Targeted Protein Degradation of HaloTag® Fusion Proteins for Phenotypic Studies
In this webinar, you will learn:
- The mechanism of HaloPROTAC3 targeted protein degradation
- Options for the expression of HaloTag® fusions for HaloPROTAC3 degradation
- Methods to detect the degradation of HaloTag® fusions after HaloPROTAC3 treatment
Summary
Targeted protein degradation has emerged as an exciting new therapeutic modality by specifically removing key target proteins from the cell, using small-molecule degrader compounds. In addition, these degraders can be used to understand temporal protein-loss phenotype and function, without the need to knock out proteins or mRNA using genomic modification approaches or siRNA, respectively. One class of degradation compounds are proteolysis-targeting chimeras (PROTACs). These heterobifunctional molecules recruit target proteins to E3 ligase components to ubiquitinate and degrade the target protein via the ubiquitin proteasomal pathway (UPS).
In this webinar, we present HaloPROTAC3, a small-molecule degrader that specifically binds to and degrades the HaloTag® protein and its fusion partners in live cells. HaloPROTAC3 binds irreversibly to the HaloTag® protein, recruiting it via co-engagement with von Hippel Lindau (VHL) protein, an E3 ligase component. This engagement activates E2/E3 ubiquitin ligase complexes, resulting in ubiquitination and subsequent degradation of the HaloTag®-target fusion by the proteasome. We outline an approach using this fusion tag PROTAC, in conjunction with CRISPR/Cas9 edited cells containing an endogenous HaloTag®-target fusion, to degrade targets from multiple cellular locations. Once degradation is accomplished phenotypic studies can be completed, as demonstrated in a case study using the Wnt3a signaling pathway after HaloPROTAC3 mediated degradation of an endogenous β-catenin HaloTag® fusion. HaloPROTAC3 is also effective in mouse studies, providing the opportunity to study protein loss in vivo related to disease treatments for cancer or otherwise lethal approaches like embryonic gene knock-outs.
Speaker
Elizabeth A Caine, PhD
Senior Research Scientist
Elizabeth Caine received her Ph.D. from the University of Wisconsin-Madison where she characterized vaccines and studied the pathogenesis of human enteroviruses that cause hand, foot, and mouth disease. She then worked as a postdoctoral scholar at Washington University in St. Louis researching the immune response in the female reproductive tract and effects on fertility after Zika virus infection. Most recently she began her role as a Senior Scientist at Promega developing approaches to enable the targeted degradation of traditionally undruggable protein targets, allowing for phenotypic studies and assessing the potential for target-specific PROTAC development.