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

Abstract

Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling and inflammatory events. Caspases are named because they are cysteine class enzymes shown to cleave after aspartate residues. In the past decade, we and others have developed unbiased proteomic methods that collectively identified ~2000 native proteins cleaved during apoptosis after the signature aspartate residues. Here, we explore non-aspartate cleavage events and identify 100s of substrates cleaved after glutamate in both human and murine apoptotic samples. The extended consensus sequence patterns are virtually identical for the aspartate and glutamate cleavage sites suggesting they are cleaved by the same caspases. Detailed kinetic analyses of the dominant apoptotic executioner caspases-3 and -7 show that synthetic substrates containing DEVD↓ are cleaved only twofold faster than DEVE↓, which is well within the 500-fold range of rates that natural proteins are cut. X-ray crystallography studies confirm that the two acidic substrates bind in virtually the same way to either caspases-3 or -7 with minimal adjustments to accommodate the larger glutamate. Lastly, during apoptosis we found 121 proteins cleaved after serine residues that have been previously annotated to be phosphorylation sites. We found that caspase-3, but not caspase-7, can cleave peptides containing DEVpS↓ at only threefold slower rate than DEVD↓, but does not cleave the unphosphorylated serine peptide. There are only a handful of previously reported examples of proteins cleaved after glutamate and none after phosphorserine. Our studies reveal a much greater promiscuity for cleaving after acidic residues and the name 'cacidase' could aptly reflect this broader specificity.

Figure 1

There is strong similarity between iceLogos for substrates cleaved after (a) P1 aspartate and (b) glutamate in apoptotic human samples. The iceLogos for P1 aspartate and P1 glutamate are virtually identical in the extended P4-P4' sequence around the cleavage site. (a) Reproduced with permission from Crawford et al.

Figure 2

Overlap of P1 glutamate and aspartate substrates in the caspases-3 and -7 pockets show minimal structural rearrangements to accommodate the extra methylene in P1 glutamate. (a–c) Human caspase-3 was labeled with Ac-DEVE-cmk, crystallized and X-ray structure solved to 2.65  Å (PDB: 5IC4). (d–f) Human caspase-7 was labeled with Ac-DEVE-cmk, crystallize and X-ray structure solved to 2.7 Å (PDB: 5IC6). The P1 glutamate labeled protein (blue) has minimal changes when overlayed with the P1 aspartate labeled protein caspase-3 (2DKO, gray) or caspase-7 (3H1P, gray). The differences between the glutamate and aspartate structures are localized to the binding pocket and substrate itself

Figure 3

Cleavage of P1-phosphoserine sites are frequently found in the Degrabase and caspase-3 can cleave synthetic substrate containing a P1 phosphoserine, biotin-WDEV(pS)↓SGVDEK(DNP). (a–d) The iceLogos for the combined group of three phospho-sites (a), phosphoserine (b), phospho-threonine (c) and phospho-tyrosine (d), do not match the typical caspase cleavage motif. (e) Caspase-3 can cleave the biotin-WDEV(pS)↓SGVDEK(DNP) substrate only threefold slower than the biotin-WDEV(D)↓SGVDEK(DNP) substrate. Error bars represent S.E.M

Figure 4

P1 aspartate and glutamate cleavage sites are not strongly conserved throughout the broader metazoan evolutionary record. Human caspase substrates with a P1 aspartate (a) and glutamate sites (b) are compared with all metazoan homologs for site conservation of a P1 aspartate or P1 glutamate in the sequence. The dashed red line indicates maximal conservation of 100% acidic residue (Asp+Glu). For both aspartate and glutamate sites, there is a tendency to remain itself as the data is skewed to the bottom left of the chart and minimal activity switching to the other acidic residue