Spatial Control of Proteolysis in Dorsoventral Polarity

描述（由申请人提供）：果蝇胚胎的背腹侧轴是通过腹侧激活Toll信号通路建立的，导致背侧转录因子的分级核摄取。途径激活依赖于卵发生过程中通过Pipe（一种假定的硫酸肝素2-O-硫转移酶）在未知碳水化合物底物上的作用合成的腹侧线索。在胚胎中，受体Toll的配体是由丝氨酸蛋白酶级联的4个成员（Nude1、原肠胚形成缺陷（GD）、Snake和Easter）参与的蛋白水解加工反应产生的。已发表的研究表明，早期级联事件独立于管道活动而发生，不受腹侧限制，而新的数据表明，复活节蛋白酶的激活需要管道，并且可能受到腹侧限制。这些观察结果表明，空间控制发生在蛋白酶级联中。为了研究pipe调控级联反应的机制，我们提出了三个目的来验证由pipe修饰的糖胺聚糖（GAGs）作为促进GD和/或Snake腹侧活性的辅助因子的假设。这些包括：(1)通过Western blotting和活性蛋白酶的定位，确定体内蛋白酶级联中的空间调节发生在哪里；(2)利用纯化的蛋白酶在体外表征GAG与GD和Snake相互作用的功能后果，并确定这些相互作用的特异性是否与管道修饰的GAGs的预期一致；(3)在体内鉴定参与管道调节蛋白酶的gag和蛋白聚糖，利用新的选择策略分离出在没有管道的情况下结构改变的物种。这项工作有助于理解蛋白酶级联反应的调控，例如那些参与先天免疫反应、止血、伤口愈合和突触可塑性的蛋白酶级联反应。此外，了解糖胺聚糖微异质性是如何促进模式形成的，对于理解硫酸肝素蛋白聚糖在脊椎动物和无脊椎动物发育中的许多新角色非常重要，例如，敲除与管道相关的小鼠基因会导致肾脏发育不全、骨骼和眼睛缺陷。

DESCRIPTION (provided by applicant): 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-O- 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, Nude1, 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.