Grant: $150,053 - National Institutes of Health - Sep. 18, 2009
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Award Description: The dorsoventral axis of the Drosophila embryo is established by ventral activation of the Toll signaling pathway, resulting in the graded nuclear uptake of the Dorsal transcription factor. Pathway activation relies on a ventral cue synthesized during oogenesis through the action of Pipe, a Golgi-localized protein having sequence similarity to glycosaminoglycan (GAG) sulfotransferases. In the embryo, the Toll ligand is generated as the last step in a serine protease cascade comprising the Nudel, Gastrulation Defective (GD), Snake and Easter proteases. We have shown that activation of Easter requires Pipe activity, while activation of Nudel and GD and cascade-dependent processing of Snake do not. We have also shown that the heparin sulfate composition of the eggshell matrix is shifted from a tri-sulfated to a mono-sulfated form when pipe is mutated, suggesting that Pipe does not modify GAGs, and perhaps GAG-like molecules, despite genetic studies that have appeared to rule out a role for GAGs in dorsoventral patterning. Here we propose three aims to test aspects of the hypothesis that Snake, perhaps in complex with GD, activates Easter ventrally in a reaction requiring a sulfated Pipe target as a cofactor: 1) To identify processed and active forms of Snake using activity labeling reagent and immunological methods in S2 cells and in embryo extracts; 2) To characterize the glycan- and matrix-binding profiles of GD and Snake using the CFG glycan array and ELISA and blot-based assays; and 3) To determine the roles of a candidate Pipe target core protein and of glycoaminoglycans in patterning using clone analysis, enzymatic disruption of a potential sulfated sugar structure, and biochemical assay of target glycan structures. The dorsoventral protease cascade provides a robust and manipulable model for studying the critical regulation of serine protease cascades and of growth factor distribution and activity, thus providing broad developmental and biomedical significance to these studies. This project will advance our understanding of the regulation of proteases by extracellular matrix proteins and sugars during development and is relevant to inflammatory diseases.
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This award's data was last updated on Sep. 18, 2009. Help expand these official descriptions using the wiki below.