Summary: Our objective is to develop general principles for engineering proteases that can control signaling in cells. This requires seven steps: 1) Choice of signaling proteins (Human RAS isoforms and oncogenic mutants); 2) Identification of dynamic regions within the signaling proteins; 3) Identification of a target sequence within dynamic regions; 4) Engineering/evolving high specificity against the target sequence; 5) Engineering zymogen activation that is catalyzed by the target protein; 6) Testing regulated RAS degradation in E. coli; 7) Testing control of signaling in human cell culture. We will complete these steps using a multi-disciplinary approach that includes computational and in vitro selection-based protein engineering, enzymology, multi- dimensional NMR, X-ray crystallography, and cell-biology. The three Specific Aims are: 1) Evolve protein-specific proteases that target active RAS; 2) Engineer zymogen activation catalyzed by active RAS; 3) Test promising proteases in cell model systems. This project will develop general principles of recognition, folding, and catalysis based on detailed understanding of structure and mechanism and use this knowledge to develop tools to control signaling in a cell. The protease and its regulators will comprise an enzymatic system that will sense the presence of active RAS and transduce a controlled release of the active RAS-specific protease, thereby modulating RAS signaling. The ability to cleave RAS in cell-based systems and thereby alter RAS signaling will represent a significant milestone towards establishing the utility of protein-specific proteases as exogenous signaling regulators. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Narrative: Our objective is to develop general principles for engineering proteases that can control signaling in cells. In accomplishing this objective we will develop knowledge to communicate fluently in a signaling language, develop tools for controlling signaling in a cell, and ultimately create avenues to advanced therapeutics.