Project Summary The broad, long-term objective of this research program is to develop general methods to conditionally regulate protein function at the level of the protein molecules rather than by targeting the DNA or mRNA precursors that encode a protein-of-interest. These experimental methods are highly specific for the targeted proteins and provide rapid and tunable control of protein function using cell- permeable small molecules. The goal is to engineer small protein domains called destabilizing domains that are rapidly degraded when expressed in mammalian cells. The instability of destabilizing domains is faithfully conferred to partner proteins fused to these small domains, allowing researchers to predictably control the levels of any protein-of-interest. One specific aim of this research program will provide new destabilizing domains that are derived from human proteins and regulated by FDA- approved drugs. A second aim of this research is to develop a new method for destabilizing domains to regulate the secretion of therapeutically useful cytokines and hormones from human cells. A third aim of this research is the development of a new method for the partner proteins regulated by the destabilizing domains to be liberated from the regulatory domain only when the entire fusion protein is stabilized by its cognate ligand. The fourth aim builds upon the biophysics underpinning the destabilizing domains to develop a new class of genetically encoded biosensors for intracellular second messenger analytes. The current lack of safe and effective methods to regulate the expression and biological activity of gene-based therapeutics seriously limits the number and types of diseases that physicians and scientists can contemplate targeting using cell and gene therapy. New regulation methods such as the destabilizing domains that are safe and effective will dramatically expand the universe of diseases that can be targeted for treatment through cell and gene therapy, thus opening new frontiers for treating human diseases that cannot be addressed using existing methods.