©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of an Interleukin-1 Converting Enzyme-related Cysteine Protease That Cleaves Sterol Regulatory Element-binding Proteins between the Leucine Zipper and Transmembrane Domains (*)

(Received for publication, April 13, 1995; and in revised form, May 22, 1995)

Xiaodong Wang (1)(§) Jih-tung Pai (1) Elizabeth A. Wiedenfeld (1)(¶) Julio C. Medina (3) Clive A. Slaughter (2) Joseph L. Goldstein (1) Michael S. Brown (1)

From the  (1)Department of Molecular Genetics and (2)Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and (3)Tularik, Inc., South San Francisco, California 90408

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

Animal cells control their content of cholesterol in part by regulating transcription of the genes for the low density lipoprotein (LDL)()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) .

The two known SREBPs, designated SREBP-1 and SREBP-2, are closely related in sequence and are 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.()

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-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) .

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-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.


EXPERIMENTAL PROCEDURES

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.


Figure 9: Labeling of 20-kDa subunit of SCA with [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-terminal Sequence Analysis of Radioactively Labeled SREBPs

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-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.


RESULTS

To screen for a protease that cleaves SREBP, we translated the mRNA for SREBP in vitro in the presence of 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.

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 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).


Figure 1: Cleavage of in vitro translated SREBP-1a and SREBP-2 by SCA. Aliquots of in vitro synthesized, 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.



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-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).


Figure 2: Identification of NH-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.



Fig. 3shows an experiment in which the size of the NH-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).


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-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.



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, 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.



Fig. 5shows that the NH-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.


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 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.



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 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.




Figure 6: NH-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).



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, 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.



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) MeSO. The final concentration of MeSO 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.



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 [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.

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 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) .

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.


DISCUSSION

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 [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) .

ICE was originally isolated from a human monocytic cell line as an enzyme that cleaves the precursor of interleukin-1 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.

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-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.

ICE cleaves interleukin-1 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) .

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 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.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant HL20948 and the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Postdoctoral Fellowship from the Damon Runyon-Walter Winchell Cancer Research Fund (1156).

Supported by Medical Scientists Training Grant GM08014.

The abbreviations used are: LDL, low density lipoprotein; DTT, dithiothreitol; ICE, interleukin-1-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.

X. Hua, M. S. Brown, Y. K. Ho, and J. L. Goldstein, manuscript in preparation.


ACKNOWLEDGEMENTS

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.


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