Collaborative Research: EAGER: Uncovering the role of Golgi organization on function

合成细胞工程有可能揭示生物细胞和细胞器功能背后的基本化学和物理过程，并推进生物启发设备和材料的设计，其应用范围从医学到生物技术。近年来，人们发现生物细胞中许多蛋白质和脂质的功能依赖于蛋白质合成后的翻译后修饰，即蛋白质表面的共价添加。大多数这些修饰是由高尔基体进行的，高尔基体是哺乳动物细胞中的一种中央膜细胞器，具有独特的动态变化结构。然而，高尔基体促进这些高度复杂的化学结构合成的特征，以及这些反应是如何在空间和时间上受到控制的，都没有得到充分的了解。为了更好地理解高尔基体结构的调控机制，本研究将在体外系统中重构高尔基体的化学和物理特征。该建议将开发新的策略来揭示膜的物理特征如何影响基本的翻译后修饰，糖基化。具体来说，它将使用微流体和基于囊泡的平台在体外重建脂质载体的多步骤糖基化修饰模型，并揭示概括翻译后修饰过程所需的膜空间和物理特征规则。这些研究的成功完成将揭示设计规则，这些规则将通过这些反应的重组来创建其他翻译后修饰，包括合成修饰。有了这些见解，将有可能扩展这些发现，以影响修饰蛋白和脂质的活性和功能，从而影响生物过程。本项目将结合膜生物物理学、膜蛋白重构、蛋白质工程、微流体学、输运现象和化学动力学等多种跨学科技术，以实现项目目标。这里开发的知识将有利于生物技术和制药工业，并在设计治疗化合物方面具有潜在的生物制造应用。该项目由生物科学理事会分子和细胞生物科学部细胞动力学、功能、系统和合成生物学集群以及工程理事会化学、生物工程、环境和运输系统部细胞和生化工程计划共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Synthetic cell engineering has the potential to reveal insights into the fundamental chemical and physical processes that underlie the function of biological cells and organelles as well as advance the design of biologically-inspired devices and materials with applications ranging from medicine to biotechnology. Recently, it has become clear that the function of many proteins and lipids in biological cells depends on posttranslational modifications, covalent additions to the surface of proteins after their synthesis. A majority of these modifications are performed by the Golgi apparatus, a central membrane organelle in mammalian cells with a unique and dynamically changing structure. Yet neither the features of the Golgi that facilitate the synthesis of these highly complex chemical structures nor how those reactions are spatially and temporally controlled is fully understood. To better understand the regulatory mechanisms of the Golgi structure, this proposal will reconstitute chemical and physical features of the Golgi in an in vitro system. This proposal will develop new strategies to uncover how physical features of membranes influence an essential posttranslational modification, glycosylation. Specifically, it will recreate a model multi-step glycosylation modification of lipid carriers in vitro using both microfluidic and vesicle-based platforms and uncover the rules of membrane spatial and physical features required to recapitulate the posttranslational modification process. Successful completion of these studies will reveal design rules that will enable creation of other posttranslational modifications through re-organization of these reactions, including synthetic modifications. With these insights, it will be possible to extend these findings to influence the activity and function of modified proteins and lipids in order to impact biological processes. This project will bridge a variety of interdisciplinary techniques in membrane biophysics, membrane protein reconstitution, protein engineering, microfluidics, transport phenomena, and chemical kinetics necessary to realize the project goals. The knowledge developed here will benefit the biotechnology and pharmaceutical industries and have potential biomanufacturing applications in the design of therapeutic compounds. This project is jointly funded by the Cellular Dynamics and Function and Systems and Synthetic Biology Clusters, Division of Molecular and Cellular Biosciences, Directorate for Biological Sciences and by the Cellular and Biochemical Engineering Program, Division of Chemical, Bioengineering, Environmental and Transport Systems, Directorate for Engineering.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.