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.

Toll 受体信号传导研究首次发现其在胚胎背侧/腹侧 (DV) 模式形成中的作用 果蝇使我们对先天免疫和炎症的理解取得了重大进展 人类以及真核基因表达的调节。在早期的单细胞果蝇胚胎中，Toll 是 分布于整个质膜，但仅在腹侧激活。这是由三人调解的 顺序作用的细胞外丝氨酸蛋白酶，其中最后一个，复活节，裂解丝氨酸的前体形式 Toll 配体位于胚胎腹侧。 Easter 的腹侧激活是由 Pipe（高尔基体）控制的。 局部磺基转移酶，在围绕滤泡上皮的腹侧细胞中表达 正在发育的卵母细胞。管道产生硫酸化线索，嵌入蛋壳腹侧。许多 该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 的底物（蛋白质、碳水化合物还是脂质？），以及 Pipe 靶标的引导机制 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 发育中卵母细胞周围的管道表达导致胚胎具有残留的 DV 极性，表明 存在另一种以前未被发现的机制可以定义胚胎 DV 极性。这 应用程序请求支持一个试点项目，其目标是利用一种新颖的方法，即邻近性 通过体内生物素化标记，识别 Toll 受体信号通路的新成分。这 理由是，因为大多数已知成分都是在严格母体效应的筛选中鉴定出来的 突变体，编码女性成年生育能力所需的途径成分的基因很可能 被忽视了。邻近标记识别与蛋白质相互作用或共定位的因素 兴趣并且不限于生存所必需的蛋白质。将实现以下具体目标 追求的目标： 1. 确定丝氨酸蛋白酶的新调节因子和效应因子 我们将在雌性种系中将它们表达为与新开发的高度融合的卵周空间 活性生物素连接酶，然后在后代胚胎中进行邻近标记。 2. 鉴定蛋白质 通过 Pipe 与高尔基体相互作用或共定位，我们将使用 Pipe-生物素连接酶进行邻近标记 融合在卵巢卵泡细胞中表达。对于这两个具体目标，已确定的假定的子集 将测试相互作用因子对胚胎 DV 模式的影响。以前不被重视的身份 途径组件将进一步深入了解 Toll 活动的控制并激发新的途径 研究。此外，该项目还将作为邻近程度的可行性研究 标记可以有效识别发育中胚胎中真正的蛋白质/蛋白质相互作用及其程度 有用的遗传背景的可用性，例如改变亚细胞的突变体 复活节或管道的本地化，可以增强这种方法的有效性。