UCSF

New Engineered Proteins for Signaling

New engineered proteins for signaling is one focus of our research.

Our goals

Once signals are initiated from the cell membrane, they trigger cascades of post-translational and protein-protein interactions that lead to observable phenotypic changes.

Identifying cellular substrates

A primary goal of this project is to use protein and cellular engineering to identify cellular substrates of proteases, kinases, and ubiquitin ligases. This line of inquiry has lead us to the design of subtiligase and its recent improvements, a peptide ligase to tag the N-terminus of cleaved proteins cut by proteases such as caspases (see Degrabase); split-kinases (sKins), to directly activate proliferative kinases such as cAbl and identify immediate downstream consequences by phospho-proteomics; and the NEDDylator, a proximity-based tagger used for direct affinity labeling of substrates of E3 ubiquitin ligases such as inhibitors of apoptosis (IAPs) and for identification of targets for small molecule drugs.

diagram

Engineering proteins to trigger events

A second goal is to engineer proteins to trigger specific cellular events, such as proteolysis. For instance, we use the Single-Nick in Proteome (SNIPer), a split-TEV protease inducible by classic CID using rapamycin or Tet-on TEV, to probe single proteolytic events in apoptosis. To ablate specific neurons in mice, we developed a TEV-sensitive caspase allele initiated by an Adeno-Cre system with Nirao Shah, PhD (Stanford University), and, more recently, we developed a light-activated caspase-3 to study neurodegeneration in flies with Grae Davis, PhD (UCSF). To trigger specific kinase activities, we have engineered split-kinases, sKins, and to control gene editing, we have designed a small molecule activated Cas9 for gene silencing or activation. Genes for these engineered proteins are available upon request.

diagram

Recently we have also developed a new generation of CIDs called AbCID (antibody-based chemically-inducible dimerization). AbCIDs can be activated with approved drugs that are dosed at much lower toxicity than conventional small molecules. Using human protein drug targets, combined with human antibodies engineered to recognize the drug-protein complex, reduces potential immunogenicity. AbCIDs are highly suitable for small molecule controlled T-cell activation and gene silencing.

Our many collaborators

For this project we have benefited tremendously from close collaborations with professors including: