Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition

Abstract

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of non-obvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.

Caspases rule the intracellular trafficking cartel

“…Indeed, the engineering of the 'best' motif in a structurally well-exposed loop in a model protein resulted in a cleavage rate that still falls short of some known endogenous substrates, which has led to the proposal that other determinants, such as exosites, allow further improvement of catalysis [19]. In fact, usage of exosites by caspases has been demonstrated for cleavage of PARP-1 by caspase-7 [20], and there is evidence that cleavage of lamin A/C by caspase-6 also employs an exosite [21]. Taken together, features including adequate primary and secondary structures and the potential use of exosites allow tailoring (speed, completeness, timing) of substrate cleavage efficacy.…”

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

“…Although the effector caspases are relatively closely related, caspases-3 and -7 are characterized as group II specificity, while caspase-6 shows group III specificity. The selection at P4 (D vs V) results in overlapping but nonidentical substrate profiles based on degradome analyses [27][28][29][30]. In the evolution of chordates, new caspase substrate specificities were important in developmental stages of the brain and nervous systems [31][32][33][34], so what may appear to be subtle changes in enzyme selection have large consequences in cellular development.…”

“…Generally, substitutions from horizontal studies lack evolutionary context, where protein epistasis affects the specific combination of amino acids along discrete evolutionary paths [39,40]. Directed evolutionary approaches expand the sequence space that can be examined, and such studies identified a combination of amino acids that relax specificity in caspase-7, resulting in a shift in substrate cleavage profiles in cellulo of evolved-caspase-7 enzymes [30]. Evolutionary biochemical methods further expand the sequence space to include the entire protein, yet the methods simultaneously narrow the scope of the problem by also examining changes that occurred between common ancestral proteins [41].…”

Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms

“…Cleaved executioners can also experience a reversal to the zymogen-like state by small molecules (Hardy et al, 2004), which forces a loop rearrangement that traps L2’ over the dimer interface and results in the expulsion of loop 3 from the active site pocket. Following activation in the cell, each executioner caspase cleaves over a hundred substrates (Agard et al, 2012; Hill et al, 2016) to propagate the well-ordered termination of the dying cell.…”