The molecular mechanism and evolutionary divergence of caspase 3/7-regulated gasdermin E activation

Reviewer #1 (Public Review):

Summary
In this study, Xu et al. provide insights into the substrate divergence of CASP3 and CASP7 for GSDME cleavage and activation during vertebrate evolution vertebrates. Using biochemical assays, domain swapping, site-directed mutagenesis, and bioinformatics tools, the authors demonstrate that the human GSDME C-terminal region and the S234 residue of human CASP7 are the key determinants that impede the cleavage of human GSDME by human CASP7.

Strengths
The authors made an important contribution to the field by demonstrating how human CASP7 has functionally diverged to lose the ability to cleave GSDME and showing that reverse-mutations in CASP7 can restore GSDME cleavage. The use of multiple methods to support their conclusions strengthens the authors' findings. The unbiased mutagenesis screen performed to identify S234 in huCASP7 as the determinant of its GSDME cleavability is also a strength.

Weaknesses
While the authors utilized an in-depth experimental setup to understand the CASP7-mediated GSDME cleavage across evolution, the physiological relevance of their findings are not assessed in detail. Additional methodology information should also be provided.

Specific recommendations for the authors
1. The authors should expand their evaluation of the physiological relevance by assessing GSDME cleavage by the human CASP7 S234N mutant in response to triggers such as etoposide or VSV, which are known to induce CASP3 to cleave GSDME (PMID: 28045099). The authors could also test whether the human CASP7 S234N mutation affects substrate preference beyond human GSDME by testing cleavage of mouse GSDME and other CASP3 and CASP7 substrates in this mutant.
2. It would also be interesting to examine the GSDME structure in different species to gain insight into the nature of mouse GSDME, which cannot be cleaved by either mouse or human CASP7.
3. The evolutionary analysis does not explain why mammalian CASP7 evolved independently to acquire an amino acid change (N234 to S234) in the substrate-binding motif. Since it is difficult to experimentally identify why a functional divergence occurs, it would be beneficial for the authors to speculate on how CASP7 may have acquired functional divergence in mammals; potentially this occurred because of functional redundancies in cell death pathways, for example.
4. For the recombinant proteins produced for these analyses, it would be helpful to know whether size-exclusion chromatography was used to purify these proteins and whether these purified proteins are soluble. Additionally, the SDS-PAGE in Figure S1B and C show multiple bands for recombinant mutants of TrCASP7 and HsCASP7. Performing protein ID to confirm that the detected bands belong to the respective proteins would be beneficial.
5. For Figures 3C and 4A, it would be helpful to mention what parameters or PDB files were used to attribute these secondary structural features to the proteins. In particular, in Figure 3C, residues 261-266 are displayed as a β-strand; however, the well-known α-model represents this region as a loop. Providing the parameters used for these callouts could explain this difference.
6. Were divergent sequences selected for the sequence alignment analyses (particularly in Figure 6A)? The selection of sequences can directly influence the outcome of the amino acid residues in each position, and using diverse sequences can reduce the impact of the number of sequences on the LOGO in each phylogenetic group.
7. For clarity, it would help if the authors provided additional rationale for the selection of residues for mutagenesis, such as selecting Q276, D278, and H283 as exosite residues, when the CASP7 PDB structures (4jr2, 3ibf, and 1k86) suggest that these residues are enriched with loop elements rather than the β sheets expected to facilitate substrate recognition in exosites for caspases (PMID: 32109412). It is possible that the inability to form β-sheets around these positions might indicate the absence of an exosite in CASP7, which further supports the functional effect of the exosite mutations performed.

Reviewer #2 (Public Review):

The authors wanted to address the differential processing of GSDME by caspase 3 and 7, finding that while in humans GSDME is only processed by CASP3, Takifugu GSDME, and other mammalian can be processed by CASP3 and 7. This is due to a change in a residue in the human CAPS7 active site that abrogates GSDME cleavage. This phenomenon is present in humans and other primates, but not in other mammals such as cats or rodents. This study sheds light on the evolutionary changes inside CASP7, using sequences from different species. Although the study is somehow interesting and elegantly provides strong evidence of this observation, it lacks the physiological relevance of this finding, i.e. on human side, mouse side, and fish what are the consequences of CASP3/7 vs CASP3 cleavage of GSDME.

Fish also present a duplication of GSDME gene and Takifugu present GSDMEa and GSDMEb. It is not clear in the whole study if when referring to TrGSDME is the a or b. This should be stated in the text and discussed in the differential function of both GSDME in fish physiology (i.e. PMIDs: 34252476, 32111733 or 36685536).