Purification of an interleukin-1β converting enzyme-related cysteine protease that cleaves sterol regulatory element-binding proteins between the leucine zipper and transmembrane domains

X. Wang, J. T. Pai, E. A. Wiedenfeld, J. C. Medina, C. A. Slaughter, J. L. Goldstein, M. S. Brown

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)18044-18050
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number30
DOIs
StatePublished - Jan 1 1995

Fingerprint

Sterol Regulatory Element Binding Proteins
Leucine Zippers
Caspase 1
Cysteine Proteases
Purification
Sterol Regulatory Element Binding Protein 1
Peptide Hydrolases
Sterols
Aldehydes
Cricetinae
Iodoacetic Acid
Apoptosis
Peptides
Ethylmaleimide
Conserved Sequence
LDL Receptors
Metalloproteases
Enzymes
Transcription
Viperidae

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification of an interleukin-1β converting enzyme-related cysteine protease that cleaves sterol regulatory element-binding proteins between the leucine zipper and transmembrane domains. / Wang, X.; Pai, J. T.; Wiedenfeld, E. A.; Medina, J. C.; Slaughter, C. A.; Goldstein, J. L.; Brown, M. S.

In: Journal of Biological Chemistry, Vol. 270, No. 30, 01.01.1995, p. 18044-18050.

Research output: Contribution to journalArticle

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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.",
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AU - Pai, J. T.

AU - Wiedenfeld, E. A.

AU - Medina, J. C.

AU - Slaughter, C. A.

AU - Goldstein, J. L.

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AB - 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.

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