The Ubiquitin proteasome system (UPS) plays essential roles in all eukaryotic cells, and has been implicated in many human diseases including cancer, HIV, diabetes and others. In recent years, the UPS-mediated degradation of transcription factors both in the dark and light was shown to play central roles in light signaling pathways in plants. The long-term goal of our research is to contribute essential information toward fundamental understanding of regulated proteolysis in eukaryotic cells. Our objective in this application is to elucidate the primary biochemical function of a novel sensory photoreceptor family phytochrome in plants. The phytochrome family (phyA-phyE in the model plant Arabidopsis) modulates developmental programs in response to changes in ambient light conditions throughout the plant life cycle. In response to light, phytochromes undergo changes in structure from an inactive form to an active form and migrate into nucleus, where the activated phytochromes physically attach to a small family of basic helix-loop-helix (bHLH) transcription factors called Phytochrome Interacting Factors (PIFs). PIFs function as negative regulators of photomorphogenesis in the dark. Upon direct physical interaction with phytochromes, PIFs are rapidly degraded through the UPS. The removal of PIFs after light exposure results in large-scale changes in gene expression that promote photomorphogenic development of plants. Although, strong progress has been made in understanding phytochrome signaling pathways in recent years, the primary biochemical mechanism by which phytochromes transduce light signal is still unknown. Moreover, the kinase necessary for the light- induced degradation of PIFs remain elusive. Because direct physical interactions with phytochromes are necessary for the light-induced degradation of PIFs, our central hypothesis is that phytochromes function as substrate recognition components of E3 Ubiquitin ligase enzymes for the rapid polyubiquitylation and degradation of PIFs in response to light. To gather evidence in support of this concept, the specific objectives are to: (1) Test the function of phytochromes as substrate recognition components of E3 Ubiquitin ligase enzymes, and (2) Identify and functionally characterize new factors that are involved in phytochrome signaling pathways. The approaches include examining if phytochromes form E3 Ubiquitin ligase enzyme complexes by various interaction and functional assays, and identifying new factors by targeted interaction assays as well as a novel genetic screen designed to isolate factors involved in degradation of PIFs in response to light. The conceived hypothesis in this proposal challenges the conventional theories about phytochrome signaling mechanisms and is expected to redirect the focus of this field.
Understanding the fundamental mechanisms in regulated proteolysis has strong implications in human health. Upon completion of this project, it is our expectation that we will have a better mechanistic view of how a novel light-switchable E3 Ubiquitin ligase enzyme regulates photomorphogenesis in plants. The significance and positive impact of this research will not only be shifting paradigm in understanding a basic eukaryotic signaling pathway but also in developing optogenetics tools useful to probe and/or prevent human diseases.
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