title: Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini.
abstract: The nearly 600 proteases in the human genome regulate a diversity of biological processes, including programmed cell death. Comprehensive characterization of protease signaling in complex biological samples is limited by available proteomic methods. We have developed a general approach for global identification of proteolytic cleavage sites using an engineered enzyme to selectively biotinylate free protein N termini for positive enrichment of corresponding N-terminal peptides. Using this method to study apoptosis, we have sequenced 333 caspase-like cleavage sites distributed among 292 protein substrates. These sites are generally not predicted by in vitro caspase substrate specificity but can be used to predict other physiological caspase cleavage sites. Structural bioinformatic studies show that caspase cleavage sites often appear in surface-accessible loops and even occasionally in helical regions. Strikingly, we also find that a disproportionate number of caspase substrates physically interact, suggesting that these dimeric proteases target protein complexes and networks to elicit apoptosis.
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
authors: Sami
authors: S
authors: Mahrus
authors: Jonathan C
authors: JC
authors: Trinidad
authors: David T
authors: DT
authors: Barkan
authors: Andrej
authors: A
authors: Sali
authors: Alma L
authors: AL
authors: Burlingame
authors: James A
authors: JA
authors: Wells
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000737:chemistry / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics; D013379:Substrate Specificity
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D011506:Proteins; D005047:Etoposide; D020169:Caspases