Structural and kinetic determinants of protease substrates.
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.
Timmer
John C
JC
Apoptosis and Cell Death Research Program at the Burnham Institute for Medical Research, La Jolla, California, USA.
Zhu
Wenhong
W
Pop
Cristina
C
Regan
Tim
T
Snipas
Scott J
SJ
Eroshkin
Alexey M
AM
Riedl
Stefan J
SJ
Salvesen
Guy S
GS
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
D010447:Peptide Hydrolases; D053148:Caspase 3