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 NH2-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 beta-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 degrees 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 [14C]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.
Highlights
II Supported by Medical Scientists Training Grant GM08014. 1 The abbreviations used are: LDL, low density lipoprotein; DTT, dithiothreitol; interleukin-1/J-converting enzyme (ICE), interleukin-1J3-converting enzyme; NEM, N-ethylmaleimide; oxysterol binding protein (OSBP), oxysterol-binding protein; SREBP cleavage activity (SCA), sterol regulatory element-binding proteins (SREBPs) cleavage activity; SRE-1, sterol-regulatory element-1; SREBP, sterol regulatory element-binding protein; MES, 4-morpholineethanesulfonic acid; SC, temal sources, and enzymes, such as 3-hydroxy-3-methylglutaryl CoA synthase, which determine the rate of cholesterol synthesis within the cell (1)
1 The abbreviations used are: LDL, low density lipoprotein; DTT, dithiothreitol; ICE, interleukin-1J3-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, temal sources, and enzymes, such as 3-hydroxy-3-methylglutaryl CoA synthase, which determine the rate of cholesterol synthesis within the cell (1)
To identify the cleavage products of the 8CA reaction, we translated the mRNA for 8REBP-2 in the absence of labeled amino acids, digested it with 8CA, performed 8D8-polyacrylamide gel electrophoresis, and immunoblotted with antibodies directed against either the NH2-terminal or COOH-terminal domains of 8REBP-2 (Fig. 2).The lower band, with an apparent molecular mass of 55 kDa, reacted with the antibody against the NH2 terminus, and the upper band, with an apparent molecular mass of 70 kDa, reacted with the antibody directed against the COOH terminus
Summary
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.; Nethylmaleimide (NEM) from Sigma; and Ac-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) from BACHEM Bioscience, Inc. Step 1 (14 g of protein) was incubated at 30 oc 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. 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 "pa rti ally purified SCA" and is used in the experiments of Fig. 9 and Table II. After passage through the G-25 Sephadex column (see above), 100-ILl aliquots of each labeled SREBP were incubated at 30 °C for 30 min with 20 ILg of SCA partially purified from HeLa cells (Superdex 75 fraction ) in a final volume of200 ILl of buffer A with the protease mixture. The final product was obtained after deprotection of the carboxylic acid residues by hydrogenolysis and deprotection of the aldehyde functionality with acetic acid and form aldehyde and was purified by high pressure liquid chromatography
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