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

合成细胞工程有可能揭示构成生物细胞和细胞器功能的基本化学和物理过程，并推动生物启发设备和材料的设计，应用范围从医学到生物技术。最近发现，许多蛋白质和脂质在生物细胞中的功能依赖于翻译后修饰，即蛋白质合成后在表面的共价加成。这些修饰大部分是由高尔基体完成的，高尔基体是哺乳动物细胞中的一个中央膜细胞器，具有独特的动态变化的结构。然而，促进这些高度复杂的化学结构合成的高尔基体的特征，以及这些反应是如何在空间和时间上受到控制的，都没有被完全理解。为了更好地了解高尔基体结构的调节机制，这一提议将在体外系统中重建高尔基体的化学和物理特征。这项提议将开发新的策略来揭示膜的物理特征如何影响必要的翻译后修饰-糖基化。具体地说，它将利用微流控和基于囊泡的平台在体外重建脂质载体的多步糖基化修饰模型，并揭示概括翻译后修饰过程所需的膜空间和物理特征的规律。这些研究的成功完成将揭示设计规则，这些规则将通过重新组织这些反应来创建其他翻译后修饰，包括合成修饰。有了这些洞察力，就有可能扩大这些发现，以影响修饰的蛋白质和脂类的活性和功能，从而影响生物过程。该项目将在膜生物物理、膜蛋白质重组、蛋白质工程、微流体学、传输现象和化学动力学方面架起实现项目目标所需的各种跨学科技术的桥梁。这里开发的知识将使生物技术和制药行业受益，并在治疗化合物的设计中具有潜在的生物制造应用。该项目由生物科学局分子和细胞生物科学部的细胞动力学和功能与系统和合成生物学集群以及工程局的化学、生物工程、环境和运输系统部门的细胞和生化工程计划共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.