Manipulating Cellular Adaptive Response by Engineering Novel and Evolutionarily Conserved Allosteric Sites in Caspase‐3

Caspases are cysteine‐dependent aspartate‐specific proteases that are regulators of carrying out cell death via apoptosis; however, at sub‐apoptotic threshold levels, they regulate other adaptive responses such as terminal erythroid differentiation. Caspase‐3 is an executioner caspase that exists as a stable zymogen until cleaved at the intersubunit linker (IL) by initiator caspases, which causes rearrangement of the active site loops (L1–L4 and L2′) to form a stable active site with a productive substrate binding pocket. There are two major pathways that both converge to activate caspase‐3 and commit the cell to apoptosis; the intrinsic pathway and the extrinsic pathway. During adaptive responses the cell fine tunes caspase activity to prevent apoptosis, leading to non‐apoptotic phenotypes. One of the mechanisms to control caspase‐3 activity in the cell is through reduction or loss of its catalytic activity by phosphorylation at the highly conserved Ser150, at the base of helix 3, or other novel allosteric sites such as Thr152, Ser249, and Thr245. The mechanisms behind how these modifications modulate caspase‐3 activity are poorly understood. The goals of this project are to quantify the structural and biophysical characteristics these posttranslational modifications have on caspase‐3 and reveal how the cell utilizes this process during apoptosis and erythroid maturation. Crosstalk between the kinome and the caspase cascade, and how phosphorylation regulates caspase‐3 is addressed in the following experiments. We mutated caspase‐3 at Ser150 as well as other predicted phosphorylation sites to mimic phosphorylation events, and using in vitro cleavage assays we found that Ser150A/D had no significant effect on the activity of the protein while Thr152D and Ser249D completely abolished activity. We solved the structures of the mutants using high‐resolution X‐ray crystallography and saw no significant structural changes in Ser150Ala/Asp. The Thr152Asp mutant, however, disrupts a hydrophobic pocket with L2 which directly disorders the catalytic cysteine (Cys163) from being in its optimal orientation, which is restored when Thr152 is mutated to a valine. To determine if these modifications have phenotypic effects on apoptosis and adaptive response pathways we performed transient transfections in K562 cells, a chronic myeloid leukemia (CML) cell line that can be chemically induced to undergo erythroid differentiation. These data reveal that our mutants have phenotype changes that both promote survival of the cell and modulate erythroid differentiation. These data supports the hypothesis that the kinome and caspase cascade interact to regulate adaptive responses. Understanding how these pathways interact to regulate adaptive responses could reveal novel targets for differentiation therapy of diseases such as leukemia.Regulation of caspase activity and subsequent phenotypic pathways in the cell. A. Clay Clark; Chem. Rev. 2016, 116, 6666–6706. DOI: 10.1021/acs.chemrev.5b00540