Spatial Control of Proteolysis in Dorsoventral Polarity

背腹极性蛋白水解的空间控制

• 批准号: 7484258
• 负责人: ELLEN K LEMOSY
• 金额: $ 24.41万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7484258
• 关键词: Antibodies; Binding; Biochemical; Biological Assay; Carbohydrates; Cells; Class; Cleaved cell; Confocal Microscopy; Cues; Data; Defect; Deposition; Development; Dorsal; Drosophila genus; Embryo; Endopeptidases; Epitopes; Event; Extracellular Matrix; Eye; Generations; Genes; Glycosaminoglycans; Hemostatic function; Heparan Sulfate Proteoglycan; Heparin; Heparin Binding; Heparitin Sulfate; Immune response; In Vitro; Instruction; Invertebrates; Kidney; Knock-out; Knowledge; Ligands; Methods; Modeling; Modification; Mus; Nuclear Grade; Oocytes; Oogenesis; Pathway interactions; Pattern; Peptide Hydrolases; Phage Display; Proteoglycan; Proteolysis; Proteolytic Processing; Publishing; Range; Reaction; Regulation; Research Personnel; Role; Serine Protease; Signal Pathway; Signal Transduction; Skeletal system; Snakes; Specificity; Synaptic plasticity; Testing; Thinking; Western Blotting; Work; Wound Healing; base; carbohydrate structure; cofactor; extracellular; fly; in vivo; inhibitor/antagonist; member; mutant; novel; programs; receptor; sulfation; sulfotransferase; transcription factor; uptake

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 putative heparan sulfate 2-0- sulfotransferase, on an unknown carbohydrate substrate. In the embryo, the ligand for the receptor Toll is generated by a proteolytic processing reaction involving 4 members of a serine protease cascade, Nudel, Gastrulation Defective (GD), Snake and Easter. Published work has shown that early cascade events occur independently of pipe activity and are not ventrally restricted, while new data shows that activation of the Easter protease requires pipe and is probably ventrally restricted. These observations establish that spatial control occurs within the protease cascade. To investigate the mechanism by which pipe regulates the cascade, three aims are proposed to test the hypothesis that glycosaminoglycans (GAGs) modified by Pipe serve as cofactors promoting the ventral activity of GD and/or Snake. These include: (1) determining where spatial regulation occurs in the protease cascade in vivo, by Western blotting and localization of active proteases; (2) characterizing the functional consequences of GAG interactions with GD and Snake in vitro using purified proteases, and determining whether the specificity of these interactions is consistent with that expected for Pipe-modified GAGs; and (3) identifying GAGs and proteoglycans involved in protease regulation by pipe in vivo, using novel selection strategies to isolate species structurally altered in the absence of pipe. This work is relevant to understanding the regulation of protease cascades, such as those involved in the innate immune response, hemostasis, wound healing, and synaptic plasticity. Additionally, knowledge of how glycosaminoglycan microheterogeneity contributes to patterning is important to understanding the many emerging roles of heparan sulfate proteoglycans in vertebrate and invertebrate development, where, for example, a knock-out of a mouse gene related to pipe results in renal agenesis and skeletal and eye defects.

果蝇胚胎的背腹轴是通过腹侧激活Toll信号而建立的 途径，导致Dorsal转录因子的分级核摄取。通路激活依赖于 在卵子发生过程中通过Pipe的作用合成的腹侧线索上，Pipe是一种假定的硫酸乙酰肝素2-0- 磺基转移酶，在一个未知的碳水化合物底物。在胚胎中，受体Toll的配体是 通过涉及丝氨酸蛋白酶级联的4个成员，Nudel， 消化不良（GD），蛇和复活节。已发表的研究表明，早期级联事件发生在 独立于管道活动，且不受腹侧限制，而新数据显示， 复活节蛋白酶需要管道，可能是腹侧限制。这些观察结果表明， 控制发生在蛋白酶级联中。为了研究pipe调节细胞凋亡的机制， 级联，提出了三个目标来检验假设，即Pipe修饰的糖胺聚糖（GAG） 作为辅助因子促进GD和/或Snake的腹侧活动。其中包括：（1）确定 通过蛋白质印迹和活性蛋白的定位， 蛋白酶;（2）表征GAG与GD和Snake在体外相互作用的功能后果 使用纯化的蛋白酶，并确定这些相互作用的特异性是否与 预期用于Pipe修饰的GAG;和（3）鉴定涉及蛋白酶的GAG和蛋白聚糖 调节管在体内，使用新的选择策略，以隔离物种结构改变，在缺乏 管。这项工作与理解蛋白酶级联反应的调节有关，例如参与蛋白酶级联反应的那些。 先天免疫反应、止血、伤口愈合和突触可塑性。此外，知识 糖胺聚糖的微异质性如何促进模式化对于理解许多 硫酸乙酰肝素蛋白聚糖在脊椎动物和无脊椎动物发育中的新作用， 例如，敲除与pipe相关的小鼠基因会导致肾发育不全以及骨骼和眼睛缺陷。