UC Santa Barbara

Proteases regulate many biological processes through their ability to activate or inactivate their target substrates and therefore present unique opportunities for therapeutic application. Because proteases catalytically turnover proteins, they could potentially be used at lower doses in therapy, reducing the cost of treatment. However, many proteases are capable of cleaving multiple physiological substrates. Therefore their activity, expression, and localization are tightly controlled to prevent unwanted proteolysis that could lead to side effects. Currently approved protease therapeutics rely on naturally evolved specificities and are often used for protease replacement therapy in genetically deficient patients. The clinical use of proteases in replacement therapy has been successful due to the narrow substrate specificity of these enzymes, which limits their toxicity. However, the application of proteases in therapy could be extended beyond their native biological functions. The emergence of methods for engineering proteases with new activities and narrow specificities toward substrates relevant in disease could greatly expand their therapeutic potential.

Here we have developed a novel intracellular screen in yeast for redesigning the specificity of human secreted proteases which we have termed protease evolution via cleavage of an intracellular substrate (PrECISE). Using PrECISE, a protease library and a target substrate flanked by fluorescent proteins CyPet and YPet, capable of Fӧrster resonance energy transfer (FRET), are co-expressed in the endoplasmic reticulum (ER) of yeast. The ER provides an oxidizing environment for the formation of disulfide bonds common to human secreted proteases and the co-localization of the protease and substrate promotes cleavage. Protease activity on the selection substrate leads to loss of intracellular FRET and increases cyan fluorescence enabling screening of large protease libraries using fluorescence activated cell sorting (FACS). As a model system, we screened randomly mutated and rationally designed libraries of the secreted protease human kallikrein 7 (hK7) using PrECISE to isolate variants with improved selectivity toward the hydrophobic core of the amyloid beta peptide (Aβ8: KLVF↓F↓AED).

Sequential rounds of low error rate random mutagenesis were found to be most effective in altering protease selectivity by incrementally introducing and screening for beneficial substitutions. Findings from our work emphasize the importance of screening large libraries during protease evolution since multiple substitutions were required to alter hK7 selectivity for Aβ. The substitutions found to improve hK7 selectivity would be impossible to predict since the majority were located far from the hK7 active site. Interestingly, improvements in selectivity were accompanied by a reduction in toxicity of the protease variant toward mammalian cells and improved resistance to wild-type inhibitors. Analysis of the crystal structures of improved variants provided insights to the potential mechanisms that affected hK7 activity and selectivity. The PrECISE method and techniques developed here can be broadly applied to evolve human proteases for specific degradation of toxic proteins involved in disease to enable their greater use in therapy.