Abstract
Caspases are a family of cysteinyl proteases that control programmed cell death and maintain homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because all caspases are produced as zymogens (procaspases [PCPs]) that must be activated to gain full activity; the protein structures are conserved through hundreds of millions of years of evolution; and some allosteric features arose with the early ancestor, whereas others are more recent evolutionary events. The apoptotic caspases evolved from a common ancestor (CA) into two distinct subfamilies: monomers (initiator caspases) or dimers (effector caspases). Differences in activation mechanisms of the two subfamilies, and their oligomeric forms, play a central role in the regulation of apoptosis. Here, we examine changes in the folding landscape by characterizing human effector caspases and their CA. The results show that the effector caspases unfold by a minimum three-state equilibrium model at pH 7.5, where the native dimer is in equilibrium with a partially folded monomeric (PCP-7, CA) or dimeric (PCP-6) intermediate. In comparison, the unfolding pathway of PCP-3 contains both oligomeric forms of the intermediate. Overall, the data show that the folding landscape was first established with the CA and was retained for >650 million years. Partially folded monomeric or dimeric intermediates in the ancestral ensemble provide mechanisms for evolutionary changes that affect stability of extant caspases. The conserved folding landscape allows for the fine-tuning of enzyme stability in a species-dependent manner while retaining the overall caspase–hemoglobinase fold.
Highlights
Of dimeric proteins compared with monomeric proteins [2, 9], two-thirds of proteins form a multimeric assembly [10, 11]
Effector caspase-3, caspase-6, and caspase-7 have a significant role in apoptosis and serve overlapping but nonredundant functions [35], and multiple studies have examined enzyme specificity and regulation of extant caspases [21, 33, 36, 37]
We have previously examined evolutionary changes resulting in amino acid substitutions that affect enzyme specificity [15, 38] and allosteric regulation [26, 39, 40], but there is a dearth of information regarding changes in the caspase folding landscape
Summary
Of dimeric proteins compared with monomeric proteins [2, 9], two-thirds of proteins form a multimeric assembly [10, 11]. In the case of PCP-7, the equilibrium folding/unfolding data were well described by the three-state equilibrium model, discussed previously for data at pH 7.5, in which the native dimer is in equilibrium with a monomeric intermediate (Equation 2) (Fig. S5).
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