Heparan sulfate proteoglycans in signaling and development

硫酸乙酰肝素蛋白多糖在信号传导和发育中的作用

• 批准号: 10608092
• 负责人: Hiroshi Nakato
• 金额: $ 33.92万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10608092
• 关键词: Affect; Animal Model; Animals; Biological; Biology; Bone Morphogenetic Proteins; Carbohydrates; Cell Communication; Cell Line; Cell surface; Cells; Chondroitin Sulfate A; Chondroitin Sulfate Proteoglycan; Development; Drosophila genus; Enzymes; Erinaceidae; Event; Goals; Growth Factor; Heparan Sulfate Proteoglycan; Integral Membrane Protein; Knowledge; Leucine-Rich Repeat; Ligand Binding; Link; Mediating; Metabolic; Modeling; Molecular; Morphogenesis; Output; Pathway interactions; Pattern; Pattern Formation; Phenotype; Physiological; Play; Proteins; Proteoglycan; Proteomics; Radiolabeled; Research; Role; Signal Transduction; Structure; Structure-Activity Relationship; System; Trachea; genetic approach; in vitro Model; in vivo; in vivo Model; interdisciplinary approach; model organism; morphogens; mutant; novel; receptor; receptor function; stem cell niche

Project Summary The long-term goal of our research is to understand fundamental principles of cell communications mediated by heparan sulfate proteoglycans (HSPGs). HSPGs are a special type of carbohydrate-modified proteins that serve as co-receptors for various growth factors, including bone morphogenetic proteins, Wnt/Wingless, and Hedgehog. These HSPG co-receptors play critical roles in quantitative and robust control of signaling output. We study in vivo functions of HSPGs using the Drosophila model. Our earlier research has established critical roles of HSPGs in development, namely in morphogen signaling and gradient formation. In addition, we have demonstrated that HSPGs regulate the stem cell niche as a universal factor. Although proteoglycan biology has made significant progress, several major questions still remain to be elucidated. For example, the molecular mechanisms of co-receptor activities of HSPGs are poorly understood. It is also unknown how distinct HS structures regulate specific signaling and patterning events. It is now critical to develop an interdisciplinary approach, which can directly link detailed HS structural motifs, ligand binding, quantitative signaling output, and developmental/physiological phenotypes. Our previous studies suggested that HSPGs cooperate with other factors to exert co-receptor activity on the cell surface. To reveal the molecular basis for co-receptor function, we use proteomic and genetic approaches to identify these missing players. We recently found that Windpipe (Wdp), a transmembrane protein containing four leucine-rich repeats, acts as a novel negative co-receptor for Hh signaling. We also showed that Wdp is a chondroitin sulfate proteoglycan (CSPG). Thus, the Hh pathway is controlled by two classes of proteoglycan co-receptors: HSPGs (positive regulators) and Wdp (CSPG, a negative regulator). We will elucidate how such a "dual PG co-receptor system" achieves quantitatively controlled signaling output and precise pattern formation. To understand how a change in HS structure affects signaling and morphogenesis, we will establish an "in vitro" model using Drosophila cells. Despite the many strengths of the Drosophila model for in vivo studies, information on Drosophila HS structure is limited. This is mainly due to the difficulty of metabolic radio-labeling of HS in vivo using Drosophila animals. To fill this gap, we have recently generated novel Drosophila cell lines mutant for five HS modifying enzymes. Our studies using these cell lines will provide a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic information obtained over the last two decades using this animal model. Together, this application will advance our field by: (1) defining fundamental molecular mechanisms of co-receptor function and (2) comprehensive understanding of the structure-function relationship of HS.

项目摘要 我们研究的长期目标是了解细胞通信的基本原理 由硫酸乙酰肝素蛋白聚糖（HSPGS）介导。 HSPG是一种特殊类型的碳水化合物修饰 作为各种生长因子的共受体的蛋白质，包括骨形态发生蛋白， Wnt/无翼和刺猬。这些HSPG共受体在定量和稳健中起关键作用 控制信号输出。我们使用果蝇模型研究HSPG的体内功能。我们的早期 研究已经确定了HSPG在开发中的关键作用，即形态学信号传导和 梯度形成。此外，我们已经证明HSPG将干细胞生态位调节为A 通用因素。尽管蛋白聚糖生物学取得了重大进展，但仍有几个主要问题 仍然有待阐明。例如，HSPG的共受体活性的分子机制是 理解不佳。尚不清楚不同的HS结构如何调节特定的信号和图案 事件。现在，开发一种跨学科方法至关重要，该方法可以直接链接详细的HS 结构基序，配体结合，定量信号输出和发育/生理表型。 我们以前的研究表明，HSPG与其他因素合作以发挥共受体 细胞表面的活性。为了揭示共受体功能的分子基础，我们使用蛋白质组学和 识别这些失踪参与者的遗传方法。我们最近发现Windpipe（WDP），一个 包含四个含亮氨酸的重复序列的跨膜蛋白充当HH的新型负共受体。 信号。我们还表明，WDP是硫酸软骨素蛋白聚糖（CSPG）。因此，HH途径是 由两类的蛋白聚糖共受体控制：HSPG（正调节器）和WDP（CSPG，A，A 负调节器）。我们将阐明这样的“双PG共受体系统”如何定量实现 受控的信号输出和精确的模式形成。 要了解HS结构的变化如何影响信号传导和形态发生，我们将建立 使用果蝇细胞的“体外”模型。尽管果蝇模型具有许多优势 研究，果蝇HS结构的信息有限。这主要是由于代谢的困难 使用果蝇动物对HS在体内的无线电标记。为了填补这一空白，我们最近产生了小说 果蝇细胞系突变体用于五个HS修饰酶。我们使用这些细胞系的研究将提供 果蝇HS的详细结构信息与生物学知识之间的直接联系 在过去的二十年中，使用该动物模型获得了表型信息。 共同通过：（1）定义基本分子机制来推进我们的领域 共受体功能以及（2）对HS结构功能关系的全面理解。

项目成果

Generation of Drosophila Heparan Sulfate Mutant Cell Lines from Existing Fly Strains.

• DOI: 10.1007/978-1-0716-1398-6_47
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Nakato E;Bowden N;Nakato H
• 通讯作者: Nakato H

Molecular Genetic Techniques for the Proteoglycan Functions in Drosophila.

• DOI: 10.1007/978-1-0716-1398-6_32
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Bowden N;Takemura M;Nakato H
• 通讯作者: Nakato H

Endogenous epitope tagging of a JAK/STAT ligand Unpaired1 in Drosophila.

• DOI: 10.17912/micropub.biology.000387
• 发表时间: 2021
• 期刊: microPublication biology
• 作者: Takemura M;Lu YS;Nakato E;Nakato H
• 通讯作者: Nakato H

In vivo activities of heparan sulfate differentially modified by NDSTs during development.

硫酸乙酰肝素的体内活性在开发过程中受到 NDST 的差异修饰。

• DOI: 10.1002/pgr2.17
• 发表时间: 2024
• 期刊: Proteoglycan research
• 作者: Nakato,Eriko;Baker,Sarah;Kinoshita-Toyoda,Akiko;Knudsen,Collin;Lu,Yi-Si;Takemura,Masahiko;Toyoda,Hidenao;Nakato,Hiroshi
• 通讯作者: Nakato,Hiroshi