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CAREER: Regulation of cargo selection and ubiquitination by protein trafficking adaptors

职业：通过蛋白质运输适配器调节货物选择和泛素化

基本信息

批准号：
1553143
负责人：
Allyson O'Donnell
金额：
$ 105.23万
依托单位：
Duquesne University
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2018-11-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1553143&HistoricalAwards=false
关键词：
CAREER Regulation cargo selection ubiquitination

项目摘要

In response to a changing environment, proteins are reshuffled in the cell - specific proteins are removed from the cell surface while others are selectively targeted to the surface. This protein reshuffling is referred to as protein trafficking and cells must make the correct 'decisions' to control protein trafficking and ensure optimal cell growth and survival. The goal of this research is to understand how the cellular decisions that dictate protein trafficking are made. This multidisciplinary project pairs cell and molecular biology with powerful new computational biology approaches to define the parameters that determine how proteins are selectively relocalized in response to environmental changes. Key players in regulating these cellular decisions are a recently described, but poorly understood, family of proteins called the alpha-arrestins. Alpha-arrestins act as cellular 'mail carriers' ensuring that proteins are delivered to the right location at the right time. In spite of their important role in protein trafficking, there remain critical, yet unanswered questions about alpha-arrestin function including: 1) how do alpha-arrestins recognize the discreet subset of proteins they regulate? and 2) what factors control when and where alpha-arrestins interact with the proteins they traffic? In other words, how do these molecular mail carriers know which letters to pick up, when to pick them up and where to deliver them? Understanding the answers to these questions is essential; defects in the trafficking decision making process have catastrophic consequences for the cell. The interdisciplinary nature of this research project ensures that trainees at all levels - including high school, undergraduate and graduate students - gain exposure to a wide-array of scientific approaches. The research objectives of this project will be integrated into an undergraduate laboratory course to ensure that undergraduate researchers get to experience the thrill of scientific discovery while learning science fundamentals. Undergraduate and high school student summer internships will also be created to allow students to contribute to advancing the boundary of knowledge in the dynamic field of protein trafficking. The goal of the research is to define principles that govern selective protein trafficking using alpha-arrestins, an exciting new class of trafficking adaptor, as a model. The alpha-arrestins, conserved from yeast to humans and related to the well-characterized and clinically important mammalian beta-arrestins, are a recently described class of trafficking adaptor that play a critical role in selective protein trafficking. While we have yet to appreciate the breadth of alpha-arrestin function, in yeast they interact with the ubiquitin ligase Rsp5 to regulate the trafficking fate of cargo proteins. The alpha-arrestins are little studied and the experiments proposed here will define key posttranslational regulation of, and novel biological functions for, these critical regulators of protein trafficking. Specifically, this research will: 1) Define how disruption of the alpha-arrestin-Rsp5 interface impairs the ubiquitin ligase efficiency of Rsp5; 2) Determine how ubiquitination regulates alpha-arrestin-mediated trafficking and determine how Rsp5 activity is restricted to permit alpha-arrestin mono-ubiquitination; and 3) Comprehensively identify alpha-arrestin cargo proteins and define motifs that dictate alpha-arrestin-cargo interaction using a robust, new computational approach that employs evolutionary signatures to infer functional relationships. The results of this project will yield new paradigms for how trafficking adaptors are regulated, ubiquitin conjugation is restricted and selective cargo trafficking is achieved. Through the use of novel computational methods and complementary genetic and biochemical approaches, this research will establish the rules that govern trafficking adaptor function. Trafficking adaptors dictate cargo localization in all eukaryotes, thus the 'decision-making' rules identified from these experiments will provide vital information to researchers with diverse interests.

为了应对环境的变化，蛋白质在细胞内重新洗牌--特定的蛋白质从细胞表面移除，而其他蛋白质选择性地定位于细胞表面。这种蛋白质重组被称为蛋白质运输，细胞必须做出正确的‘决定’来控制蛋白质运输，并确保最佳的细胞生长和存活。这项研究的目标是了解决定蛋白质运输的细胞决策是如何做出的。这个多学科项目将细胞生物学和分子生物学与强大的新计算生物学方法相结合，以定义决定蛋白质如何随着环境变化而选择性重新定位的参数。调节这些细胞决定的关键角色是最近被描述但知之甚少的一种蛋白质家族，称为α-拦截素。α-拦截素作为细胞的“邮件载体”，确保蛋白质在正确的时间被送到正确的位置。尽管它们在蛋白质运输中扮演着重要的角色，但关于α-arrestin的功能仍然存在一些关键但尚未回答的问题，包括：1)α-拦阻蛋白如何识别它们调节的谨慎的蛋白质子集？2)是什么因素控制着阿尔法-抑制素何时何地与它们所运输的蛋白质相互作用？换句话说，这些分子邮递员如何知道要取哪些信，什么时候取，送到哪里？了解这些问题的答案是至关重要的；贩运决策过程中的缺陷对该组织来说是灾难性的后果。这一研究项目的跨学科性质确保了各级受训人员--包括高中生、本科生和研究生--能够接触到各种各样的科学方法。该项目的研究目标将被整合到本科实验室课程中，以确保本科研究人员在学习科学基础的同时体验科学发现的刺激。还将设立本科和高中生暑期实习，使学生能够为推进蛋白质贩运动态领域的知识边界做出贡献。这项研究的目标是确定管理选择性蛋白质贩运的原则，使用阿尔法-拦截素作为模型，这是一种令人兴奋的新型贩运适应因子。α-拦阻蛋白是最近发现的一类运输适配子，在选择性蛋白质运输中起着关键作用，从酵母到人类都是保守的，与具有良好特性和临床意义的哺乳动物的β-拦阻蛋白有关。虽然我们还没有意识到α-arrestin功能的广泛性，但在酵母中，它们与泛素连接酶Rsp5相互作用，调节货物蛋白的运输命运。对α-拦阻蛋白的研究很少，这里提出的实验将定义这些蛋白质运输的关键调控因子的关键翻译后调节和新的生物学功能。具体地说，这项研究将：1)确定α-arrestin-Rsp5界面的中断如何损害RSP5的泛素连接酶效率；2)确定泛素化如何调节α-arrestin介导的交易，并确定RSP5的活性如何受到限制，以允许α-arrestin单一泛素化；以及3)全面识别α-arrestin货物蛋白，并使用一种稳健的新计算方法来定义决定α-arrestin-Cargo相互作用的基序，该方法使用进化特征来推断功能关系。该项目的成果将为如何管制贩运适应因子、限制泛素结合和实现有选择的货物贩运提供新的范例。通过使用新的计算方法和互补的遗传和生化方法，这项研究将建立管理运输适配功能的规则。运输适配子决定了所有真核生物中的货物定位，因此从这些实验中确定的“决策”规则将为具有不同兴趣的研究人员提供重要信息。