Determinants of Protein Degradation Dynamics in Cell Signaling

University Of Washington, Seattle WA

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Abstract

DESCRIPTION (provided by applicant): Regulated turnover of diverse proteins is critical during many cellular signaling processes and is frequently dysregulated in cancerous cells, yet we know remarkably little about what controls the turnover rates. These gaps impede understanding of cellular processes at play during both normal function and cancerous growth (e.g., cell cycle progression), and limit the design of interventions for cancerous cell states. Ubiquitin E3 ligases are enzymes that target proteins for turnover. Small molecule E3 disruptors are being pursued as anticancer agents and may be significantly less toxic than the more general ubiquitin-targeted therapies currently in use. The SKP1-Cullin-F-box (SCF) class of E3s uses F-box proteins as modular substrate-recognition subunits and is highly conserved between humans and plants. One of the best characterized E3s acts in plants to respond to the hormone auxin. The auxin pathway has unique advantages for studying E3 function, including: auxin receptors (AFBs) are themselves F-box proteins; substrate (Aux/IAA) degradation is small-molecule-triggered rather than requiring substrate phosphorylation; and evolution has provided sequence variants with distinct properties for both F-boxes (6 Arabidopsis AFBs) and substrates (29 Arabidopsis Aux/IAAs). Most importantly, our lab has engineered auxin-induced degradation in yeast to enable rapid, quantitative analysis of substrate degradation dynamics. This research project aims to rigorously characterize how F-box:substrate interactions determine the dynamics of protein turnover. My central hypothesis is that the dynamics of regulated substrate degradation are encoded within specific sequence motifs in both the substrate and the E3, and that these motifs regulate protein-protein interactions. To explore this hypothesis, a highly efficient and quantitative synthetic auxin-degradation system in yeast will be used in combination with in vitro and in planta studies to identify determinants in substrates and F-boxes that control substrate degradation dynamics and investigate the evolution of F-box small-molecule binding. A long-range goal is a complete understanding of how degradation dynamics are encoded within E3:substrate complexes and how they influence normal and dysfunctional signaling networks. This research will provide a mechanistic framework for understanding the dynamics of regulated protein turnover and offer insights into fundamental properties of E3:substrate interactions. Such knowledge can guide future design of small molecule drug candidates to modulate dysfunctional E3 activities in cancer cells. Thus, this research will both improve basic understanding of mechanisms that drive cancer and aid in designing therapeutics that specifically target dysfunctional signaling pathways in cancer cells.