(Received for publication, April 13, 1995; and in revised form, May 22, 1995)
From the
We describe the characterization and purification of a protease
that cleaves sterol regulatory element-binding protein-1 (SREBP-1) and
SREBP-2 in vitro. Cleavage occurs between the basic
helix-loop-helix-leucine zipper and the first transmembrane domain of
each SREBP. This is the region in which the SREBPs are cleaved
physiologically by a sterol-regulated protease that releases an
NH Animal cells control their content of cholesterol in part by
regulating transcription of the genes for the low density lipoprotein
(LDL) The two
known SREBPs, designated SREBP-1 and SREBP-2, are closely related in
sequence and are In sterol-depleted cells a protease clips
SREBP-1a and SREBP-2 at a site between the leucine zipper and the first
membrane attachment region(4, 5, 7) . This
frees the NH In cultured cells SREBP-1 and SREBP-2 are regulated coordinately;
they appear to act in a redundant fashion, and there is no evidence
that heterodimers are required(2, 3) . In hamster
liver SREBP-1 and -2 appear to be regulated independently and not
coordinately(8) . The proteases that process SREBP-1 and -2 in
cultured cells or in liver have not been identified. In the current
experiments we have identified and purified an intracellular protease
that cleaves SREBP-1a and -2 selectively in the region between the
leucine zipper and the first transmembrane domain. The protease has
properties consistent with those of a new family of cysteine proteases
as defined by the interleukin-1
Figure 9:
Labeling of 20-kDa subunit of SCA with
[
To screen for a protease that cleaves SREBP, we translated
the mRNA for SREBP in vitro in the presence of We eventually settled upon a strategy in which cells were
homogenized in a buffer containing a mixture of inhibitors of serine,
aspartyl, and metalloproteases in order to prevent nonspecific
cleavage. Under these conditions we observed a trace amount of specific
SCA in the cytosolic fraction of HeLa cells. The activity was markedly
increased when the cytosolic fraction was preincubated for 2 h at 30
°C. Using the preincubated cytosol as a starting material, we were
able to obtain a partial purification of SCA from HeLa cells and
hamster liver by sequential column fractionation as outlined under
``Experimental Procedures.'' Fig. 1demonstrates
the ability of partially purified SCA from HeLa cells to cleave in
vitro translated
Figure 1:
Cleavage of in
vitro translated SREBP-1a and SREBP-2 by SCA. Aliquots of in
vitro synthesized,
To identify the
cleavage products of the SCA reaction, we translated the mRNA for
SREBP-2 in the absence of labeled amino acids, digested it with SCA,
performed SDS-polyacrylamide gel electrophoresis, and immunoblotted
with antibodies directed against either the NH
Figure 2:
Identification of NH
Fig. 3shows an experiment in which the size of the
NH
Figure 3:
Comparison of SREBP-1a cleaved in
vitro and in vivo. Lanes 1 and 2, 40
µg of HeLa cell nuclear extract in 10 µl of high salt buffer (4) were incubated at 37 °C for 1 h with 30 µl of 50
mM Tris-HCl (pH 8.5) and 1 mM sodium EDTA in the
absence (lane 1) or presence (lane 2) of 20 units of
calf intestine alkaline phosphatase. Lanes 3-5, in
vitro synthesized SREBP-1a (unlabeled) was incubated in 25 µl
of buffer A for 30 min at 30 °C with (lanes 4 and 5) or without (lane 3) 0.2 µg of partially
purified SCA from HeLa cells, followed by incubation at 37 °C for 1
h in 30 µl of the above Tris/EDTA buffer in the absence (lanes
3 and 4) or presence (lane 5) of 20 units of
alkaline phosphatase. Samples were subjected to SDS-PAGE and then
transferred to a nitrocellulose filter. The filter was incubated with a
rabbit antiserum against SREBP-1 directed at the
NH
As a further test for specificity, we compared the
specific cleavage of SREBP-2 by SCA and by two nonspecific proteases,
trypsin and protease V-8 (Fig. 4). The two specific products of
cleavage were observed when 50 ng of partially purified SCA were used (lane 3). Increasing the amount of SCA 10-fold to 500 ng
yielded more of the same cleavage products, but there was no evidence
of further breakdown to smaller fragments (lane 4). Treatment
with 5 ng of trypsin partially cleaved SREBP-2 to a variety of
fragments (lane 6), and 50 ng completely destroyed the protein (lane 7). Similar results were obtained with V8 protease (lanes 9-12). In this and other experiments, we were
unable to find a concentration of trypsin or V8 protease that cleaved
either SREBP-2 or SREBP-1a selectively at the site of SCA cleavage.
Figure 4:
Cleavage
of SREBP-2 by various proteases. Aliquots of in vitro synthesized,
Fig. 5shows that the NH
Figure 5:
DNA binding by in vitro synthesized SREBP-2 after cleavage with SCA. Aliquots of in
vitro translated SREBP-2 (unlabeled) were incubated for 30 min at
30 °C in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 0.2 µg of partially purified
SCA from HeLa cells. We then added a
The properties of SCA suggested that it
might be a cysteine protease of the ICE family (see
``Discussion''). These enzymes are distinguished by their
ability to cleave proteins at Asp residues(9) . Accordingly, we
scanned the sequence of SREBP-1a and SREBP-2 in the region between the
leucine zipper and the first transmembrane domain in search of
conserved Asp residues. We noted the sequences indicated in Table 1which contain a conserved Asp (Asp-460 in SREBP-1a and
Asp-468 in SREBP-2). We hypothesized that the conserved Asp in these
sequences was the site of cleavage by SCA. To test this hypothesis, we
translated the SREBP-1a and SREBP-2 mRNAs in the presence of specific
Figure 6:
NH
To further test
the proposed site of cleavage, we used the technique of in vitro mutagenesis to change Asp-468 to Ala in SREBP-2 (D468A mutation).
We then translated the wild-type and D468A mutant versions of SREBP-2
and cleaved with SCA. As shown in Fig. 7, SCA cleaved wild-type
SREBP-2, but not the D468A mutant. A similar result was obtained for
the D460A mutant of SREBP-1a (data not shown).
Figure 7:
Abolition of cleavage of SREBP-2 by
replacement of Asp-468. Aliquots of in vitro synthesized,
ICE and members of
its family are sensitive to inhibition by sulfhydryl-modifying agents,
including NEM(9) . Fig. 8A shows that NEM
inhibited SCA at a concentration of 10 µM. The inhibition
was prevented by the inclusion of DTT in the reaction (lane
7).
Figure 8:
Inhibition of SCA by N-ethylmaleimide (panel A) and tetrapeptide aldehydes (panel B). Panel A, aliquots (0.2 µg of protein)
of partially purified SCA from HeLa cells were incubated with NEM at
the indicated concentration (lanes 1-7) in 20 µl of
buffer containing no DTT. In lane 7, DTT at a final
concentration of 1 mM was added to the reaction before the
addition of NEM. Panel B, aliquots (0.2 µg of protein) of
SCA were incubated in 20 µl with varying concentrations of the
indicated tetrapeptide aldehyde (lanes 1-10) dissolved
in buffer A containing 3% (v/v) Me
ICE is known to be inhibited by the tetrapeptide aldehyde
Ac-YVAD-CHO (9) . This inhibitor was designed by changing His
at the P2 position to Ala, which improved its affinity as an
inhibitor(9, 21) . Based on this precedent, we
synthesized the tetrapeptide Ac-DEAD-CHO, which corresponds to the
cleavage site in SREBP-2 with Ala substituted for Pro at the P2
position. Fig. 8B shows that Ac-DEAD-CHO inhibited SCA
cleavage of SREBP-2 at 0.1 µM. In contrast, Ac-YVAD-CHO
failed to inhibit at 1 µM. To identify the protein band
corresponding to SCA, we used a [ To obtain a partial amino acid
sequence of SCA, we prepared two peptides from the 20-kDa protein
isolated from the SDS-polyacrylamide gels. We also obtained the
sequence of the NH The relative degree of purification of the SCA preparations used in
the current studies was difficult to determine because of the
semiquantitative nature of the cleavage assay and because the total
amount of enzyme activity appeared to increase during purification,
perhaps owing to the removal of inhibitors. Sterol-regulated cleavage of SREBP is a central mechanism for
the control of cholesterol levels in animal
cells(4, 7, 8) . In this report we have
identified a cytosolic protease, designated SCA, that makes a single
cut in SREBP-1 and SREBP-2 near the site at which these proteins are
cleaved by sterol-regulated proteolysis in vivo. At present,
however, we have no evidence that this SCA cleaves either of the SREBPs
in intact cells. The properties of SCA suggest that it is a cysteine
protease related to the ICE family. These features (9) include:
1) absolute requirement for cleavage after an aspartic acid; 2)
presence in the cytosol in an inactive form that could be activated by in vitro incubation; 3) resistance to inhibitors of serine,
aspartyl, and metalloproteases; 4) sensitivity to inhibition by
sulfhydryl modifying agents such as NEM and by a tetrapeptide
terminating in an aspartic aldehyde; and 5) covalent labeling by
[ ICE was originally isolated
from a human monocytic cell line as an enzyme that cleaves the
precursor of interleukin-1 ICE exists in the cytosol of monocytes as an inactive
45-kDa precursor (9, 22) . The protein is cleaved at
several positions to create an NH ICE cleaves interleukin-1 The
amino acid sequence of human ICE was found to resemble the predicted
amino acid sequence of Ced-3, a protein of Caenorhabditis
elegans(23) . The Ced-3 gene is essential for
programmed cell death, or apoptosis, in that organism. Overexpression
of ICE in animal cells triggers apoptosis, and this can be blocked by
overexpression of the crmA gene, a specific ICE
inhibitor(23) . Whether ICE is a required participant in
apoptosis in animal cells is not yet established. Mice homozygous for a
targeted disruption of the ICE gene have no apparent defect in
apoptosis(24) . The CPP-32 gene was identified when
a sequence tagged site in the human genome was observed to resemble the
sequence of the gene encoding ICE(10) . A cDNA encoding CPP-32
was cloned from a human lymphocyte library, and the sequence predicted
a 277-amino acid protein with 30% identity to human ICE. The QACRG
sequence containing the active site Cys was conserved, and CPP-32 was
postulated to be a cysteine protease of the ICE type(10) . The
predicted molecular mass of CPP-32 is 32 kDa. On the basis of its
resemblance to ICE, CPP-32 was postulated to be cleaved in vivo into 20- and 10-kDa subunits. Overexpression of cDNAs encoding
both of these subunits caused apoptosis in insect Sf9
cells(10) . Whether CPP32 has protease activity and the nature
of its putative substrates are unknown. In contrast to ICE, which is
present primarily in monocytes and macrophages, CPP-32 appears to be
expressed in other cell types as determined by Northern
blotting(10) . The sequences of the NH SCA cuts SREBP-1a at
the sequence SEPDSP between the D-S bond, and it cuts SREBP-2 at
the sequence DEPDSP between the D-S bond. These data suggest that
SCA may prefer negatively charged residues at the P3 or P4 position in
contrast to the bulky hydrophobic residues that are preferred by ICE.
Interestingly, one of the internal cleavage sites in ICE has a Glu at
the P3 position (FEDDA)(9, 21) . In addition, the
postulated cleavage site within CPP-32 has a Glu at the P3 position
(IETDS)(10) . Whether SCA/CPP32 is capable of activating ICE,
and whether it is capable of undergoing autocatalytic cleavage, remains
to be demonstrated. Recently, poly(ADP-ribose) polymerase was shown
to be a substrate for an ICE-like family member, but not for ICE
itself(25) . This enzyme is rapidly cleaved during the early
stages of apoptosis. The cleavage site in poly(ADP-ribose) polymerase
has a Glu at the P3 position (EVDG), raising the possibility that its
cleavage enzyme is SCA/CPP32. Like ICE, SCA can be inhibited by a
tetrapeptide aldehyde. The aldehyde Ac-DEAD-CHO is more than 10 times
as active as Ac-YVAD-CHO against SCA. We have not yet performed studies
to define the amino acids in Ac-DEAD-CHO that are essential for its
inhibitory activity. SCA cleaves SREBP-1a and SREBP-2 at a site
between the leucine zipper and the first transmembrane region, which is
the general region where the enzyme is cleaved by the sterol-regulated
protease(4, 5) . We have no evidence as yet that SCA
participates in sterol-regulated cleavage of SREBP in vivo. We
have assayed for SCA activity in HeLa cells grown in the absence or
presence of sterols. In both cases enzyme activity was undetectable,
and it required activation by in vitro incubation before it
would cut SREBP (data not shown). The current data also raise the
possibility that SREBP-1a and SREBP-2 are cleaved by SCA/CPP32 during
apoptosis. The physiologic function of such cleavage, if it occurs, is
unknown.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal fragment that activates transcription of the
genes for the low density lipoprotein receptor and
3-hydroxy-3-methylglutaryl CoA synthase. The cleavage enzyme,
designated SREBP cleavage activity (SCA), belongs to a new class of
cysteine proteases of the interleukin-1
-converting enzyme (ICE)
family, all of which cleave at aspartic acid residues. Like ICE, SCA
was inactive in cytosol, and it was activated in vitro by
incubation at 30 °C. SCA was resistant to inhibitors of serine,
aspartyl, and metalloproteases, but it was sensitive to N-ethylmaleimide. The enzyme cleaved SREBP-1 and SREBP-2
between the Asp and Ser of a conserved sequence (S/DEPDSP). The
activity was blocked by a tetrapeptide aldehyde,
Ac-Asp-Glu-Ala-Asp-aldehyde (Ac-DEAD-CHO). A purified preparation of
SCA from hamster liver contained a prominent 20-kDa polypeptide that
could be labeled with [
C]iodoacetic acid.
Labeling was blocked by Ac-DEAD-CHO. Partial amino acid sequence of
this polypeptide revealed that it was the hamster equivalent of human
CPP32, a putative protease whose cDNA was recently identified by virtue
of sequence homology to ICE. CPP32 and ICE have been implicated in
apoptosis in animal cells. Whether SCA/CPP32 participates in vivo in the sterol-regulated activation of SREBP, or whether it
activates SREBPs during apoptosis, remains to be determined.
(
)receptor, which supplies cholesterol
from external sources, and enzymes, such as 3-hydroxy-3-methylglutaryl
CoA synthase, which determine the rate of cholesterol synthesis within
the cell(1) . Transcription is regulated by proteins designated
sterol regulatory element-binding proteins (SREBPs) that are bound to
the membranes of the endoplasmic reticulum and nuclear
envelope(2, 3, 4, 5) .
1150 amino acids in
length(2, 3) . Human SREBP-1 is expressed in several
forms as a result of alternative splicing(2, 6) . The
most extensively studied isoform is SREBP-1a. The
NH
-terminal regions of both SREBP-1a and -2 are classic
transcription factors of the basic helix-loop-helix-leucine zipper
family. This region terminates approximately at residue 400 of each
protein. This is followed by a spacer region of
90 amino acids,
followed by a pair of hydrophobic sequences that anchor the proteins to
membranes, followed by a long COOH-terminal extension of
500 amino
acids(5, 7) . The proteins are oriented so that the
NH
-terminal and COOH-terminal regions both extend into the
cytoplasm. The small loop between the two membrane attachment regions
is believed to protrude into the lumen of the ER.
(
)
-terminal segment, which enters the nucleus and
binds to sterol-regulatory elements in the promoters of the LDL
receptor and 3-hydroxy-3-methylglutaryl CoA synthase genes. The SREBPs
activate transcription by virtue of acidic domains at their NH
termini(5) . When sterols accumulate in cells, cleavage
of both SREBPs is reduced, and any residual SREBP in the nucleus is
rapidly degraded by a proteolytic enzyme that is sensitive to
inhibition by N-acetyl-leucyl-leucyl-norleucinal(4) .
-converting enzyme
(ICE)(9) . Peptide sequencing of the purified protease reveals
it to be the hamster homologue of human CPP32, a member of the ICE
family that was cloned by virtue of its DNA sequence resemblance to
ICE, but which has not been studied for proteolytic activity in
vitro(10) . We here describe the purification and
properties of this protease as it relates to the cleavage of SREBP-1a
and -2.
General Methods and Materials
We obtained bovine pancreatic trypsin (sequencing grade),
protease V8, calf intestine alkaline phosphatase, and all protease
inhibitors from Boehringer Mannheim; individual unlabeled amino acids
from Life Technologies, Inc.; radioactive amino acids from Amersham
Corp.; N-ethylmaleimide (NEM) from Sigma; and
Ac-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) from BACHEM Bioscience, Inc.
Site-specific mutagenesis of the cDNAs for human SREBP-2 (Asp-468
Ala) and SREBP-1a (Asp-460
Ala) were performed as
described by Kunkel et al.(11) using the Muta-gene
Phagemid kit (Bio-Rad). cDNA clones for human SREBP-1a(2) ,
human SREBP-2(3) , and rabbit oxysterol binding protein (OSBP) (12) were described in the indicated reference. Human HeLa S3
cells were grown in spinner culture as described
elsewhere(13) . Male Golden Syrian hamsters (100-120 g),
obtained from Sasco (Omaha, NE), were exposed to a 12-h light/12-h dark
cycle and fed a standard mouse/rat chow diet (Teklad, Madison, WI).
In Vitro Translation of SREBP mRNA
SREBP-1a cloned into the SalI site of pBluescript
SK(+), SREBP-2 cloned into the EcoRI site of
EXlox(+) vector, and OSBP cloned into the EcoRI/SmaI site of pGEM-4 were prepared using a
Maxiprep kit (Promega) and translated in the TNT T3 (for SREBP-1a) or
SP6 (for SREBP-2 and OSBP) coupled reticulocyte lysate system
(Promega). Each coupled transcription-translation reaction contained
5-10 µg of plasmid DNA in a final volume of 400 µl
according to the manufacturer's instructions. After incubation at
30 °C for 2 h, each translated SREBP was purified by passing the
transcription-translation mixture through a 10-ml Sephadex G-25 gel
filtration column equilibrated with buffer A (10 mM Hepes-KOH
at pH 7.5, 1.5 mM MgCl
, 10 mM KCl, 1
mM sodium EDTA, 1 mM sodium EGTA, 0.5-1 mM dithiothreitol (DTT)) supplemented with a mixture of protease
inhibitors that consisted of 0.1 mM Pefabloc, 5 µg/ml
pepstatin A, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 25
µg/ml N-acetyl-leucyl-leucyl-norleucinal. The translated
proteins contained within the exclusion volume of the column were used
in assays described below.
Antibodies and Immunoblot Analysis
Rabbit polyclonal antibodies directed against human SREBP-1a
(amino acids 31-175) (2) and human SREBP-2 (amino acids
48-403) (3) were prepared as described in the indicated
reference. A monoclonal antibody IgG-1C6 (IgG subclass 1) was produced
by immunizing a female Balb/C mouse with a fusion protein encoding
amino acids 833-1141 of human SREBP-2 (3) followed by the FLAG
octapeptide DYKDDDDK (14) . IgG-1C6 was purified
from ascites fluid by protein G-Sepharose affinity chromatography.
Immunoblot analysis was performed as described previously(4) .
The primary antibody was detected with horseradish
peroxidase-conjugated anti-rabbit IgG (made in donkeys) using the
enhanced chemiluminescence (ECL) Western blotting detection kit
(Amersham Corp.).
Assay for SREBP Cleavage Activity (SCA)
SREBP-1a or SREBP-2 was translated as described above in a
methionine-free amino acid mixture supplemented with 750 µCi/ml
[S]methionine (>1000 Ci/mmol) according to
the instructions of the manufacturer (Promega). After passage through
the G-25 Sephadex column (see above), 5 µl of the translation
mixture were incubated at 30 °C for varying times with enzyme
fractions in a final volume of 25 µl of buffer A supplemented with
the mixture of protease inhibitors. At the end of the incubation, 25
µl of 2
SDS sample buffer (15) were added to each
tube, after which each sample was boiled for 3 min and then subjected
to electrophoresis on an 8% SDS-polyacrylamide gel. The gel was
subsequently dried and exposed at room temperature to Reflection film
(DuPont NEN) for 16 h.
Purification of SCA from HeLa Cells
All purification steps were performed at 4 °C except for
the SP-Sepharose chromatography, which was done at room temperature.Step 1: Preparation of Activated S-100 Fraction
Cell
pellets from 100 liters of HeLa cells were suspended in 5 volume
of buffer A with protease inhibitors, disrupted by Dounce
homogenization, and centrifuged at 1000
g for 10 min.
The supernatant was centrifuged at 10
g for 1 h in a Sorvall AH629 rotor. The resulting supernatant (S-100
fraction) was incubated at 30 °C for 2 h before fractionation. This
step was necessary to activate the cleavage enzyme.
Step 2: SP-Sepharose Chromatography
The pH of the
activated S-100 fraction (7.5 g of protein) was adjusted to 6.5 with
concentrated HCl, and the mixture was applied to an SP-Sepharose column
(100-ml bed volume) equilibrated with buffer B (buffer A adjusted to pH
6.5 and supplemented with 0.1 mM Pefabloc). The column was
washed with 2 column volumes of buffer B and then eluted with 2 column
volumes of buffer A containing 0.5 M NaCl. The column eluate
was dialyzed overnight against two changes of 2 liters of buffer A
containing 0.1 mM Pefabloc.Step 3: Mono Q Chromatography
The pH of the
dialyzed 0.5 M NaCl eluate from Step 2 (480 mg of protein) was
adjusted to 8.3 with 1 N NaOH, and the mixture was passed
through a 0.22-µm filter. One-half of the preparation was loaded
onto a Mono Q 10/10 column equilibrated in buffer C (buffer A adjusted
to pH 8.3 and supplemented with 0.1 mM Pefabloc). The column
was washed with 2 column volumes of buffer C and then eluted in 4-ml
fractions with an 80-ml linear salt gradient (0 to 0.3 M NaCl). The other half of the preparation was processed on an
identical column. Fractions containing SCA activity (eluting at
150 mM NaCl; fractions 8-10) from the two columns
were pooled and concentrated in a Centriprep 10 concentrator (Amicon).
Step 4: Superdex 75 Gel Filtration
The
concentrated pooled fraction from Step 3 (15 mg of protein) was applied
to a Superdex 75 gel filtration column equilibrated with buffer A with
0.1 mM Pefabloc. After filtration, the active fractions were
pooled and stored in multiple aliquots at -80 °C. This
fraction is referred to as ``partially purified SCA'' and is
used in all experiments except Fig. 9and Table 2.
C]iodoacetic acid. Panel A, aliquots
(
20 µg) of partially purified SCA from hamster liver were
incubated in 30 µl of buffer A (minus DTT) with the indicated
concentration of the indicated tetrapeptide aldehyde at 30 °C for
15 min, followed by the addition of 10 µl of 2 mM
[2-
C]iodoacetic acid (54 mCi/mmol). After an
additional 15 min at 30 °C, the samples were subjected to
electrophoresis on a 10-20% SDS gradient gel and then transferred
to a nitrocellulose filter. The filter was first stained with 0.2%
Ponceau S (Panel B) and then exposed to film at room
temperature for 60 days (Panel A). An arrow denotes
the 20-kDa protein whose
C-radiolabeling was inhibited by
the tetrapeptide aldehyde, Ac-DEAD-CHO.
Purification of SCA from Hamster Liver
All purification steps were carried out at 4 °C except
for the SP-Sepharose chromatography, which was done at room
temperature.Step 1: Preparation of S-100 Fraction
Livers from 25
hamsters (125 g each), rinsed once with 700 ml of cold
phosphate-buffered saline and once with 700 ml of cold buffer A with
protease mixture, were homogenized for 15 s in the same buffer (0.4
g/ml) in a specially designed Waring blender (16, 17) followed by three strokes of a motor-driven
homogenizer. The homogenates were centrifuged at 10
g for 1 h in a Sorvall AH 629 rotor. The resulting supernatant
(S-100 fraction) was filtered through a layer of cheese cloth and
dialyzed overnight against three changes of 6 liters of buffer A with
0.1 mM phenylmethylsulfonyl fluoride.
Step 2: SP-Sepharose Chromatography
The S-100
fraction from Step 1 (14 g of protein) was incubated at 30 °C for 2
h, after which it was adjusted to pH 6.5 with concentrated HCl.
One-half of the preparation was loaded onto a 200-ml SP-Sepharose
column equilibrated with buffer B. After washing with 2 column volumes
of buffer B, the column was eluted with buffer A containing 0.5 M NaCl. The other one-half of the preparation was processed on
an identical column. The active fractions were pooled (500 ml) and
dialyzed overnight against two changes of 6 liters of buffer A with 1
mM phenylmethylsulfonyl fluoride.Step 3: Q-Sepharose Chromatography
The dialyzed
fractions from Step 2 (2.56 g of protein) were adjusted to pH to 8.3
with 1 N NaOH and loaded onto a 100-ml Q-Sepharose column
equilibrated with buffer C. The column was eluted with a 500-ml linear
salt gradient (0-0.3 M NaCl), and 10-ml fractions were
collected. Fractions containing SCA (eluting at 175 mM NaCl; fractions 17-44) were pooled and used directly in the
next step.
Step 4: Hydroxylapatite Chromatography
The pooled
fractions from Step 3 in buffer C (392 mg of protein) were loaded onto
a 10-ml hydroxylapatite column (Macro-Prep ceramic hydroxylapatite,
Bio-Rad) equilibrated with 10 mM potassium phosphate, pH 6.8.
The column was washed with 2 column volumes of 0.1 M potassium
phosphate at pH 6.8 and eluted with an 80-ml linear gradient
(0.1-0.4 M potassium phosphate), and 4-ml fractions were
collected. Fractions containing SCA (eluting at 0.3 M potassium phosphate; fractions 3-12) were pooled and
dialyzed against buffer D (20 mM Tris-HCl at pH 8.0, 10 mM KCl, 1 mM sodium EDTA, 1 mM sodium EGTA, 1
mM DTT, and 0.1 mM Pefabloc).
Step 5: Mono Q Chromatography
The pooled fractions
from Step 4 (50 mg of protein) were loaded onto a Mono Q 10/10 fast
protein liquid chromatography column equilibrated with buffer D. The
column was washed with 2 column volumes of buffer D and eluted with a
120-ml linear salt gradient (0-0.3 M NaCl). Fractions of
5 ml were collected. Fractions containing SCA (eluting at 150
mM NaCl; fractions 6-9) were pooled and concentrated to
2.25 ml with a Centricon 10 (Amicon).
Step 6: Superdex 75 Gel Filtration
The pooled
fractions from Step 5 (12 mg of protein) were loaded onto a
Superdex 75 gel filtration column equilibrated with buffer A with 0.1
mM Pefabloc and eluted with the same buffer. The active
fractions were pooled and dialyzed against buffer E (50 mM MES-HCl at pH 6.0, 10 mM KCl, 1 mM sodium EDTA,
1 mM sodium EGTA, 1 mM DTT; 0.1 mM Pefabloc).
Step 7: Mono S Chromatography
The pooled fractions
from Step 6 (6 mg of protein) were loaded onto a Mono S 5/5 fast
protein liquid chromatography column. SCA was eluted in 2.5-ml
fractions with a 50-ml linear salt gradient (0-1 M NaCl). Fractions containing SCA (eluting at
325 mM NaCl; fractions 4-9) were pooled and concentrated.
Step 8: Superdex 200 Gel Filtration
The pooled
fractions from Step 7 (2 mg of protein) were loaded onto a
Superdex 200 column equilibrated with buffer A with 0.1 mM Pefabloc. The column was eluted with the same buffer, and the
active fractions were pooled and concentrated with a Centricon 10
(
0.5 mg protein). This fraction is referred to as ``partially
purified SCA'' and is used in the experiments of Fig. 9and Table 2.
Gel Mobility Shift Assay
In vitro translated SREBP-2 (unlabeled) was
incubated with a polymerase chain reaction-generated P-labeled oligonucleotide containing two tandem copies of
the wild-type or mutant SRE-1 element of the LDL receptor promoter.
Probes were synthesized and purified, and the DNA binding reactions and
electrophoresis were performed as described
previously(13, 17) .
NH
The mRNAs encoding SREBP-1 and -2 were translated in
vitro in a reticulocyte lysate supplemented with an amino acid
mixture devoid of one amino acid (arginine, proline, valine, or
leucine). The translation reaction was supplemented with the
corresponding radioactive amino acid: 250 µCi/ml
[2,3,4,5--terminal Sequence Analysis of
Radioactively Labeled SREBPs
H]leucine (150 Ci/mmol), 250 µCi/ml
[2,3,4,5-
H]proline (57 Ci/mmol), 250 µCi/ml
[2,3,4,5-
H]arginine (58 Ci/mmol), or 12.5
µCi/ml [U-
C]valine (265 mCi/mmol). After
passage through the G-25 Sephadex column (see above), 100-µl
aliquots of each labeled SREBP were incubated at 30 °C for 30 min
with 20 µg of SCA partially purified from HeLa cells (Superdex 75
fraction) in a final volume of 200 µl of buffer A with the protease
mixture. The reaction mixtures were subjected to electrophoresis on an
8% SDS-polyacrylamide gel and transferred to poly(vinylidene fluoride)
membranes (Immobilon-P
, Millipore). After drying, the
membranes were exposed to an imaging plate and scanned in a Fuji X Bas
1000 PhosphorImager. The bands containing the COOH-terminal product of
the cleavage reaction were excised and subjected directly to multiple
cycles of Edman degradation on an Applied Biosystems model 477A
sequencer. Fractions from each cycle (250 µl) were collected, and a
200-µl aliquot was counted in a scintillation counter.
Partial Amino Acid Sequence of Enzyme That Cleaves SREBPs
Partially purified SCA from hamster liver (Step 8, 0.25
mg) was subjected to electrophoresis in a 10-20% SDS gradient gel
and then transferred onto a piece of poly(vinylidene fluoride) membrane
(Immobilon-P
, Millipore). The 20-kDa subunit was
visualized by Coomassie Blue staining and excised for direct
NH
-terminal sequencing and solid-phase tryptic digestion (18) . Sequence analysis was carried out as described
previously(19) .
Synthesis of Tetrapeptide Aldehyde Inhibitor of SCA
Ac-Asp-Glu-Ala-Asp-aldehyde (Ac-DEAD-CHO) was synthesized
from the known Boc-Asp(OBzl)-HO(20) . The aldehyde
functionality of Boc-Asp(OBzl)-HO was protected as the semicarbazone
(SC) and the Boc group removed under standard acidic conditions to give
Asp(OBzl)-SC. Condensation of this semicarbazone with Boc-Ala affords
Boc-Ala-Asp(OBzl)-SC. The Boc protecting group was removed under acidic
conditions and the resulting Ala-Asp(OBzl)-SC was coupled to
Ac-Asp(OBzl)-Glu(OBzl)-OH to yield Ac-Asp(OBzl)-Glu(OBzl)-Ala-Asp
(OBzl)-SC. The final product was obtained after deprotection of the
carboxylic acid residues by hydrogenolysis and deprotection of the
aldehyde functionality with acetic acid and formaldehyde and was then
purified by high pressure liquid chromatography.
S-labeled amino acids and incubated the protein with
cytosolic and membrane extracts from sterol-depleted cells. We searched
for an activity that would cleave SREBP so as to generate an
NH
-terminal fragment similar in size to the nuclear form of
the protein as determined by SDS-polyacrylamide gel electrophoresis.
All of our initial attempts were unsuccessful. Either we failed to
observe proteolysis or we observed multiple fragments of inappropriate
size.
S-labeled SREBP-1a and SREBP-2, as
monitored by SDS-gel electrophoresis and autoradiography. In vitro translation of the SREBP-1a and SREBP-2 mRNAs produced precursor
proteins that migrated in the range of 125 kDa (lanes 1 and 3). Incubation with SCA generated two major cleaved products
of
55 and 70 kDa from either SREBP-1a or SREBP-2 (lanes 2 and 4). In addition, a minor cleaved product just below
the 55-kDa band was observed in the case of SREBP-2 (lane 4).
As a control for specificity, we used another in vitro translated protein, OSBP. SCA caused no appreciable cleavage of
this protein (lanes 5 and 6).
S-labeled SREBP-1a (lanes 1 and 2), SREBP-2 (lanes 3 and 4), and
OSBP (lanes 5 and 6) were incubated in the absence (lanes 1, 3, and 5) or presence (lanes
2, 4, and 6) of 0.2 µg of SCA that was
partially purified from HeLa cells as described under
``Experimental Procedures.'' After incubation at 30 °C
for 30 min, the samples were subjected to SDS-PAGE. The gel was exposed
to film for 16 h at room temperature.
-terminal or
COOH-terminal domains of SREBP-2 (Fig. 2). The lower band, with
an apparent molecular mass of 55 kDa, reacted with the antibody against
the NH
terminus (lane 2), and the upper band, with
an apparent molecular mass of 70 kDa, reacted with the antibody
directed against the COOH terminus (lane 4).
-terminal
and COOH-terminal fragments of SREBP-2 after in vitro cleavage. Aliquots of in vitro synthesized SREBP-2
(unlabeled) were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 0.2 µg
of partially purified SCA from HeLa cells for 30 min at 30 °C. The
samples were subjected to SDS-PAGE and then transferred to a
nitrocellulose filter. The filter was incubated with a rabbit antiserum
against the NH
-terminal portion of SREBP-2 (1:2000
dilution) (lanes 1 and 2) or with IgG-1C6 (5
µg/ml), a monoclonal antibody against the COOH-terminal portion of
SREBP-2 (lanes 3 and 4). Bound antibodies were
visualized with horseradish peroxidase-conjugated anti-rabbit IgG using
the ECL Western blot detection system kit as described under
``Experimental Procedures.'' The film was exposed at room
temperature for 5 s (lanes 1 and 2) or 10 s (lanes 3 and 4). N, NH
-terminal; C, COOH-terminal.
-terminal fragment of SREBP-1a that was generated in
vitro was compared with that isolated from HeLa cell nuclear
extracts. The in vivo nuclear protein migrated as a doublet at
65 kDa (lane 1). This was reduced to the range of
55
kDa after treatment with alkaline phosphatase (lane 2). The in vitro translated SREBP-1a was cleaved by SCA to generate an
NH
-terminal fragment with an apparent molecular mass of
55 kDa (lane 4), which was within the range of the
phosphatase-treated protein from the nuclear extracts (lane
2). Treatment of the in vitro translated SREBP-1a with
alkaline phosphatase caused no change in molecular weight (lane
5). We conclude from this experiment that SCA cuts SREBP-1a at a
site that is close to the site that generates the nuclear fragment in
intact cells. These data also provide the first evidence that SREBP-1a
from nuclear extracts is phosphorylated. Similar results were obtained
with respect to the NH
-terminal fragment of SREBP-2 (data
not shown).
-terminal domain (1:1000 dilution), and the bound
antibody was visualized with horseradish peroxidase-conjugated
anti-rabbit IgG using the ECL Western blot detection system kit. The
film was exposed at room temperature for 5
s.
S-labeled SREBP-2 were incubated in the
standard cleavage buffer with increasing amounts of the indicated
protease. Lanes 1-4: 0 (lane 1), 5 (lane
2), 50 (lane 3), and 500 ng (lane 4) of
partially purified SCA from HeLa cells. Lanes 5-8: 0 (lane 5), 5 (lane 6), 50 (lane 7), and 500
ng (lane 8) of trypsin. Lanes 9-12: 0 (lane
9), 5 (lane 10), 50 (lane 11), and 500 ng (lane 12) of protease V-8. The cleavage reactions were carried
out at 30 °C for 30 min, after which the samples were subjected to
SDS-PAGE. The gel was exposed to film for 16 h at room
temperature.
-terminal cleavage product
generated by SCA from SREBP-2 was active in binding in vitro to its specific DNA recognition sequence, designated SRE-1 (13) . Full-length in vitro translated SREBP-2 was
incubated with a
P-labeled oligonucleotide that contains
two copies of the 10-base pair SRE-1. Although full-length SREBP-1c has
been previously demonstrated to bind SRE-1 in
vitro(2) , no binding of SREBP-2 was observed in this
experiment apparently because the amount added was too low (lane
1). After cleavage by SCA, the released NH
-terminal
fragment of SREBP-2 retarded the mobility of the
P-labeled
oligonucleotide (lane 3). The cleaved SREBP-2 fragment did not
bind a mutant version of the SRE-1 that is transcriptionally inactive (lane 4). Thus, cleavage of SREBP-2 by SCA markedly activates
its DNA binding activity.
P-labeled, polymerase
chain reaction-derived DNA probe (94 base pairs) containing two copies
of either the wild-type SRE-1 sequence from the human LDL receptor (lanes 1 and 3) or a mutant version of the SRE-1
sequence with a substitution of A for C at position 10 of SRE-1, which
abolishes transcriptional activity(13, 17) . After 20
min at room temperature, the reaction mixtures were subjected to
electrophoresis, and the gel was exposed to film for 16 h at -80
°C with an intensifying screen. The upper arrow denotes
the band corresponding to SREBP-2 bound to SRE-1; X denotes a
contaminating protein from the reticulocyte lysate that binds both the
mutant (M) and wild-type (WT)
probes.
C- or
H-labeled amino acids. After in
vitro cleavage with SCA, we isolated the COOH-terminal fragment by
SDS-gel electrophoresis and subjected it to Edman degradation. Fig. 6shows the NH
-terminal sequences predicted for
the COOH-terminal fragments of SREBP-1a and -2, assuming that cleavage
occurred at the postulated Asp. When SREBP-1a was labeled with
[
H]proline and digested with SCA, we observed
peaks at positions 2 and 11, which are the expected positions if
SREBP-1a had been cleaved at the postulated Asp.
[
H]Arginine gave a peak at position 14, which
corresponds to the first Arg residue. The sequence of SREBP-2 revealed
two peaks for [
C]valine, two peaks for
[
H]leucine, and two peaks for
[
H]arginine, all at the appropriate positions.
These data support the hypothesis that SCA cleaves SREBP-1a at Asp-460,
and SREBP-2 at Asp-468 as shown in Table 1.
-terminal sequence of
COOH-terminal fragment of human SREBP-1a (left) and SREBP-2 (right) after cleavage by SCA. The mRNA encoding SREBP-1a or
SREBP-2 was translated in the presence of the indicated
H-
or
C-labeled amino acid and cleaved by partially purified
SCA from HeLa cells as described under ``Experimental
Procedures.'' The two cleaved products corresponding to the
NH
-terminal and COOH-terminal fragments of the SREBPs were
identified by the PhosphorImager scan of SDS gels. The slower migrating
band for both SREBP-1 and SREBP-2, which corresponded to the
COOH-terminal fragment (Fig. 2), was processed for
NH
-terminal sequence analysis as described under
``Experimental Procedures.'' The radioactivity recovered at
each cycle of Edman degradation is plotted. The amino acid sequences
shown at the top of each panel correspond to the sequences that
immediately follow the postulated sites of cleavage of SREBP-1a and -2
(amino acids 460 and 468, respectively).
S-labeled wild-type SREBP-2 (lanes 1-5) or
mutant SREBP-2 with the aspartic acid at position 468 mutated to
alanine (lanes 6-10) were incubated at 30 °C for 30
min with increasing amounts of partially purified SCA from HeLa cells:
0 (lanes 1 and 6), 0.1 (lanes 2 and 7), 0.2 (lanes 3 and 8), 0.4 (lanes 4 and 9), and 0.8 µg (lanes 5 and 10). The samples were subjected to SDS-PAGE, and the gel was
exposed to film for 16 h at room
temperature.
SO. The final
concentration of Me
SO in all lanes was 0.3%. Panels A and B, after incubation at 30 °C for 15 min, aliquots
(5 µl) of in vitro synthesized,
S labeled
SREBP-2 in buffer A were added to each 20-µl reaction (final volume
of 25 µl). After an additional 15 min (Panel A) or 60 min (Panel B) at 30 °C, the samples were subjected to
SDS-PAGE, and the gels were exposed to film for 16 h at room
temperature.
C]iodoacetic
acid labeling technique that was previously used to label
ICE(21) . The partially purified preparation of hamster SCA
contained a prominent protein that migrated at
20 kDa and another
that migrated at
10 kDa as determined by Ponceau-S staining of
SDS-polyacrylamide gels (Fig. 9B). The band at 20 kDa
was labeled when the SCA preparation was incubated with
[
C]iodoacetic acid (Fig. 9A, lane 1). This labeling was not inhibited by Ac-YVAD-CHO (lanes 2 and 3), but it was inhibited by Ac-DEAD-CHO
at concentrations as low as 10 nM (lanes 4 and 5). Ac-DEAD-CHO did not inhibit labeling of any of the other
bands on the gel. These data strongly suggest that the 20-kDa protein
contains the active site Cys, which is protected from labeling when the
site is occupied by Ac-DEAD-CHO.
terminus of this protein (Table 2). All of these sequences showed a high degree of
identity with the recently reported sequence of human CPP-32, a member
of the ICE family whose cDNA was isolated by a polymerase chain
reaction strategy based on DNA sequence homology with ICE(10) .
C]iodoacetic acid in a fashion that was
inhibited by a competing tetrapeptide aldehyde. The suspicion that SCA
is an ICE-related enzyme was confirmed when peptide sequence data
revealed that the protein is the hamster equivalent of human CPP-32, a
member of the ICE family(10) .
into its active secreted form (reviewed
in (9) ). Monocytes secrete interleukin-1
through an
unorthodox pathway that involves export from the cytosol through the
plasma membrane without involvement of the endoplasmic reticulum or the
usual secretory apparatus. Interleukin-1
is synthesized as a
31-kDa polypeptide that must be cleaved to a 17.5-kDa form in order to
be secreted. The cleavage event is catalyzed by ICE, and it is
inhibited when intact monocytes are treated with a specific ICE
inhibitor.
-terminal fragment of
20-kDa and a COOH-terminal fragment of 10-kDa. These fragments remain
associated as a heterodimer, and two heterodimers associate to form the
tetrameric active enzyme (9) . The cleavage events that produce
the 20- and 10-kDa fragments occur at Asp residues, and evidence has
been provided to indicate that these cleavages are
autocatalytic(9, 21) . The 20-kDa subunit of ICE
contains the active site Cys, which is imbedded in the highly conserved
sequence QACRG.
between the Asp and
Gly residues of a YVHDG sequence(9) . Cleavage absolutely
requires an Asp in the P1 position. There is a strong requirement for
Tyr or another bulky hydrophobic residue in the P4 position. The His in
the P2 position can be changed to Ala without loss of efficiency. There
is a preference for small aliphatic residues such as Gly or Ala in the
P1` position. ICE is inhibited by the tetrapeptide aldehyde
Ac-YVAD-CHO, which forms a hemithioacetal with the active site Cys.
Inhibition is competitive and is slowly reversible(9) .
terminus
and the two peptides from purified SCA obtained in the present study
indicate that it is the hamster homologue of human CPP-32 (Table 2). The degree of sequence identity is much higher than
that observed between the SCA peptides and different members of the ICE
family, including ICE, Ced-3, and ICH-1/Nedd-2.
-converting enzyme; NEM, N-ethylmaleimide;
OSBP, oxysterol-binding protein; SCA, SREBP cleavage activity; SRE-1,
sterol-regulatory element-1; SREBP, sterol regulatory element-binding
protein; MES, 4-morpholineethanesulfonic acid; SC, semicarbazone; PAGE,
polyacrylamide gel electrophoresis; Ac-DEAD-CHO,
Ac-Asp-Glu-Ala-Asp-aldehyde; Ac-YVAD-CHO, Ac-Tyr-Val-Ala-Asp-aldehyde;
Boc, t-butoxycarbonyl; Bzl, benzyl.
We thank Steve McKnight and Barry Rosen of Tularik,
Inc. for invaluable help in the design and synthesis of SCA inhibitors;
our colleagues Xianxin Hua and Juro Sakai for providing site-directed
mutants of SREBP; Gloria Brunschede, Marc Duderstadt, John Dawson, and
Amber Luong for excellent technical assistance; Richard Gibson for
invaluable help with animals; and Carolyn Moomaw and Steve Afendis for
excellent help with amino acid sequence analysis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.