Sequential Unfolding Mechanisms of Monomeric Caspases.

Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies with either monomeric (initiators) or dimeric (effectors) oligomeric states. The regulation of apoptosis is influenced by the activation mechanism of the two subfamilies, but the evolution of the well-conserved caspase-hemoglobinase fold into the two subfamilies is not well understood. We examined the folding landscape of monomeric caspases from two coral species over a broad pH range of 3-10.5. On an evolutionary timescale, the two coral caspases diverged from each other approximately 300 million years ago, and they diverged from human caspases about 600 million years ago. Our results indicate that both proteins have overall high stability, ∼15 kcal mol-1, near the physiological pH range (pH 6-8) and unfold via two partially folded intermediates, I1 and I2*, that are in equilibrium with the native and the unfolded state. Like the dimeric caspases, the monomeric coral caspases undergo a pH-dependent conformational change resulting from the titration of an evolutionarily conserved site. Data from molecular dynamics simulations paired with limited proteolysis and MALDI-TOF mass spectrometry show that the small subunit of the monomeric caspases is unstable and unfolds prior to the large subunit. Overall, the data suggest that all caspases share a conserved folding landscape, that a conserved allosteric site can be fine-tuned for species-specific regulation, and that the subfamily of stable dimers may have evolved to stabilize the small subunit.

Sequential unfolding mechanisms of monomeric caspases.

Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies with either monomeric (initiators) or dimeric (effectors) oligomeric states. The regulation of apoptosis is influenced by the activation mechanism of the two subfamilies, but the evolution of the well-conserved caspase-hemoglobinase fold into the two subfamilies is not well understood. We examined the folding landscape of monomeric caspases from two coral species over a broad pH range of 3 to 10.5. On an evolutionary timescale, the two coral caspases diverged from each other approximately 300 million years ago, and they diverged from human caspases about 600 million years ago. Our results indicate that both proteins have overall high stability, ∻ 15 kcal mol -1 near the physiological pH range (pH 6 to pH 8), and unfold via two partially folded intermediates, I 1 and I 2 , that are in equilibrium with the native and the unfolded state. Like the dimeric caspases, the monomeric coral caspases undergo a pH-dependent conformational change resulting from the titration of an evolutionarily conserved site. Data from molecular dynamics simulations paired with limited proteolysis and MALDI-TOF mass spectrometry show that the small subunit of the monomeric caspases is unstable and unfolds prior to the large subunit. Overall, the data suggest that all caspases share a conserved folding landscape, that a conserved allosteric site can be fine-tuned for species-specific regulation, and that the subfamily of stable dimers may have evolved to stabilize the small subunit.

Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold.

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1ß (IL-1ß). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1ß. Seeking to understand the evolutionary origin of this caspase, we utilized the large amount of data available in public databases to perform ancestral sequence reconstruction of an inflammatory caspase of a Carnivora ancestor. We expressed the catalytic domain of this putative ancestor in Escherichia coli, purified it, and compared its substrate specificity on synthetic and protein substrates to extant caspases. We demonstrated that it activates gasdermin D but has reduced ability to activate IL-1ß. Our reconstruction suggests that caspase-1 was lost in a Carnivora ancestor, perhaps upon a selective pressure for which the generation of biologically active IL-1ß by caspase-1 was detrimental. We speculate that later, a Carnivora encountered selective pressures that required the production of IL-1ß, and caspase-4 subsequently gained this activity. This hypothesis would explain why extant Carnivora possess an inflammatory caspase with caspase-1 catalytic function placed on a caspase-4 scaffold.