title: Purification of an interleukin-1 beta converting enzyme-related cysteine protease that cleaves sterol regulatory element-binding proteins between the leucine zipper and transmembrane domains.
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.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235, USA.
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: J T
authors.initials: JT
authors.lastname: Pai
authors.forename: E A
authors.initials: EA
authors.lastname: Wiedenfeld
authors.forename: J C
authors.initials: JC
authors.lastname: Medina
authors.forename: C A
authors.initials: CA
authors.lastname: Slaughter
authors.forename: J L
authors.initials: JL
authors.lastname: Goldstein
authors.forename: M S
authors.initials: MS
authors.lastname: Brown
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D020170:Caspase 1; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D016350:Leucine Zippers; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C497387:SREBF1 protein, human; C497390:SREBF2 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors; D003546:Cysteine Endopeptidases; D020170:Caspase 1