digestion Assay--
Isolated nuclei as above
were washed twice with lysis buffer, resuspended in buffer B containing
1% Triton X-100 and then incubated on ice for 5 min. After further
washing with buffer B, 1 × 106 nuclei in 100 µl
were incubated with 400 nM GrnA or PMSF-inactivated GrnA
and/or 400 nM PHAP II at 37 °C for 2 h. Micrococcal
nuclease (20 units, Roche Molecular Biochemicals) or RQ1 DNase I
(10 units, Promega) was added for 5 min or 10 min, respectively, before
the addition of stopping buffer (10 mM EDTA, 200 µg/ml
protease K, and 2% SDS in 20 mM Tris (pH 8.0)). After
overnight deproteinization, DNA was extracted with phenol and ethanol
precipitation and analyzed by ethidium bromide staining on 1.5%
agarose gels.
Granzyme Loading with Perforin--
Recombinant GrnA, Ser Klenow Incorporation Assay--
To assess single-stranded DNA
damage induced by GrnA loading, which is not detectable by terminal
dUTP nick-end labeling assay, the Klenow fragment of DNA polymerase was
used to label DNA breaks as described (13). Six h after Grn loading
with perforin, pelleted cells were lysed in an equal volume of Nonidet
P-40 lysis buffer and incubated with 5 units of Klenow (New England
Biolabs) and 10 µCi of 32P-dCTP (PerkinElmer Life
Sciences) for 1 h at 37 °C. Radiolabeled nuclei, pelleted by
centrifuging for 5 min at 2580 × g and washed twice in
5 ml of Nonidet P-40 lysis buffer, were counted after adding
scintillation fluid (Beckman). The DNA labeling index was calculated by
dividing the dpm of loaded cells by the dpm of mock-treated cells.
Histone H1 Is an In Vitro Substrate of GrnA in the Presence of
Negatively Charged Heparin Sulfate or PHAP Proteins--
Histone H1 is
an in vitro substrate of GrnA complexed to HS3
(Fig. 1A). We recently
identified two highly acidic proteins PHAP II/set/TAF-I GrnA Cleaves Histone H1 and Core Histones in Isolated
Nuclei--
To determine whether histone H1 cleavage also occurs
in situ, we treated isolated HL60 nuclei with GrnA in the
presence or absence of HS. (Fig.
2A) Within 1 h of
incubation at 37 °C with 180 nM GrnA, histone H1
(visualized as a group of bands of molecular mass of ~35 kDa) is
cleaved to ~25 kDa as seen on immunoblot. At higher concentrations,
smaller fragments are sometimes visualized, as in in vitro
assays. At higher enzyme concentrations or longer incubation times, no
immunoreactive band is visualized, suggesting complete digestion (Fig.
2, B and C). Similar results are found with
nuclei isolated from Jurkat and HeLa cells (data not shown). The
addition of HS does not enhance the cleavage, suggesting that native
molecules in the nucleus can replace negatively charged HS. Cleavage
occurs as early as 15 min after adding GrnA at concentrations of GrnA
as low as 240 nM. To determine whether core histones might also be targets of GrnA, GrnA-treated nuclei were also analyzed by
immunoblot for histones H2B and H3 (Fig. 2D). Specific
antibodies to the other core histones are not available. Both of these
core histones were also cleaved to smaller fragments, with kinetics similar to the histone H1 cleavage reaction. Cleavage of the core histones is specific, since it does not occur with mutant enzyme or in
the presence of GrnA inhibitor.
GrnA Treatment of Nuclei Enhances Chromatin Susceptibility to
Exogenous Nucleases--
Disruption of the histones in chromatin might
alter the sensitivity of DNA to degradation by endonucleases activated
during apoptosis. GrnA treatment of isolated nuclei without the
addition of exogenous nucleases shows no apparent change in DNA length on agarose gels (data not shown). To determine whether GrnA treatment enhances the nuclease susceptibility of chromatin, isolated nuclei were
permeabilized with 1% Triton X-100 and pretreated with GrnA with and
without recombinant PHAP II before brief incubation with micrococcal
nuclease (Fig. 3A) Without
GrnA pretreatment, genomic DNA is digested into oligonucleosomes; after
GrnA pretreatment, only mononucleosome-sized fragments remain. Although
PHAP II is a nucleosome assembly protein that might be expected to
alter DNA accessibility, the addition of recombinant PHAP II, either alone or with GrnA, does not alter susceptibility to micrococcal nuclease. After limited RQ1 DNase I digestion of isolated nuclei (Fig. 3B), DNA fragmentation is also more complete after
GrnA pretreatment. DNA from nuclei that were mock-treated or treated with PMSF-inactivated GrnA contains fragments greater than 1.2 kilobases in size; after GrnA treatment, the largest visualized fragments are only 300 base pairs. Therefore, GrnA loading of intact
nuclei enhances DNA nuclease digestion. GrnA enhancement of
endonuclease susceptibility requires its serine protease activity.
DNA Is an in Vitro Cofactor for GrnA Histone
Cleavage--
Although the PHAP proteins can be found in either the
nucleus or cytoplasm, they do not localize to the nucleus by
immunofluorescence microscopy and immunoblot under the exponential
growth conditions used here (data not shown). Because DNA is a
prominent acidic nuclear molecule associated with histones and because
DNA binds in a sequence-independent manner to GrnA (data not shown), we determined whether plasmid DNA can substitute in vitro as
the negatively charged moiety in the histone H1 cleavage reaction. When
plasmid DNA is added, histone H1 is completely degraded (Fig. 4A). Adding approximately
equal amounts by weight of plasmid DNA and histone H1 substrate
optimizes cleavage in solution; either more or less leads to less rapid
proteolysis (Fig. 4A and data not shown). Histone H1 begins
to be cleaved in vitro within 2 min and is completely
degraded by 1 h (Fig. 4B). Core histones contained in
purified nucleosomes stripped of linker histones, prepared as described
by Cote et al. (40), were also cut by GrnA in
vitro (Fig. 4C). When the core histones were separated from DNA, their in vitro cleavage was facilitated by
addition of plasmid DNA (Fig. 4D). Stained polyacrylamide
gels of GrnA-treated purified core histones also demonstrates that all
the core histones are GrnA targets in vitro.
Histones Are Cleaved by GrnA in Perforin-loaded Cells in the
Presence of Caspase Inhibition at GrnA Concentrations Required to
Induce Cytolysis--
The concentrations of GrnA and kinetics of
histone H1 cleavage suggest that histone H1 and possibly the core
histones might be physiologically relevant GrnA substrates. We tested
this by loading GrnA into K562, HeLa, and Jurkat cells with a sublytic concentration of perforin, the CTL granule protein required for granzyme concentration in the nucleus (6, 12, 39) (Fig. 5A). In GrnA-loaded cells, but
not in cells treated with GrnA or perforin alone, histone H1 was
completely degraded by 400 nM GrnA within 2 h.
Moreover, histone H1 was not cleaved in cells loaded with either the
enzymatically inactive Ser GrnA Cuts Histone H1 in a Core Folded Region and Cleaves the
N-terminal Tail of Histone H2B at Cleavage Sites Distinct from
Trypsin--
Although GrnA is a tryptase, it cleaved the arginine- and
lysine-rich histones at specific sites that are different from the described previously trypsin sites (Fig.
6). The N-terminal sequence SLVSKGTLVQ of
the GrnA-produced ~25-kDa histone H1 fragment places the GrnA
cleavage after Lys-86-90 of the various H1 subtypes
(GenBankTM accession numbers P10412 (histone H1.4), P16401
(H1.1), P16403 (H1.2), P16402 (H1.3) CAB11421 (H1.5), P22492 (H1T)). This is in a folded core region of histone H1 (amino
acids 36-121) that is normally protected from trypsin digestion (36), supporting the notion that the histone H1 cleavage by GrnA is specific.
The major GrnA cleavage band of purified core histones was also
analyzed by N-terminal sequencing. The N-terminal sequence KGSKKAVTK of
this band corresponds to cleavage after Lys-12 in the N-terminal tail
of Histone H2B. This GrnA cleavage site also differs from the trypsin
digests, which occur after Lys-21 and Lys-24 of H2B (44). Although the
cleavage site is different, removal of part of the N-terminal tail of
the core histones may have a similar effect as trypsin at unfolding
chromatin. A minor component of protein in this band contained the
N-terminal sequence of histone H3, suggesting that this histone may be
cut by GrnA near the C terminus.
We have shown that GrnA, a key CTL and natural killer cell
enzyme that induces cell death of virally infected or transformed cells, can disrupt chromatin and make it more susceptible to nuclease digestion. This may be due to the specific activity of this tryptase for arginine- and lysine-rich histones. Just as GrnA enhances DNA
susceptibility to exogenous endonucleases, it is also likely to make
DNA more susceptible to the caspase-activated DNase CAD triggered via
GrnB during cell-mediated lysis.
GrnA cleavage of histones may be an important factor in the synergistic
enhancement of DNA degradation by GrnA and -B (6, 7, 13). Although we
have not seen evidence for histone degradation in apoptosis induced by
GrnB loading or activation of the Fas pathway, other modifications of
histones may also open up chromatin accessibility to the
caspase-activated DNase. In fact, histone H1 is poly(ADP-ribosyl)ated
during apoptosis induced by chemotherapeutic drugs or UV irradiation
(45). Furthermore, inhibition of poly(ADP-ribosyl)ation in cells
treated with these apoptosis inducers blocks both the enhanced
accessibility of chromatin to exogenous DNases and internucleosomal DNA
fragmentation caused by activation of endogenous DNases. A role for
histone H1 interaction with CAD has also been suggested by the fact
that histone H1 facilitates the DNase activity of CAD/DFF (46). It has
also been suggested that chromatin with higher degrees of histone
acetylation is more susceptible to apoptotic nucleases (58). Since DNA
fragmentation is a hallmark of apoptosis, it should come as no surprise
that the histone proteins that maintain chromatin in a condensed
configuration and make it inaccessible need to be modified to allow
access to endonucleases, just as they are modified to open up chromatin
for transcriptional activation.
The compact structure of DNA complexed to core histones and
hypercondensation induced by histone H1 (35-37) may protect genomic DNA from endonuclease digestion. When chromatin is stripped of linker
histones (of which histone H1 is the predominant example), chromatin is
released from a compacted solenoid to an extended state. When
mononucleosomes stripped of linker histones are then treated with
trypsin, the N-terminal highly basic tails of the core histones, which
are the sites of histone acetylation for transcriptional regulation,
are removed, leaving fragments similar in size to what we observe with
GrnA treatment (44). The basic tails have little to do with the
formation of the nucleosome core but are important in stabilizing the
chromatin solenoid (47). Trypsinized polynucleosomes unfold into a more
extended structure and are degraded more rapidly by exogenous nucleases
than their untreated counterparts (47). Tryptic digestion of chromatin cleaves small N-terminal peptides of at most 27 amino acids in length
from the free N-terminal tails of histones H2A, 2B, 3, and 4 and even
smaller fragments (10 or fewer amino acids) from the C termini of only
histones H2A and H3 (44, 48-50). This digestion leaves intact the
central nucleosome core. Although GrnA cleavage of the core histones
differs in detail from the action of trypsin, both enzymes cleave
fragments at the free termini, leaving the core of the core histones
intact. The net effect of removing the basic tails of the core histones
on chromatin structure may be similar. However, structural studies of
GrnA-treated chromatin are required to prove this. In light of our
finding, it is interesting that loading trypsin into cells induces all
the nuclear features of apoptosis, including oligonucleosomal DNA
fragmentation (51).
An indirect piece of evidence of histone modification during apoptosis
comes from the prevalence of anti-histone antibodies in mice and humans
with systemic lupus erythematosis (52). Recent studies suggest that
many autoantigens are directed at proteins modified during apoptosis
(53). In fact lupus autoantibodies recognize histone H1 and the
N-terminal released fragments of the core histones, which are not the
major antigenic regions when whole core histones are injected into
animals (54).
Although initial in vitro studies suggested that
proteoglycan was required for histone H1 cleavage by GrnA,3
we found that a variety of negatively charged molecules (PHAP I, PHAP
II, and DNA) could facilitate the in vitro reaction.
Moreover, loading free GrnA into intact cells or isolated nuclei
efficiently led to histone cleavage, which suggests that DNA provides
the balancing negative charge in vivo. The granzymes are
released from cytolytic granules complexed to serglycans, which may
help enhance in vivo stability (55).3 It is
unknown whether the granzymes dissociate from the proteoglycan complex
before uptake into the target cell, in an endocytic vesicle during
uptake, (56) or sometime thereafter. A recent study showed that
proteoglycan-complexed GrnB was less active than free GrnB in cleaving
caspases-3 and -7 in vitro but was comparable in caspase activation when loaded into intact cells (55). This suggests that the
granzyme-proteoglycan complexes probably dissociate before the
granzymes encounter their cytosolic or nuclear substrates.
It is intriguing that all the substrates of GrnA that we have
identified (PHAP II, histone H1, and core histones) are known to
associate with and modify chromatin structure. However, it is unclear
what the role of PHAP II is in making DNA more accessible for
degradation or in the histone digestion we describe here. PHAP II was
first identified as a translocated gene (set) linked to the
nucleoporin can in undifferentiated leukemia (29). It was
postulated that the nucleoporin linkage facilitated the nuclear localization of the set protein (57). The nucleosome assembly protein
function of set/PHAP II (32) could then function to unwind as well as
assemble chromatin, thereby enhancing transcription for leukemogenesis.
We found that PHAP II also translocates to the nucleus of target cells
during CTL lysis.4 Similarly, we postulated that nucleosome
disassembly by PHAP II might be the first step for DNA degradation.
However, the addition of recombinant PHAP II with GrnA to isolated
nuclei does not enhance endonuclease digestion compared with adding
GrnA alone. Moreover, we also found that GrnA cleavage of PHAP II
disrupts its nucleosome assembly function.4 This makes this
hypothesis for PHAP II function in GrnA-mediated cell death less likely.
GrnA degradation of histones and subsequent enhancement of chromatin
sensitivity to DNases is likely to be an important component of
GrnA-induced DNA fragmentation and the synergistic induction of DNA
fragmentation by the two predominant granzymes (6, 7, 13).
Post-translational modifications of the histones, which modulate
chromatin structure and DNA accessibility for transcription, do not
appear to grossly alter histone susceptibility to GrnA cleavage, but
this needs to be explored further.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, fibronectin,
type IV collagen, the thrombin receptor, pro-urokinase-type plasminogen activator, and nucleolin have been identified (23-27). In most cases,
the biological significance of these substrates remains to be
demonstrated. In fact, nucleolin does not appear to be a direct
substrate of GrnA.2 We
recently identified the nucleosome assembly protein PHAP
II/set/TAF-I
/I2PP2A as a substrate of GrnA
during CTL lysis and GrnA loading with perforin (13, 28-34).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol.
Ala GrnA were produced and purified as previously
reported (13, 34). Recombinant GrnB was produced and purified as
described previously (38). Perforin was purified from the rat RNK-16
cell line as described (39) and used at a sublytic concentration that
induced <10% cytolysis in a 2-h assay, as determined independently
for each cell line.
-D-galactopyranoside (Sigma).
His-tagged PHAP II was sequentially purified by nickel resin (Novagen)
and anion exchange chromatography. The identity and proper folding of
the recombinant protein was verified by reactivity with a rabbit antiserum raised to an N-terminal PHAP II peptide as described (29) and
by its function in a nucleosome assembly
assay.4
-mercaptoethanol, and 0.4 mM PMSF). Pelleted nuclei were washed twice with lysis buffer and once with buffer A (20 mM HEPES (pH 7.5), 3 mM MgCl2, 0.2 mM EGTA, 3 mM
-mercaptoethanol, and 0.4 mM PMSF). The
nuclei were resuspended in buffer A to which was added dropwise with gentle stirring one-third of the total volume of buffer A containing 0.6 M KCl and 10% glycerol. After stirring for 10 min at
4 °C, the nuclear pellet was recovered by centrifugation for 30 min at 17,500 × g. To prepare mononucleosomes, chromatin
was isolated after Dounce homogenization, treated for 5 min at 37 °C
with micrococcal nuclease, and applied to a Sepharose CL-6B column. The
fractions were analyzed on SDS-PAGE and native agarose gels after
deproteinization. Fractions containing mononucleosomes stripped of
histone H1 were pooled. To purify histones, nuclear pellets containing
~12 mg of DNA were resuspended in 50 ml of HAP buffer (50 mM sodium phosphate (pH 6.8), 0.6 M NaCl, 1 mM
-mercaptoethanol, and 0.4 mM PMSF) and
stirred gently for 30 min at 4 °C. To this mixture was added 20 g of dry Bio-Gel HTP Hydroxylapatite powder (Bio-Rad) with stirring. The resin was packed into a column, and the flow-through containing partially pure histone H1 was collected. After further washing with 600 ml of HAP buffer, core histones were eluted from the
column with HAP buffer containing 2.5 M NaCl. Histone H1
was further purified by gel filtration.
Ala GrnA, or GrnB were loaded with perforin into K562 or Jurkat cells
as described (13). For each reaction 1 × 105 cells in
60 µl of Hanks' balanced salt solution with 1 mg/ml bovine serum
albumin, 1 mM CaCl2, and 2 mM
MgCl2 were incubated with an indicated amount of
recombinant granzymes and sublytic concentrations of perforin at
37 °C for indicated times. In some experiments, caspase activation
was blocked by preincubating effector and target cells for 30-60 min
with 100 µM z-VAD-FMK and 100 µM z-DEVD-FMK
(Calbiochem), concentrations that were maintained throughout the
experiment. Quantitation of cytolysis induced by loading was assayed in
parallel experiments by treating 51Cr-labeled cells with
GrnA and perforin, incubating at 37 °C for 2 h, and counting
51Cr released into the supernatant on a Packard Topcount as
described (13). Specific cytotoxicity was calculated by the formula:
((sample release)
(spontaneous release))/((total release)
(spontaneous release)) × 100. Cells were lysed by adding 5×
SDS-loading buffer and a mixture of described previously protease
inhibitors. Samples were boiled for 5 min and then electrophoresed on
15% SDS-PAGE and immunoblotted with antibodies against histone H1,
H2B, and H3 and Rho-GDI (Pharmingen).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/I2PP2A (calculated pI ~ 3.9) and PHAP I/I1PP2A (calculated pI ~ 3.8) as candidate participants in the GrnA cell death pathway based on
their binding to recombinant GrnA (30, 34, 41). PHAP II, which has
nucleosome assembly protein activity (32), is a GrnA substrate in
vitro after GrnA loading with perforin and after CTL attack (13,
34). Moreover, PHAP II binds to purified core histones in an ionic
interaction (data not shown and Ref. 42). We therefore tested whether
this negatively charged molecule could substitute for HS in the histone
H1 cleavage reaction (Fig. 1A). Histone H1 is cleaved by
GrnA more efficiently on a molar basis in the presence of recombinant
PHAP II compared with HS. Histone H1 is cleaved in the presence of HS
to a fragment with an apparent molecular mass of 25 kDa at the lowest
concentration of GrnA tested (7 nM). At higher
concentrations of GrnA, smaller fragments of ~22- and 18-kDa are
often seen; at the highest concentrations, no immunoreactive band is
visible. It should be noted that all the histones migrate aberrantly on
SDS-PAGE gels because of their charge. Molecular masses given in this
paper are the apparent molecular masses. The digestion in the presence
of PHAP II is more rapid and complete; none of the intermediate H1
cleavage products are detected at any of the concentrations tested.
Histone H1 isoforms of differing mobilities, due to differences in
backbone sequence or post-translational modifications (43), are all
susceptible to GrnA digestion. Histone H1 in vitro cleavage
is inhibited by increasing concentrations of salt beginning at
physiological concentrations of ~150 mM. (Fig.
1B) The PHAP II-associated acidic protein PHAP I is
comparable with PHAP II as a cofactor for GrnA in the histone H1
cleavage reaction (data not shown). The lack of specificity for the
negatively charged cofactor and the inhibition by salt suggest that
ionic interactions between these charged molecules are important.
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Fig. 1.
GrnA cleaves histone H1 in vitro
in the presence of PHAP II. A, PHAP II is more
efficient on a molar basis than HS in promoting the in vitro
cleavage of purified histone H1 by GrnA. Histone H1 (200 ng) was
treated for 4 h at 37 °C with serial dilutions of GrnA
(beginning with 960 nM at the highest concentration) in the
presence of 1 µM recombinant PHAP II or 27 µM HS. Without either PHAP II or HS, no histone cleavage
occurs; neither does it occur with enzymatically inactive Ser Ala
GrnA (960 nM). B, GrnA cleavage of histone H1 in
the presence of PHAP II is inhibited by increasing concentrations of
salt beginning at 150 mM. Immunoblots probed for histone H1
are shown. K, ×1000.
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Fig. 2.
GrnA cleaves histone H1 and core histones in
isolated nuclei without HS. A, HL60 nuclei were
incubated at 37 °C with 180 nM GrnA for 1 h in the
presence or absence of HS, and the nuclear pellet (P) and
supernatant (S) were analyzed by SDS-PAGE and histone H1
immunoblot. Histone fragmentation does not require HS in
situ in isolated nuclei. There was no detectable whole or
fragmented histone H1 released into the nuclear supernatants.
B and C, dose response for a 1-h incubation of
intact nuclei and kinetics of cleavage in situ.
D, core histones H2B and H3, visualized by immunoblot, are
also cleaved in isolated HeLa nuclei treated with GrnA.
S AGrnA, Ser
Ala GrnA.
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Fig. 3.
GrnA treatment enhances chromatin
accessibility to exogenous nucleases in isolated premeabilized
nuclei. A, COS nuclei were incubated for 2 h with
medium (mock digestion; lane 1), 400 nM PHAP II
(2), 400 nM GrnA (3), or 400 nM GrnA and 400 nM PHAP II (4) and
then incubated for 5 min with micrococcal nuclease before stopping the
reaction. B, HL60 nuclei were incubated at 37 °C for
3 h with medium (mock digestion; lane 1), 400 nM PMSF-treated GrnA (2), or 400 nM
active GrnA (3) before digestion with RQ1 DNase I for
10 min. Extracted DNA visualized by ethidium bromide staining.
Lane M, 100-base pair DNA ladder (New England
Biolabs).
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Fig. 4.
A and B, DNA
substitutes for HS or PHAP II in the in vitro cleavage
reaction of purified histone H1. Purified histone H1 (1 µg) was
digested in a 25-µl reaction volume, and the reaction was analyzed by
GelCode® staining of SDS-PAGE gels. The reaction time for
A was 1 h. C, core histones H2B and H3 in
linker histone-stripped mononucleosomes are cut by GrnA. Samples
analyzed by immunoblot. D, purified core histones (1 µg)
were also digested by GrnA in vitro. The addition of DNA
enhances the in vitro reaction. Core histones were
visualized by GelCode® staining. The first lane
contains no histones. The closed arrows in A,
B, and D indicate GrnA. K,
×1000.
Ala mutant GrnA or the homologous GrnB.
In a dose response assay, histone H1 cleavage began to be detected at
GrnA concentrations of 25-100 nM and within 30-60 min of
loading GrnA. Although GrnA loading mimicked the conditions of CTL
lysis, it is difficult to know what effective concentrations of
perforin and GrnA are present in the synaptic cleft formed by the
apposition of a CTL with its specifically recognized target cell.
However, the concentration of GrnA, which induces histone H1 cleavage,
was comparable with that which induces cell death measured by
51Cr release assay (Fig. 5B) or single-stranded
DNA damage measured by Klenow incorporation (13) (Fig. 5C).
To determine whether GrnA digestion of the core histones is
physiologically relevant, immunoblots of GrnA-loaded cells were also
probed for histones H2B and H3 (Fig. 5D). Histone H2B and H3
were cleaved to ~16-kDa fragments with kinetics and concentration
requirements similar to that for histone H1. Therefore, the core
histones, like linker histone H1, are likely to be cleaved under
physiological conditions of cytolysis induced by CTL. Although histone
H1 digestion went to completion in vivo, the core histones
were not completely degraded. Histone H1 and core histone cleavage
after perforin loading of GrnA was specific. Some other protein
substrates of the caspase pathway, such as Rho-GDI and the catalytic
subunit of DNA-dependent protein kinase (data not shown),
were not cleaved after GrnA loading but were cleaved after GrnB loading
(not shown and Ref. 13). Moreover, histone H1 and the core histones
were not degraded after loading GrnB or enzymatically inactive GrnA.
Furthermore, GrnA cleavage of histones H1, H2B, and H3 was independent
of caspase activation since it occurred in the presence of caspase
blockade with z-VAD-FMK and z-DEVD-FMK (Fig. 5D). Histone H1
was also not degraded in Jurkat cells treated with anti-Fas antibody,
an activator of the caspase pathway (data not shown).
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Fig. 5.
GrnA loading of K562 cells with perforin
induces cleavage of histone H1 and core histones, even in the presence
of caspase blockade. A, GrnA, but not GrnB or Ser Ala GrnA, loading induces histone H1 degradation. Granzymes were loaded
with a sublytic concentration of perforin into K562 cells. The granzyme
loading was performed by incubation at 37 °C for 2 h with
indicated concentrations of GrnA or 400 nM Ser
Ala GrnA
or 500 nM GrnB or by incubation for indicated times with
200 nM GrnA. B, dose response for cytotoxicity
induced by GrnA loading, measured by a 2 h 51Cr
release assay, shows that the dose of GrnA required for cutting histone
H1 parallels that for induction of cell death. C, dose
response for single-stranded nicking induced by GrnA loading, measured
by Klenow incorporation 6 h after loading, shows that the dose of
GrnA required for cutting histone H1 parallels that for DNA damage. The
DNA-labeling index compares the amount of labeling induced after
treatment compared with mock-treated cells. D, GrnA loading
leads to cleavage of histone H1 and core histones H2B and H3 in the
presence of complete caspase blockade with z-DEVD-FMK and z-VAD-FMK.
Blots of control samples, performed in parallel but not treated with
caspase inhibitors, are indistinguishable (not shown). K562 cells were
preincubated for 1 h with caspase inhibitors before
perforin-loading GrnA (200 nM and 2-fold serial dilutions)
for the indicated times. Cell lysates were analyzed by immunoblot for
the indicated proteins. The caspase substrate Rho-GDI was not cleaved.
S
AGrnA, Ser
Ala GrnA.
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Fig. 6.
GrnA cleaves histone H1 and H2B at different
sites than trypsin. A, the N-terminal sequence of the
initial cleavage product of histone H1 identifies the GrnA cleavage
site after Lys-85. This is in a core region of histone H1 (amino acids
36-121) that is protected from trypsin digestion (36). The doublet
visualized in the uncleaved and cleaved histone H1 bands corresponds to
post-translational modifications of histone H1. B, when
purified core histones are digested by GrnA in the presence of plasmid
DNA, N-terminal sequencing of the dominant band demonstrates GrnA
cleavage of histone H2B after Lys-12, as indicated by the open
arrow. The closed arrows identify the previously
reported trypsin cleavage sites (44).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Powers for the GrnA inhibitor and R. Kingston, J. Th'ng and F. Rosen for useful suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI45587.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. Tel.: 617-278-3381; Fax: 617-278-3493; E-mail: lieberman@cbr.med.harvard.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M005390200
2 M. Pasternack, unpublished information.
3 L. Wagner, W. Gartner, P. J. Beresford, L. Bensinger, Y. Ge, A. H. Greenberg, J. Lieberman, M. S. Pasternack, submitted for publication.
4 P. J. Beresford, D. Zhang, D. Oh, Z. Fan, E. L. Greer, M. Jaju, and J. Lieberman, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CTL, cytolytic
T lymphocyte;
Grn, granzyme;
GrnA, granzyme A;
Ser Ala GrnA, enzymatically inactive GrnA with Ser
Ala mutation of the active
site;
GrnB, granzyme B;
CAD/DFF, caspase-dependent DNase;
PHAP, putative HLA-associated protein;
HS, heparan sulfate;
PMSF, phenylmethylsulfonyl fluoride;
z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone;
z-DEVD-FMK, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone;
PAGE, polyacrylamide gel electrophoresis.
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