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: 10571942
• 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 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10571942
• 关键词: Adult; Biological; Biological Response Modifiers; Biotin; Biotinylation; Blood coagulation; Carbohydrates; Cell membrane; Cells; Complement; Complex; 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; Membrane; Molecular; Natural Immunity; Nature; Oocytes; Ovarian Follicle; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Perivitelline Space; Pilot Projects; Play; Process; Proteins; Reagent; Receptor Signaling; Regulation; Request for Applications; Research; Residual state; Role; Science; Serine Protease; Side; Signal Pathway; Snakes; Sulfate; Techniques; Technology; Testing; extracellular; fly; follicular epithelial cell; gastrulation; gene regulatory network; in vivo; insight; interest; mutant; novel; novel strategies; 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.

Toll受体在胚胎背腹（DV）模式中的作用首次被发现， 果蝇在我们对先天免疫和炎症的理解方面取得了重大进展， 人，以及真核基因表达的调控。在早期的单细胞苍蝇胚胎中，托尔是 分布在整个质膜，但仅在腹侧激活。这是由三个中介 顺序作用的细胞外丝氨酸蛋白酶，其中最后一个，复活节，切割前体形式的 Toll配体位于胚胎的腹侧。复活节的神经激活由高尔基体Pipe控制， 局部磺基转移酶，表达于滤泡上皮的腹侧细胞， 发育中的卵母细胞管道产生一个硫酸化的线索，成为嵌入腹在蛋壳。许多 这一途径的各个方面仍有待阐明，包括直接作用的精确分子性质。 Pipe底物（蛋白质、碳水化合物或脂质？），以及管道目标引导的机制 复活节的腹侧激活除了腹侧限制管道表达外， 在发育中的卵母细胞周围表达pipe导致胚胎具有残留的DV极性，表明 存在另一种以前未发现的机制，可以定义胚胎DV极性。这 一个应用程序请求支持一个试点项目，其目标是利用一种新的方法，即接近 通过体内生物素化标记，以鉴定Toll受体信号传导途径的新组分。的 理由是，因为大多数已知成分是在严格的母体效应筛选中鉴定的， 突变体，基因编码的途径所需的组成部分，女性的生存能力，以生育的成年可能 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， 感兴趣的并且不限于为了生存而被激活的蛋白质。以下具体目标将是 追求：1.为了鉴定丝氨酸蛋白酶的新调节子和效应子， 我们将在雌性生殖系中表达它们，作为新开发的高度融合的基因。 活性生物素连接酶，然后在后代胚胎中进行邻近标记。2.来鉴定那些 与高尔基体相互作用或与Pipe共定位，我们将用Pipe-生物素连接酶进行邻近标记 融合蛋白在卵泡细胞中表达。对于这两个具体目标，一个子集的确定假定 将测试相互作用物对胚胎DV图案化的影响。以前不受重视的身份 通路组分将提供对Toll活性控制的进一步了解，并刺激新的途径， research.此外，该项目还将作为一个可行性研究，在多大程度上接近 标记可以有效地识别发育胚胎中真正的蛋白质/蛋白质相互作用， 有用的遗传背景的可用性，例如改变亚细胞结构的突变体， 本地化的复活节或管道，可以提高这种方法的有效性。