Intracellular switching using genetically engineered protein microdomains

使用基因工程蛋白质微结构域进行细胞内转换

• 批准号: 8865428
• 负责人: John Andrew MacKay
• 金额: $ 31.74万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8865428
• 关键词: Adoption; Binding; Biocompatible Materials; Biological; Biological Models; Biological Process; Biology; Caveolins; Cell Size; Cell Surface Receptors; Cell membrane; Cell model; Cell physiology; Cell surface; Cells; Cellular biology; Chimeric Proteins; Clathrin; Clathrin Heavy Chains; Clathrin Light Chains; Clathrin-Coated Vesicles; Complex; Cytosol; DNA; Data; Dimerization; Disease; Dynamin; Elastin; Endocytosis; Engineering; Epidermal Growth Factor Receptor; Event; Fluorescence Microscopy; G-Protein-Coupled Receptors; Genes; Genetic Engineering; Genetic Transcription; Glass; Goals; Golgi Apparatus; Immunofluorescence Immunologic; Infection; Knock-out; Libraries; Life; Lighting; Macromolecular Complexes; Malignant Neoplasms; Mediating; Membrane; Modeling; Molecular; Molecular Structure; Mono-S; Names; Nuclear; Organelles; Pathway interactions; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Physiological; Polymers; Population; Process; Proteins; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Recovery; Regulation; Reporting; Research Project Grants; STAT protein; Series; Shapes; Signal Transduction; Small Interfering RNA; Sorting - Cell Movement; Specificity; Structure; Technology; Temperature; Testing; Therapeutic; Time; Western Blotting; base; caveolin 1; clinically relevant; copolymer; design; drug discovery; flotillin; human disease; improved; information processing; inhibitor/antagonist; insight; knock-down; novel; polypeptide; promoter; protein function; public health relevance; response; self assembly; signal processing; small molecule; stem; synthetic biology; tool; trafficking; transcription factor

 DESCRIPTION Intracellular switching using genetically engineered protein microdomains Biology is unparalleled in the replication of complex structures with diverse functions on the molecular and cellular scale; however, our ability to engineer functional materials at or below the size of the cell remains primitive. By using biological materials to assemble structures, process information, and harness energy, the emerging field of synthetic biology may bridge the gap between current technology and that needed to study and intervene in disease. Towards this futuristic goal, this project elaborates on a platform discovered by our team to control functional structures that are 10-1000 times smaller than a cell. These structures are based on polypeptides; therefore, they can be encoded in DNA and grown inside of living cells. Our group recently reported that temperature-responsive protein polymers expressed in the cytosol assemble organelle-sized structures within minutes of an increase of 1 degree Celsius. We named these structures genetically engineered protein microdomains, reported that they can either sort or co-assemble intact fusion proteins, and that their assembly can control a model cellular internalization pathway called clathrin-mediated endocytosis. Now we present preliminary evidence that these microdomains can activate a model cell-surface receptor and drive its internalization. This research project is designed to validate and expand the potential applications for these microdomains. The overall hypothesis is that through design, these microdomains can stimulate, deactivate, or respond to target cellular processes. Three aims are proposed: Aim 1) Manipulation of endocytotic pathways using microdomains; Aim 2) Interrogating cell signaling using ELP microdomains; and Aim 3) Expanding microdomain technology. This application innovates in three main ways: i) our interdisciplinary team is the first to report that intracellular ELPs generate microdomains that exert control over cellular pathways; ii) unlike traditional mechanisms for modulating protein activity, ELP microdomains can be activated or deactivated rapidly in live cells; and iii) this project will generalize these strategies so that they can be used to target a broad array of cellular functions. The successful demonstration of this approach is intended to shift the paradigm for how cellular biology studies are performed, enabling precise manipulation of biological processes that are fundamentally important to drug discovery. A comprehensive series of studies will be performed to demonstrate the breadth of potential applications for microdomain assembly within the cell. When completed, this project will deliver a biomolecular toolbox of broad utility to study biological processes associated with human disease.

 生物学在分子和细胞尺度上复制具有不同功能的复杂结构方面是无与伦比的;然而，我们在分子水平或低于分子水平上设计功能材料的能力， 细胞的大小保持原始。通过使用生物材料组装结构、处理信息和利用能量，新兴的合成生物学领域可能会弥合当前技术与研究和干预疾病所需技术之间的差距。为了实现这一未来的目标，该项目详细阐述了我们的团队发现的一个平台，以控制功能 比细胞小10-1000倍的结构。这些结构基于多肽;因此，它们可以在DNA中编码并在活细胞内生长。我们的研究小组最近报告说，细胞质中表达的温度响应性蛋白质聚合物在1摄氏度升高的几分钟内组装成细胞器大小的结构。我们将这些结构命名为基因工程蛋白质微结构域，报道了它们可以分选或共组装完整的融合蛋白，并且它们的组装可以控制称为网格蛋白介导的内吞作用的模型细胞内化途径。现在，我们提出了初步的证据，这些微区可以激活模型细胞表面受体，并驱动其内化。该研究项目旨在验证和扩展这些微域的潜在应用。总体假设是，通过设计，这些微区可以刺激，灭活或响应靶细胞过程。提出了三个目标：目的1）使用微结构域操纵内吞途径;目的2）使用ELP微结构域询问细胞信号传导;和目的3）扩展微结构域技术。该应用在三个主要方面进行了创新：i）我们的跨学科团队首次报告细胞内ELP产生对细胞通路施加控制的微结构域; ii）与传统的调节蛋白质活性的机制不同，ELP微结构域可以在活细胞中快速激活或失活; iii）该项目将推广这些策略，以便它们可以用于靶向广泛的细胞功能。这种方法的成功演示旨在改变如何进行细胞生物学研究的范式，从而能够精确操纵对药物发现至关重要的生物过程。将进行一系列全面的研究，以证明细胞内微区组装的潜在应用的广度。完成后，该项目将提供一个具有广泛实用性的生物分子工具箱，用于研究与人类疾病相关的生物过程。