The rate of protein turnover can act as a pacemaker to coordinate responses within and between cells, and is frequently dysregulated in human disease. Yet we know remarkably little about what controls substrate degradation kinetics or how these kinetics translate into downstream responses. One possible reason is the lack of available models for high- resolution structure-function analysis of degradation and transcriptional activation. The SCF class of E3 ubiquitin ligases is highly conserved among animals, plants and fungi. We propose to use an SCF involved in auxin response, at the heart of nearly every aspect of plant biology, as a model to investigate general principles underlying E3 function and connect that function to transcriptional activation and morphogenesis. The small-molecule triggered degradation in the auxin pathway offers a unique advantage for these studies, and has facilitated our engineering of auxin-induced degradation and transcriptional activation in yeast. Work with this system has led to our central hypothesis: the auxin system functions as a universal developmental timer in plants, and similar logic circuits likely act in most eukaryotes. To test this hypothesis, we propose to: (1) Define the determinants and relevance of variation in degradation rates. We have already identified several domains of interest in E3 and substrate components, and are using synthetic and computational tools to connect individual residues to degradation dynamics. (2) Quantify the impact of degradation rate on transcriptional repression. We have extended our synthetic assays to include auxin-induced transcription. We can now quantitatively track the molecular events between substrate turnover and downstream responses over time. This technology enables our study of previously intractable problems like how the removal of co-repressors is integrated with transcriptional activation. (3) Couple cellular degradation timers to developmental transitions. We have shown in transgenic plants that substrate degradation rate sets the pace of lateral organ development. We will use multiple, complementary approaches to analyze the transcriptome of these plants to elucidate how the timing of substrate turnover regulates developmental progression in a cell-type-dependent manner. Together, the proposed work will provide a mechanistic framework for E3 function in the auxin response and potentially provide insights into fundamental properties of E3:substrate interactions and downstream events. These insights can inform our understanding of E3s associated with human disease, as well as guiding future design of synthetic circuits using auxin components for therapeutic applications.
Ubiquitin-mediated protein degradation is key to cellular homeostasis and is often disrupted in human disease. The human genome encodes more E3s than protein kinases, and the specificity of E3:substrate interactions makes them attractive drug targets. A structural and biochemical understanding of the dynamic nature of E3:substrate interactions and their link to transcriptional control, especially in a readily-screenable synthetic context, might allow new breakthroughs in this area.
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