title: Complementary proteomic tools for the dissection of apoptotic proteolysis events.
abstract: Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a protein's structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques--the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range. By applying this integrated proteomic work flow to analyze proteolytic events resulting from the induction of intrinsic apoptosis in Jurkat cells via etoposide treatment, 3346 proteins were quantified, of which 360 proteins were identified as etoposide-induced proteolytic substrates, including 160 previously assigned caspase substrates. In addition to global profiling, a targeted approach using BAX HCT116 isogenic cell lines was utilized to dissect pre- and post-mitochondrial extrinsic apoptotic cleavage events. By employing apoptotic activation with a pro-apoptotic receptor agonist (PARA), a limited set of apoptotic substrates including known caspase substrates such as BH3 interacting-domain death agonist (BID) and Poly (ADP-ribose) polymerase (PARP)-1, and novel substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome were also identified.
authors.affiliation: Department of Protein Chemistry, Genentech Inc, 1 DNA Way, South San Francisco, California 94080, USA.
authors.forename: Victoria C
authors.initials: VC
authors.lastname: Pham
authors.forename: Robert
authors.initials: R
authors.lastname: Pitti
authors.forename: Veronica G
authors.initials: VG
authors.lastname: Anania
authors.forename: Corey E
authors.initials: CE
authors.lastname: Bakalarski
authors.forename: Daisy
authors.initials: D
authors.lastname: Bustos
authors.forename: Suchit
authors.initials: S
authors.lastname: Jhunjhunwala
authors.forename: Qui T
authors.initials: QT
authors.lastname: Phung
authors.forename: Kebing
authors.initials: K
authors.lastname: Yu
authors.forename: William F
authors.initials: WF
authors.lastname: Forrest
authors.forename: Donald S
authors.initials: DS
authors.lastname: Kirkpatrick
authors.forename: Avi
authors.initials: A
authors.lastname: Ashkenazi
authors.forename: Jennie R
authors.initials: JR
authors.lastname: Lill
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects*; D001224:Aspartic Acid / Q000737:chemistry; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry; D020169:Caspases / Q000737:chemistry; D019295:Computational Biology; D005047:Etoposide / Q000494:pharmacology; D045325:HCT116 Cells; D006801:Humans; D019169:Jurkat Cells; D009687:Nuclear Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000737:chemistry; D000071456:Sequestosome-1 Protein; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry; D033921:Vesicular Transport Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001224:Aspartic Acid; D051023:BH3 Interacting Domain Death Agonist Protein; D020169:Caspases; D005047:Etoposide; D009687:Nuclear Proteins; D010455:Peptides; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins; D016601:RNA-Binding Proteins; D000071456:Sequestosome-1 Protein; D014157:Transcription Factors; C494814:BID protein, human; C118585:P62 protein, human; C099718:SQSTM1 protein, human; C096163:TFG protein, human; D033921:Vesicular Transport Proteins; C063552:transcription factor BTF3; C499320:PARP1 protein, human