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.

最初发现其在胚胎背/腹侧（DV）构图中的作用，对收费受体信号的研究 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 人类和真核基因表达的调节。在早期的单细胞蝇胚胎中，通行费是 分布在整个质膜中，但仅在腹侧激活。这是由三个介导的 依次作用的细胞外丝氨酸蛋白酶，其最后一个复活节裂解了该蛋白酶的前体形式 在胚胎的腹侧收费配体。复活节的腹腹激活由管道控制，高尔基 在周围的卵泡上皮的腹腹细胞中表达的局部硫代转移酶 发展卵母细胞。管道产生一个硫酸盐的提示，该提示被腹侧嵌入蛋壳中。许多 该途径的各个方面仍有待阐明，包括直接的精确分子性质 管道（蛋白质，碳水化合物或脂质？）的底物以及管道靶标的机制 复活节的腹侧激活。除了管道表达的腹侧限制外，均匀的低水平 在发育中的卵母细胞周围的管道表达导致残留DV极性的胚胎，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 申请要求支持试点项目，其目的是利用一种新颖的方法，接近性 通过体内生物素化标记，以鉴定收费受体信号通路的新成分。这 理由是因为在屏幕上确定了大多数已知组件，以实现严格的孕产妇效应 突变体，编码女性生存到成年所需的编码途径成分的基因可能是 被忽略了。接近标签标记确定与与蛋白质相互作用或共定位的因素 兴趣，不仅限于可用于生存的蛋白质。以下具体目标将是 追捕：1。确定在运行中运行的丝氨酸蛋白酶的新调节剂和效应子 Perivitelline空间我们将在女性种系中表达为新发达，高度的融合 活性生物素连接酶，然后在后代胚胎中执行接近标记。 2。识别蛋白质 与管道中的高尔基体相互作用或共定位，我们将与管道 - 生物素连接酶进行接近标记 在卵巢卵泡细胞中表达的融合。对于两个特定目标，已确定的一部分 相互作用者将进行对胚胎DV构图的影响。以前未欣赏的身份 途径组件将进一步了解控制收费活动的控制，并刺激新的途径 研究。此外，该项目还将作为邻近程度的可行性研究 标记可以有效地识别出发育胚胎中的真正的蛋白质/蛋白质相互作用以及程度 有用的遗传背景的可用性，例如改变亚细胞的突变体 复活节或管道的定位可以提高这种方法的有效性。