<p>Plant growth and development are strictly regulated by internal hormonal signaling networks, which integrate and coordinate to promote plants’ adaptation and survival in the changing environment. Among the diverse hormones, gibberellins (GAs) are the phytohormones that regulate various processes, from seed germination to fruit development. The conserved plant-specific GRAS family protein DELLAs, key repressors in the GA signaling pathway, serve as the central coordinator of multiple signaling networks through physical interactions with many key transcription factors/regulators in other pathways. </p><p>Diverse DELLA-interacting proteins (DIPs) from different signaling pathways and various protein families have been identified in recent years. All the DIPs interact with the C-terminal GRAS domain of DELLA, however, the mechanism of how the GRAS domain interacts with diverse proteins remains a mystery. To solve this problem, I expressed a number of DELLA proteins in E.coli and obtained high-purity protein for biochemical and structural analysis.</p><p>As the central coordinator of plant growth and development, DELLA’s activity and stability are regulated by post-translational modifications. Our lab recently showed that SECRET AGENT (SEC) modulates the activity of DELLA through O-linked N-acetylglucosamine (O-GlcNAc) modification in Arabidopsis. Nevertheless, SEC’s paralog SPINDLY (SPY), a putative O-GlcNAc transferase (OGT) identified 20 years ago, does not have OGT activity, and serves as opposite role to SEC in GA signaling with an unknown mechanism. </p><p>Our lab made the breakthrough in uncovering the SPY function, and showed it promotes the O-fucosylation of DELLA in planta. I further proved that SPY is a novel protein O-fucosyltransferase through biochemical analysis. SPY specifically transfers O-fucose from GDP-fucose to its substrate peptide, and SPY mutant proteins showed reduced or abolished transferase activity. This is the first work to identify O-fucosylation of nuclear proteins in any organism. O-fucosylation of DELLA activates DELLA by promoting its interaction with DIPs, opposite to repression of interaction with O-GlcNAcylation. Previous studies showed that SPY is involved in multiple cellular pathways such as GA signaling, cytokinin signaling and the circadian clock. Thus, SPY plays an important role in regulating plant growth and development through O-fucosylation of key components in diverse intracellular pathways. </p><p>SPY orthologs are conserved in bacteria, protists, algae and plants, while SEC orthologs are also present in fungi and animals. SPY-like and SEC-like proteins share high sequence similarity, except that two key residues important for the OGT activity of SEC is missing in SPY. Structure analysis of SPY (or its orthologs) would greatly facilitate our understanding of its unique substrate specificity. Toward this goal, I expressed Arabidopsis SPY proteins (as well as bacterial SPY orthologs) in E.coli and obtained high-purity protein for structural analysis. I further identified lead conditions that produce needle-cluster crystals. While optimization would be required, these studies will ultimately reveal the structure of SPY and the architecture of the active site, to show how SPY interacts with GDP-fucose for the transferase activity.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/13413 |
| Date | January 2016 |
| Creators | Sui, Ning |
| Contributors | Sun, Tai-ping |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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