Abstract
The engineering of enzymes for the purpose of controlling their activity represents a valuable approach to address challenges in both fundamental and applied research. Here, we describe and compare different design strategies for the generation of a human rhinovirus-14 (HRV14) 3C protease-inducible caspase-3 (CASP3). We exemplify the application potential of the resulting protease by controlling the activity of a synthetic enzyme cascade, which represents an important motif for the design of artificial signal transduction networks. In addition, we use our engineered CASP3 to characterize the effect of aspartate mutations on enzymatic activity. Besides the identification of mutations that render the enzyme inactive, we find the CASP3-D192E mutant (aspartate-to-glutamate exchange at position 192) to be inaccessible for 3C protease-mediated cleavage. This indicates a structural change of CASP3 that goes beyond a slight misalignment of the catalytic triad. This study could inspire the design of additional engineered proteases that could be used to unravel fundamental research questions or to expand the collection of biological parts for the design of synthetic signaling pathways.
Highlights
Proteases are important tools for biological research and applications, such as proteomics, structural biology, or the biotechnological manufacturing of proteins
To develop a CASP3 whose activity can be induced by the 3C protease but that is not capable of autoproteolytic activation, we applied two fundamental design strategies
The first strategy replaces the endogenous cleavage site between the large (p17) and small (p12) subunit with the 3C protease cleavage site (3CS, Figure 1), the second strategy separates the CASP3 subunits with a bulky protein domain that can be excised by the 3C protease (Figure 2)
Summary
Proteases are important tools for biological research and applications, such as proteomics, structural biology, or the biotechnological manufacturing of proteins. Whereas the engineering of split or autoinhibited proteases requires careful evaluation of structural properties and often library-based selection and optimization, naturally occurring zymogens offer a more straightforward way towards custom-designed, inducible proteases Inactive precursors adopt their active form by, for instance, the removal of inhibitory peptide units or Molecules 2019, 24, 1945; doi:10.3390/molecules24101945 www.mdpi.com/journal/molecules. The location or time of enzymatic action can be precisely controlled This principle plays a crucial role in various physiological processes such as digestion, blood coagulation, complement activation, or programmed cell death. In the latter case, the activation of caspase (cysteinyl aspartic protease) cascades is key to controlling and inducing apoptosis and inflammatory events [15]. We studied effects of CASP3 mutations on cleavage efficiency and catalytic activity
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