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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

基本信息

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
项目类别：
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

项目摘要

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.

首次发现其在胚胎背/腹侧（DV）模式中的作用，研究Toll受体信号