Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

Abstract

Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (k cat /K M ) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.N-terminomics | caspase | proteolysis | proteomics | apoptosis C aspases are cysteine-class proteases that typically cleave after aspartic acid and play critical roles in cellular remodeling during development, cell differentiation, inflammation, and cell death (reviewed in refs. 1-3). There are 12 caspases in humans alone, which have been classically grouped on the basis of sequence homology, domain architecture, and cell biology as inflammatory (caspase-1, caspase-4, caspase-5, and caspase-11), apoptotic initiators (caspase-2, caspase-8, caspase-9, and caspase-10), or executioners (caspase-3, caspase-6, and caspase-7) (4, 5). Identifying the natural protein substrates for caspases can provide important insights into their cellular roles. A series of large-scale proteomic studies from several laboratories has collectively identified nearly 2,000 native proteins cleaved after an aspartic acid during apoptosis of human cells (recently reviewed in refs. 6-8). By adding individual caspases to extracts, it has been possible to begin to identify caspasespecific substrates, ranging from just a few, in the case of caspase-4, caspase-5, and caspase-9 (9), to several hundred for caspase-1, caspase-3, caspase-7, and caspase-8 (10, 11). Some of these substrates overlap, but others are unique to specific caspase...

Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues

“…The rates of individual caspase substrates vary over 500-fold so this twoto eightfold rate differences between aspartate and glutamate cleavages is almost biologically indistiguishable. 14,21 Our studies indicate that P1 glutamate cleavages are biologically relevant and isofunctional with P1 aspartate cleavages.…”

“…[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In addition to providing unbiased information about which proteins are cleaved, in some cases, these experiments locate the precise sites and quantify the rates of cleavage.…”

“…14,19,20 This large database of aspartate-cleaved proteins and their conservation in metazoans has helped to reveal the pathways and nodes that drive apoptosis.…”

“…[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In addition to providing unbiased information about which proteins are cleaved, in some cases, these experiments locate the precise sites and quantify the rates of cleavage. [13][14][15][16][17] Using the subtiligasebased N-terminomics approach, we identified more than 1700 aspartate cleavage events distributed among about 1200 different protein substrates upon induction of apoptosis across seven human cell lines (http://wellslab.ucsf.edu/degrabase/). 18 These findings and others revealed structural preferences for cleavage in loops4helices4sheets.…”

“…18 These findings and others revealed structural preferences for cleavage in loops4helices4sheets. 14,19,20 This large database of aspartate-cleaved proteins and their conservation in metazoans has helped to reveal the pathways and nodes that drive apoptosis.…”