Expressed Artificial Proteins That Induce Degradation of the Platelet-Derived Growth Factor Beta Receptor

Author

Emily Branham, Yale University Graduate School of Arts and SciencesFollow

Date of Award

Spring 2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

DiMaio, Daniel

Abstract

Targeted protein degradation (TPD) is a rapidly advancing field with diverse applications spanning from fundamental research to therapeutic interventions. With considerable opportunity for scientific creativity, emerging degradation strategies promise to expand the reach of this field to challenging targets that often elude conventional, small molecule-based degraders. Of particular interest in this report, transmembrane (TM) proteins represent more than half of all drug targets, yet their intrinsic properties present challenges for standard degradation approaches. Here, we describe a novel genetic approach to TPD, presenting the first example of expressible protein degraders: traptamer-based targeting chimeras (TRAPTACs). As their name implies, these innovative degraders leverage short, artificial TM proteins known as traptamers as target-binding ligands, thereby broadening the scope of TPD capabilities by affording enhanced access to TM protein targets. Traptamers, which are themselves membrane-embedded, bind specifically to the TM domain of a protein target to induce a specific phenotype, such as receptor activation. By appending a downregulation-inducing sequence to the N-terminus of a traptamer, we are able to convert a TM protein binder into a TM protein degrader. Notably, as expressible protein degraders, TRAPTACs offer unique advantages over small molecule-based degraders, such as rapid design and testing, along with amenability to genetic engineering. Our research focused on E5-based TRAPTACs. The 44-amino acid bovine papillomavirus E5 oncoprotein is the model TM protein upon which all traptamers are designed. It binds the TM domain of the platelet-derived growth factor beta receptor (PDGFR), a therapeutically-relevant receptor tyrosine kinase. As a proof-of-concept, we designed TRAPTACs that incorporate E5 with the goal of degrading PDGFR. TRAPTACs were initially designed to recruit von Hippel-Lindau (VHL) E3 ubiquitin ligase by fusing the LAPYI recruiting sequence to E5 by a flexible 5- or 10-amino acid glycine/serine linker. These TRAPTACs robustly and specifically downregulate PDGFR. Surprisingly, our studies show VHL is not required for receptor downregulation by E5-based TRAPTACs, as evidenced by TRAPTAC activity in cells lacking VHL. Further investigation of the TRAPTAC mechanism through inhibitor studies, biochemical assays, and pulse-chase analysis confirmed that the downregulation of PDGFR induced by TRAPTAC expression occurs via receptor degradation, although the degradation pathway used remains unidentified. Subsequent mutational analyses of both TRAPTAC and PDGFR revealed various requirements for TRAPTAC action. Binding between the TRAPTAC and PDGFR is required but not sufficient to induce receptor degradation, and part of the receptor outside of the TM domain is required for its downregulation to occur. TRAPTACs do not require the LAPYI or glycine/serine linker sequences, although not all sequences produce an active TRAPAC when appended to E5. Further, the identity of appended amino acids as well as the position along E5 at which they are appended affects TRAPTAC activity. We also identified a minimal sequence modification to E5 that produces an active TRAPTAC: strikingly, a very short, two glycine insertion at the N-terminus of E5 effectively converts the receptor-activating traptamer into a receptor-degrading TRAPTAC. A single glycine inserted at the same position, however, does not produce an active TRAPTAC. Thus, robust, steady-state downregulation of PDGFR can be switched on and off by the addition of a second glycine residue. These results emphasize the sensitivity of TRAPTAC action to slight sequence alterations. As a further demonstration of this principle, we discovered that, despite the marked decrease in steady-state receptor levels in TRAPTAC-expressing cells compared to cells expressing E5, PDGFR is similarly destabilized by TRAPTAC expression and by E5 expression. To explain our observations, we propose that there is a balance between receptor activation and degradation that can be tipped by slight traptamer sequence changes. Our E5-based TRAPTAC studies illustrate the potential of expressible protein degraders. The use of traptamers as target-binding ligands expands the reach of TPD technology, facilitating TM protein targeting. Further, applying a genetics approach to TPD has the potential improve upon conventional small molecule-based degrader design, development, and testing strategies.

Recommended Citation

Branham, Emily, "Expressed Artificial Proteins That Induce Degradation of the Platelet-Derived Growth Factor Beta Receptor" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1431.
https://elischolar.library.yale.edu/gsas_dissertations/1431