title: Structural and kinetic determinants of protease substrates.
abstract: Two fundamental questions with regard to proteolytic networks and pathways concern the structural repertoire and kinetic threshold that distinguish legitimate signaling substrates. We used N-terminal proteomics to address these issues by identifying cleavage sites within the Escherichia coli proteome that are driven by the apoptotic signaling protease caspase-3 and the bacterial protease glutamyl endopeptidase (GluC). Defying the dogma that proteases cleave primarily in natively unstructured loops, we found that both caspase-3 and GluC cleave in alpha-helices nearly as frequently as in extended loops. Notably, biochemical and kinetic characterization revealed that E. coli caspase-3 substrates are greatly inferior to natural substrates, suggesting protease and substrate coevolution. Engineering an E. coli substrate to match natural catalytic rates defined a kinetic threshold that depicts a signaling event. This unique combination of proteomics, biochemistry, kinetics and substrate engineering reveals new insights into the structure-function relationship of protease targets and their validation from large-scale approaches.
authors.affiliation: Apoptosis and Cell Death Research Program at the Burnham Institute for Medical Research, La Jolla, California, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Timmer
authors.forename: Wenhong
authors.initials: W
authors.lastname: Zhu
authors.forename: Cristina
authors.initials: C
authors.lastname: Pop
authors.forename: Tim
authors.initials: T
authors.lastname: Regan
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Snipas
authors.forename: Alexey M
authors.initials: AM
authors.lastname: Eroshkin
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: Riedl
authors.forename: Guy S
authors.initials: GS
authors.lastname: Salvesen
mesh_terms: D001671:Biochemistry / Q000379:methods; D053148:Caspase 3 / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008968:Molecular Conformation; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D010447:Peptide Hydrolases; D053148:Caspase 3