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

• 批准号: 1902859
• 负责人: Allyson O'Donnell
• 金额: $ 55.12万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-22 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1902859&HistoricalAwards=false
• 关键词: CAREER; Regulation; cargo; selection; ubiquitination

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.

为了响应变化的环境，蛋白质在细胞中重新洗牌-特异性蛋白质从细胞表面去除，而其他蛋白质选择性地靶向表面。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。这项研究的目的是了解决定蛋白质运输的细胞决定是如何做出的。这个多学科项目将细胞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选择性地重新定位以应对环境变化的参数。调节这些细胞决定的关键参与者是最近描述的，但知之甚少的蛋白质家族，称为α-arrestins。α-抑制蛋白作为细胞的“邮递员”，确保蛋白质在正确的时间被运送到正确的位置。尽管它们在蛋白质运输中起重要作用，但仍然存在关于α-抑制蛋白功能的关键但未回答的问题，包括：1）α-抑制蛋白如何识别它们调节的蛋白质的离散子集？以及2）什么因素控制α-抑制蛋白与它们运输的蛋白质相互作用的时间和地点？换句话说，这些分子邮递员如何知道该取哪些信件，何时取信以及将信件送到哪里？理解这些问题的答案是至关重要的;贩运决策过程中的缺陷会给细胞带来灾难性的后果。该研究项目的跨学科性质确保了各级学员-包括高中，本科和研究生-接触到广泛的科学方法。该项目的研究目标将被纳入本科实验室课程，以确保本科研究人员在学习科学基础知识的同时体验科学发现的快感。还将创建本科生和高中生暑期实习机会，让学生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使用α-arrestins（一种令人兴奋的新型贩运适配器）作为模型来管理选择性蛋白质贩运的原则。α-抑制蛋白，从酵母到人类都是保守的，并且与充分表征的和临床上重要的哺乳动物β-抑制蛋白相关，是最近描述的一类在选择性蛋白质运输中起关键作用的运输衔接子。虽然我们还没有认识到α-抑制蛋白功能的广度，但在酵母中，它们与泛素连接酶Rsp 5相互作用以调节货物蛋白的运输命运。α-arrestins研究很少，这里提出的实验将定义关键的翻译后调节，和新的生物学功能，这些关键的调节蛋白质运输。具体而言，本研究将：1）确定α-arrestin-Rsp 5界面的破坏如何损害Rsp 5的泛素连接酶效率; 2）确定泛素化如何调节α-arrestin介导的运输，并确定Rsp 5活性如何被限制以允许α-arrestin单泛素化;和3）使用稳健的，一种新的计算方法，采用进化签名来推断功能关系。该项目的结果将产生新的模式，如何贩运适配器进行调节，泛素共轭是有限的，并实现选择性货物贩运。通过使用新的计算方法和互补的遗传和生物化学方法，这项研究将建立管理贩运适配器功能的规则。贩运适配器决定货物定位在所有真核生物中，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。