Maternal Control of the Drosophila Embryonic Dorsal/Ventral Axis

果蝇胚胎背/腹轴的母体控制

• 批准号: 8446476
• 负责人: DAVID S. STEIN
• 金额: $ 29.81万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-15 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8446476
• 关键词: Affect; Bacteria; Behavior; Binding; Biological; Biological Models; Blood Clot; Blood Coagulation Disorders; Blood coagulation; Carbohydrates; Cells; Cleaved cell; Cues; Data; Development; Disease; Dorsal; Drosophila genus; Elements; Embryo; Epithelium; Etiology; Future; Gene Expression; Generations; Glycoproteins; Glycosaminoglycans; Goals; Growth Factor; Growth Factor Receptors; Health; Homologous Gene; Human; Immune; Infection; Inorganic Sulfates; Investigation; Knowledge; Ligands; Lighting; Mediating; Membrane; Mission; Molecular; Nature; Neurons; Oocytes; Oogenesis; Organism; Outcome; Parasites; Parasitic infection; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Perivitelline Space; Predisposition; Process; Property; Proteins; Public Health; Receptor Signaling; Regulation; Research; Role; Serine; Serine Protease; Side; Signal Pathway; Signal Transduction; Snakes; Structure; Testing; Therapeutic; Unspecified or Sulfate Ion Sulfates; Viral; Virus Diseases; Vitelline Membrane; Work; base; burden of illness; cell motility; egg; gastrulation; innovation; insight; microbial; novel; receptor; sulfotransferase; sulfurtransferase; vitelline membrane proteins

DESCRIPTION (provided by applicant): Sulfated carbohydrates such as glycosaminoglycans control many, diverse processes affecting human health including blood clotting, immune and neural cell migration, viral and microbial infection, and growth factor/receptor signaling. However, for many of these processes, how the sulfated carbohydrates mediate their biological effects at the mechanistic level is not understood, presenting a barrier to our ability to harness the properties of these molecules for therapeutic purposes. Drosophila embryo dorsal-ventral polarity is determined by a sulfated carbohydrate embedded in the eggshell that influences serine proteolytic activity in the perivitelline space between the embryonic membrane and the eggshell. Gastrulation Defective (GD) processes and activates Snake, Snake processes and activates Easter, and Easter cleaves the Spaetzle precursor into a ligand for the transmembrane receptor Toll. Activation of Easter occurs solely on the ventral side of the egg under the control of Pipe, a homologue of vertebrate glycosaminoglycan sulfating enzymes that is expressed in ventral follicle cells of the developing oocyte. Pipe transfers sulfate to the carbohydrate side chains of glycoproteins that become incorporated into the inner vitelline membrane layer of the eggshell and constitute a ventral cue that promotes processing of Easter by activated Snake. The long-term goal of this project is to determine how the sulfated ventral cue controls the spatially restricted processing of Easter, and in turn, the activation of Toll. Th central hypothesis, which is based on the recent observation that GFP-tagged GD becomes ventrally localized in the perivitelline space, is that processed GD binds to the sulfated ventral cue and once concentrated there, facilitates an interaction between Snake and Easter that results in Easter processing. The proposed investigations pursue three specific aims: 1) To elucidate the mechanism that controls the ventral localization of GD within the perivitelline space of the egg by determining the extent to which GD processing is required for its localization and by identifying determinants within GD that are required for localization and processing. 2) To determine how localized GD mediates the cleavage of Easter by Snake by identifying the GD domains that are required for its interactions with Easter and Snake, and by determining the extent to which GD promotes Snake protease activity, Easter susceptibility to cleavage, or productive interaction between the two proteins. 3) To elucidate the structure of the Pipe-modified carbohydrates that comprise the ventral cue and determine how these molecules facilitate GD localization and Easter processing by isolating VML, a key eggshell protein modified by Pipe, examining its associated carbohydrates by mass spectrometric analysis, and identifying proteins that interact with it in a Pipe/ventral cue-dependent manner. The proposed studies are significant because in the course of elucidating the molecular mechanism underlying this paradigmatic developmental pathway, they will yield new insights into the ways in which serine proteases and other medically important proteins are influenced by their interactions with sulfated carbohydrates.

描述（由申请方提供）：硫酸化碳水化合物（如糖胺聚糖）控制着影响人类健康的许多不同过程，包括凝血、免疫和神经细胞迁移、病毒和微生物感染以及生长因子/受体信号传导。然而，对于许多这些过程，硫酸化碳水化合物如何在机制水平上介导其生物学效应尚不清楚，这对我们利用这些分子的性质用于治疗目的的能力构成了障碍。果蝇胚胎背腹极性由嵌入蛋壳中的硫酸化碳水化合物决定，该碳水化合物影响胚胎膜和蛋壳之间的卵黄周间隙中的丝氨酸蛋白水解活性。Gastrulation Defective（GD）处理并激活Snake，Snake处理并激活Easter，Easter将Spaetzle前体切割成跨膜受体Toll的配体。复活节的激活仅发生在卵的腹侧，在Pipe的控制下，Pipe是脊椎动物糖胺聚糖硫酸化酶的同系物，在发育中的卵母细胞的腹侧卵泡细胞中表达。Pipe将硫酸盐转移到糖蛋白的碳水化合物侧链上，糖蛋白的碳水化合物侧链被并入蛋壳的内部卵黄膜层中，并构成腹侧线索，促进激活的Snake处理复活节。本项目的长期目标是确定硫酸化腹侧线索如何控制复活节的空间限制加工，进而激活Toll。中心的假设，这是基于最近的观察，GFP标记的GD成为腹侧本地化在卵周空间，是加工GD绑定到硫酸化的腹侧线索，一旦集中在那里，促进蛇和复活节之间的相互作用，导致在复活节处理。建议的调查追求三个具体的目标：1）通过确定GD加工所需的程度，并通过确定GD内的决定因素，所需的定位和处理，以阐明控制GD腹侧定位在卵周间隙内的机制。2)通过鉴定GD与Easter和Snake相互作用所需的GD结构域，并通过确定GD促进Snake蛋白酶活性、Easter对切割的敏感性或两种蛋白质之间的有效相互作用的程度，确定局部GD如何介导Snake对Easter的切割。3)为了阐明Pipe修饰的碳水化合物的结构，包括腹侧线索，并确定这些分子如何促进GD定位和复活节处理分离VML，一个关键的蛋壳蛋白修饰的Pipe，检查其相关的碳水化合物的质谱分析，并确定蛋白质，与它相互作用的Pipe/腹侧线索依赖的方式。拟议的研究是重要的，因为在阐明这一典型的发展途径的分子机制的过程中，他们将产生新的见解丝氨酸蛋白酶和其他医学上重要的蛋白质的影响的方式与硫酸化碳水化合物的相互作用。