Purification of an Interleukin-1 Converting Enzyme-related Cysteine Protease That Cleaves Sterol Regulatory Element-binding Proteins between the Leucine Zipper and Transmembrane Domains

doi:10.1074/jbc.270.30.18044

Purification of an Interleukin-1 Converting Enzyme-related Cysteine Protease That Cleaves Sterol Regulatory Element-binding Proteins between the Leucine Zipper and Transmembrane Domains (*)

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

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-1β-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 [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.

Footnotes

↵^§ Recipient of a Postdoctoral Fellowship from the Damon Runyon-Walter Winchell Cancer Research Fund (1156).
↵^¶ Supported by Medical Scientists Training Grant GM08014.
↵* 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.
↵¹ The abbreviations used are:

LDL

low density lipoprotein

DTT

dithiothreitol

ICE

interleukin-1β-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.
↵²X. Hua, M. S. Brown, Y. K. Ho, and J. L. Goldstein, manuscript in preparation.
- Received April 13, 1995.
- Revision received May 22, 1995.