Identification of new components of the Toll receptor signaling pathway in Drosophila

果蝇Toll受体信号通路新成分的鉴定

• 批准号: 10360133
• 负责人: DAVID S. STEIN
• 金额: $ 7.93万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-15 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360133
• 关键词: Adult; Biological; Biological Response Modifiers; Biotin; Biotinylation; Blood coagulation; Carbohydrates; Cell membrane; Cells; Complement; Cues; Data; Dorsal; Drosophila genus; Effectiveness; Embryo; Event; Exhibits; Feasibility Studies; Female; Fibrinolysis; Gene Expression; Genes; Genetic; Goals; Golgi Apparatus; Health; Human; Immunity; Inflammation; Inflammatory; Investigation; Label; Ligands; Ligase; Lighting; Lipids; Mediating; Molecular; Natural Immunity; Nature; Oocytes; Ovarian Follicle; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Perivitelline Space; Pilot Projects; Play; Precipitation; Process; Proteins; Reagent; Receptor Signaling; Regulation; Regulator Genes; Request for Applications; Research; Residual state; Role; Science; Serine Protease; Side; Signal Pathway; Snakes; Sulfate; Techniques; Technology; Testing; base; extracellular; fly; follicular epithelial cell; gastrulation; in vivo; insight; interest; mutant; novel; novel strategies; protein protein interaction; receptor; sample fixation; success; sulfotransferase; transcription factor

First discovered for its role in embryonic dorsal/ventral (DV) patterning, studies of Toll receptor signaling in Drosophila have led to major advances in our understanding of innate immunity and inflammation in humans, and of the regulation of eukaryotic gene expression. In the early single-cell fly embryo, Toll is distributed throughout the plasma membrane, but only activated ventrally. This is mediated by three sequentially-acting extracellular serine proteases, the last of which, Easter, cleaves a precursor form of the Toll ligand on the ventral side of the embryo. Ventral activation of Easter is controlled by Pipe, a Golgi- localized sulfotransferase that is expressed in ventral cells of the follicular epithelium that surrounds the developing oocyte. Pipe generates a sulfated cue that becomes embedded ventrally in the eggshell. Many aspects of this pathway remain to be elucidated, including the precise molecular nature of the direct substrate of Pipe (protein, carbohydrate, or lipid?), and the mechanism through which the Pipe target directs the ventral activation of Easter. In addition to the ventral restriction of pipe expression, uniform low levels of pipe expression around the developing oocyte results in embryos with residual DV polarity, indicating that there exists another, previously undetected mechanism that can define embryonic DV polarity. This application requests support for a pilot project, the objective of which is to utilize a novel approach, proximity labeling by in vivo biotinylation, to identify new components of the Toll receptor signaling pathway. The rationale is that because most of the known components were identified in screens for strict maternal effect mutants, genes encoding pathway components required for viability of females to fertile adulthood are likely to have been overlooked. Proximity labeling identifies factors that interact or colocalize with proteins-of- interest and is not limited to proteins that are dispensable for survival. The following specific aims will be pursued: 1. To identify new regulators and effectors of the serine proteases operating in the perivitelline space we will express them in the female germline as fusions to newly-developed, highly active biotin ligases, then perform proximity labeling in progeny embryos. 2. To identify proteins that interact with or colocalize in the Golgi with Pipe we will perform proximity labeling with Pipe-biotin ligase fusions expressed in ovarian follicle cells. For both specific aims, a subset of the identified presumed interactors will be tested for effects upon embryonic DV patterning. The identity of previously unappreciated pathway components will provide further insight into the control of Toll activity and stimulate new avenues of research. In addition, this project will also serve as a feasibility study of the extent to which proximity labeling can effectively identify bona fide protein/protein interactions in developing embryos and the extent to which the availability of useful genetic backgrounds, for example mutants that alter the subcellular localization of Easter or Pipe, can enhance the effectiveness of this approach.

首次发现Toll受体在胚胎背侧/腹侧(DV)模式中的作用，对Toll受体信号转导的研究 果蝇在我们对先天性免疫和炎症的理解方面取得了重大进展 人类，以及真核基因表达的调控。在早期单细胞果蝇胚胎中，Toll是 分布于整个质膜，但仅激活腹侧。这是由三个人调解的 顺序作用的胞外丝氨酸蛋白酶，最后一个，复活节，裂解一种前体形式 胚胎腹侧的Toll配体。复活节的腹侧兴奋是由一种高尔基体--管道控制的。 定位的磺基转移酶，表达在围绕滤泡上皮的腹侧细胞中 发育中的卵母细胞。管道产生一种硫酸盐化的线索，嵌入蛋壳中的腹侧。许多 这一途径的一些方面仍有待阐明，包括直接的分子性质 PIPE的底物(蛋白质、碳水化合物或脂肪？)，以及PIP靶的导向机制 复活节的腹侧活动。除了PIPE表达的腹侧限制外，一致的低水平 在发育中的卵母细胞周围表达管道会导致胚胎具有残留的DV极性，这表明 还有另一种以前未被发现的机制可以定义胚胎DV的极性。这 应用程序请求支持一个试点项目，该项目的目标是利用一种新的方法--邻近性 体内生物素化标记，以确定Toll受体信号通路的新成分。这个 其基本原理是，因为大多数已知的成分在筛选中被确定为严格的母体效应 突变，编码雌性成年期存活所需的途径组件的基因很可能 被忽视了。邻近标记识别与蛋白质相互作用或共定位的因子 兴趣，并不局限于生存所必需的蛋白质。以下是具体目标 研究方向：1.确定新的丝氨酸蛋白酶的调节者和效应者。 卵黄周空间我们将在雌性生殖系中将它们表达为新开发的、高度 活性生物素连接酶，然后在子代胚胎中进行邻近标记。2.鉴定具有 与管道在高尔基体内相互作用或共定位我们将使用管道-生物素连接酶进行邻近标记 融合基因在卵泡细胞中表达。对于这两个特定目标，确定的推定的子集 将测试交互作用对胚胎DV模式的影响。以前不被赏识的身份 途径组件将进一步深入了解Toll活性的控制，并刺激新的途径 研究。此外，该项目还将作为对接近程度的可行性研究。 标记可以有效地识别胚胎发育过程中真正的蛋白质/蛋白质相互作用及其程度 对此有用的遗传背景的可用性，例如改变亚细胞的突变体 本地化的复活节或烟斗，可以增强这一方法的有效性。