| Feature | Human CASP3 summary | Evidence |
|---|---|---|
| Verified identity | CASP3 encodes human caspase-3, an executioner/effector caspase in the caspase family; UniProt P42574 aligns with literature describing a cysteine-dependent, aspartate-specific protease central to apoptosis. | (pqac-00000000, pqac-00000001, pqac-00000003) |
| Enzyme classification | EC 3.4.22.56; proteolytic enzyme that hydrolyzes peptide bonds in protein substrates during apoptosis and other stress responses. | (pqac-00000001, pqac-00000003) |
| Protease type | Cysteine-dependent, aspartate-specific protease (cysteine aspartyl protease). Caspases cleave almost exclusively C-terminal to Asp at the P1 position. | (pqac-00000001, pqac-00000003, pqac-00000006) |
| Primary biochemical function | CASP3 is the major executioner protease that performs downstream substrate cleavage after activation by initiator caspases, thereby driving apoptotic dismantling of the cell; it also contributes to selected non-apoptotic stress-adaptation pathways. | (pqac-00000000, pqac-00000003, pqac-00000009, pqac-00000012) |
| Catalytic mechanism | Activated initiator caspases cleave procaspase-3 at the intersubunit linker to generate the active executioner enzyme; executioner caspases are inactive homodimers that require cleavage to form active tetramers that mediate downstream substrate cleavage. Proteolysis occurs at the scissile bond between P1 and P1′, with strict requirement for Asp at P1. | (pqac-00000001, pqac-00000003, pqac-00000006) |
| Preferred cleavage motif | Deep substrate profiling in native human cell lysates identified a clear caspase-3 consensus of **DEVD↓(G/S/A)** for P4-P1↓P1′; broader tolerated motifs exist, indicating cleavage depends on both sequence and local structural context. | (pqac-00000004, pqac-00000007) |
| Minimal sequence rule | Preference is often summarized as DXXD↓X, but the strongest observed motif for human caspase-3 is DEVD↓(G/A/S); P4 contributes importantly to specificity. | (pqac-00000004, pqac-00000006, pqac-00000007) |
| Importance of substrate context | Sequence alone is insufficient to explain cleavage; local accessibility and structural context strongly affect whether a potential motif is actually cleaved in proteins. | (pqac-00000004, pqac-00000006) |
| Scale of substrate repertoire | Reverse N-terminomics identified **906 putative protein substrates** and **1126 cleavage sites** for caspase-3 in native lysates, representing one of the most comprehensive substrate maps reported. | (pqac-00000003, pqac-00000004) |
| Novelty of substrate map | Of the observed caspase-3 cleavage events, **577 cleavage sites** had not previously been found in the apoptosis DegraBase, and **257 substrates** were newly reported. | (pqac-00000004) |
| Major substrate classes | CASP3 cleaves structural, regulatory, DNA-repair, cytoskeletal, chromatin, and signaling proteins, consistent with its role in morphological remodeling, DNA fragmentation, and apoptotic execution. | (pqac-00000005, pqac-00000007, pqac-00000009) |
| Key substrate example: PARP1 | PARP1 is a canonical bona fide caspase-3 substrate and a standard readout of caspase-3 activity during apoptosis; cleavage of PARP1 is repeatedly cited as a hallmark of executioner caspase activation. | (pqac-00000007, pqac-00000012, pqac-00000013) |
| Key substrate example: ICAD/DFF45 | CASP3 cleaves the inhibitor of caspase-activated DNase (ICAD), enabling CAD-mediated genomic DNA fragmentation during apoptosis. | (pqac-00000011, pqac-00000007) |
| Key substrate example: GSDME | Human GSDME contains a DxxD motif recognized by CASP3; recent work emphasizes that human GSDME is cleaved by CASP3 but not efficiently by human CASP7, illustrating non-redundant substrate discrimination between the two executioner caspases. | (pqac-00000007) |
| Key substrate example: CAD (carbamoyl-phosphate synthetase/aspartate transcarbamylase/dihydroorotase) | Recent work shows CAD must be cleaved by caspase-3 at Asp1371 prior to degradation, linking CASP3 activity to chemosensitivity and pyrimidine synthesis control in cancer cells. | (pqac-00000005) |
| Other validated/representative substrate themes | Caspase-3 substrates include proteins involved in apoptosis, stress adaptation, autophagy modulation, DNA-damage signaling, and inflammatory restraint; cleavage landscapes differ under lethal vs non-lethal stress. | (pqac-00000012, pqac-00000013) |
| Subcellular distribution of substrates | In the N-terminomics dataset, caspase-3 substrates were enriched in cytoplasmic and nuclear proteins: ~49% cytoplasmic and ~48% nuclear, with smaller fractions in mitochondria, ER, membrane, and secreted compartments. | (pqac-00000004) |
| Comparison with caspase-7: shared features | CASP3 and CASP7 are closely related executioner caspases with overlapping preferences and a shared preference for DEVD-like motifs; both are activated downstream of initiator caspases in apoptosis. | (pqac-00000000, pqac-00000007) |
| Comparison with caspase-7: key difference | Despite shared DxxD recognition, CASP3 and CASP7 are not fully redundant. Human GSDME is cleaved by CASP3 but not by human CASP7, and structural work attributes this to differences in the CASP7 p10 subunit/prime-side substrate recognition. | (pqac-00000007) |
| Comparison with caspase-7: proteolytic breadth | Caspase-3 is described as the most proteolytically proficient executioner caspase and has a very broad substrate repertoire; some recent assay work and reviews continue to treat CASP3 as the dominant executioner enzyme. | (pqac-00000007, pqac-00000005) |


*Table: This table summarizes the primary enzymatic activity, cleavage preferences, substrate scope, and mechanism of human caspase-3, with a focused comparison to caspase-7. It is useful for quickly identifying what CASP3 cleaves, how it recognizes substrates, and why it is considered the dominant executioner caspase.*