A rapid, reversible switch for controlling intracellular trafficking

用于控制细胞内运输的快速、可逆开关

• 批准号: 7978859
• 负责人: John Andrew MacKay
• 金额: $ 24.3万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7978859
• 关键词: Anabolism; Area; Behavior; Biological; Biological Process; Biology; Cell physiology; Cells; Cellular biology; Clathrin; Clathrin-Coated Vesicles; Complex; Development; Disease; Dissection; Dominant-Negative Mutation; Elastin; Endocytosis; Engineering; Environment; Family; Figs - dietary; Fluorescence Microscopy; Future; Gene Targeting; Genes; Genetic; Goals; Green Fluorescent Proteins; Human; Infection; Investigation; Life; Macromolecular Complexes; Malignant Neoplasms; Mammalian Cell; Mediating; Methodology; Methods; Microscope; Microscopy; Molecular; Molecular Weight; Nanostructures; Nanotechnology; National Institute of Biomedical Imaging and Bioengineering; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Phase; Phase Transition; Problem Solving; Process; Property; Proteins; Recombinants; Regulation; Research; Research Project Grants; Scientist; Shock; Signal Pathway; Signal Transduction; Small Interfering RNA; Solutions; Specificity; Stimulus; System; Techniques; Technology; Temperature; Time; Transition Temperature; Tropoelastin; aqueous; biophysical chemistry; design; improved; innovation; mathematical model; nanoscale; novel; polypeptide; programs; protein purification; public health relevance; response; self assembly; success; therapy development; tool; tool development; trafficking; uptake

DESCRIPTION (provided by applicant): There are few techniques that can both rapidly and reversibly manipulate components inside living cells; furthermore, the development of tools capable of doing so will significantly enhance the ability of scientists to understand biology and treat disease. The long-term goal of this research is to engineer new molecular tools that can be assembled and operated inside biological environments such as the cell. Towards this goal, this proposal describes an investigation of the phase transition of environmentally-responsive polypeptides (ERPs). The hypothesis is that ERPs can sequester key factors inside cells and that they can reversibly switch on and off molecular pathways inside live cells. To demonstrate the feasibility of this approach, ERPs will be designed to control a ubiquitous cellular process called clathrin-mediated endocytosis. This process is important in the regulation of many diseases, including cancer and infection. The following specific aims are proposed: 1) Biosynthesis and biophysical chemistry of ERP switches: A biophysical approach will be used to characterize the behavior of ERPs and ERP-fusion in solution and mathematical modeling will be developed that permits these systems to be designed to response to any desired temperature. The intracellular behavior of ERPs will be observed inside human cells that produce ERPs fused to a fluorescent protein that can be viewed under a microscope. Using this construct, we will evaluate how quickly the ERPs self-associate inside the cell. A phase diagram describing the parameters that influence the intracellular ERP phase transition will be compared to that in free solution. 2) ERP switching of clathrin-mediated endocytosis: ERPs will be fused to a key protein involved with the uptake of factors in the cellular environment. This research project is intended to culminate in a general strategy for selectively halting cellular pathways. This proposal is innovative in three main ways: (i) the specific ERP behavior proposed here has never been observed inside of live human cells; (ii) this approach is expected to be a rapid, reversible technique that can potentially switch on or off specific cell pathways; and (iii) this approach can be generalized to target any cellular pathway for which a known protein interacts. 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 the treatment of disease. PUBLIC HEALTH RELEVANCE: Understanding the process by which diseases, such as cancer or infection, proceed at a cellular level is critical to the development of new treatments. This proposal describes the exploration of a novel, enabling technology that is intended to rapidly turn on and off critical cellular process involved with disease. Success of this project will catalyze numerous future studies of many disease processes and culminate with improved treatments for illnesses.

描述（由申请人提供）：很少有技术可以快速和可逆地操纵活细胞内部的组件。此外，能够这样做的工具的开发将显着增强科学家了解生物学和治疗疾病的能力。这项研究的长期目标是设计可以在细胞等生物学环境中组装和操作的新分子工具。为了实现这一目标，该提案描述了对环境响应性多肽（ERP）的相变的研究。假设是ERP可以隔离细胞内部的关键因素，并且它们可以在活细胞内部的分子途径上可逆地打开和关闭分子途径。为了证明这种方法的可行性，ERP将被设计为控制一个无处不在的细胞过程，称为网格蛋白介导的内吞作用。此过程在调节许多疾病（包括癌症和感染）中很重要。提出了以下特定目的：1）ERP开关的生物合成和生物物理化学：将使用生物物理方法来表征ERP和ERP融合在溶液中的行为和数学模型，以允许这些系统设计用于对任何所需温度的响应。将在人类细胞内观察到ERP的细胞内行为，该细胞产生与荧光蛋白融合的ERP，可以在显微镜下观察。使用此构建体，我们将评估ERP在细胞内部自我关联的速度。描述影响细胞内ERP相变的参数的相图将与自由溶液中的参数进行比较。 2）网格蛋白介导的内吞作用的ERP切换：ERP将融合到与细胞环境中因子摄取的关键蛋白质融合。该研究项目旨在最终以一种有选择地停止细胞途径的一般策略。该提议在三种主要方面具有创新性：（i）从未观察到在活细胞内部提出的特定ERP行为； （ii）这种方法有望是一种快速，可逆的技术，可以潜在地打开或关闭特定的细胞途径； （iii）该方法可以推广以靶向任何已知蛋白相互作用的细胞途径。这种方法的成功演示旨在改变对细胞生物学研究的范式的范式，从而可以精确地操纵对疾病治疗至关重要的生物学过程。 公共卫生相关性：了解癌症或感染等疾病在细胞水平上进行的过程对于新疗法的发展至关重要。该提案描述了一种探索一种新型技术，该技术旨在快速打开和关闭与疾病有关的关键细胞过程。该项目的成功将促进许多疾病过程的未来研究，并通过改进的疾病治疗方法达到顶峰。