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: 8094406
• 负责人: John Andrew MacKay
• 金额: $ 19.46万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8094406
• 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; Fluorescence Microscopy; Future; Gene Targeting; Genes; Genetic; Goals; Green Fluorescent Proteins; Human; Infection; Investigation; Lead; 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的成分;此外，能够这样做的工具的开发将显着提高科学家理解生物学和治疗疾病的能力。这项研究的长期目标是设计新的分子工具，可以在细胞等生物环境中组装和操作。为了实现这一目标，该建议描述了环境响应多肽（ERPs）的相变的调查。该假说认为，ERPs可以隔离细胞内的关键因子，并且它们可以可逆地打开和关闭活细胞内的分子通路。为了证明这种方法的可行性，ERPs将被设计为控制一个普遍存在的细胞过程，称为网格蛋白介导的内吞作用。这一过程在许多疾病的调节中很重要，包括癌症和感染。提出了以下具体目标：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溶液中ERP和ERP融合的行为，并将开发数学建模，以允许这些系统被设计为响应于任何期望的温度。将在产生与荧光蛋白融合的ERPs的人类细胞内观察ERPs的细胞内行为，所述荧光蛋白可以在显微镜下观察。使用这个结构，我们将评估ERPs在细胞内自我关联的速度。将描述影响细胞内ERP相变的参数的相图与游离溶液中的相图进行比较。2)网格蛋白介导的内吞作用的ERP转换：ERP将融合到参与细胞环境中因子摄取的关键蛋白质。这个研究项目的目的是在一个选择性地停止细胞通路的一般策略达到高潮。该提议在三个主要方面具有创新性：（i）本文提出的特定ERP行为从未在活的人类细胞内观察到;（ii）这种方法预计是一种快速，可逆的技术，可以潜在地打开或关闭特定的细胞通路;（iii）这种方法可以推广到靶向已知蛋白质相互作用的任何细胞通路。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疾病治疗至关重要的生物过程。 公共卫生相关性：了解疾病（如癌症或感染）在细胞水平上进行的过程对于开发新的治疗方法至关重要。该提案描述了对一种新的使能技术的探索，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，并最终改善疾病的治疗方法。