RUI: Understanding headgroup-acylated glycerophospholipid biosynthesis and function in Escherichia coli

RUI：了解大肠杆菌中头基酰化甘油磷脂的生物合成和功能

• 批准号: 1516805
• 负责人: Teresa Garrett
• 金额: $ 53.53万
• 依托单位: Vassar College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2019-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1516805&HistoricalAwards=false
• 关键词: RUI; Understanding; headgroup; acylated; glycerophospholipid

This project presents an integrated research and education program to engage undergraduate students in original research on the structure, function and biosynthesis of a class of understudied lipids called headgroup-acylated glycerophospholipids (GPLs) in the bacterium Escherichia coli. Lipids are a key part of living cells and together with other macromolecules such as proteins, they form the membrane that surrounds all cells. Lipids support essential cell functions such as cell division and maintaining the membrane that surrounds and delimits the cell. The two main research objectives are: to understand the functional properties of two proteins that affect headgroup-acylated GPL levels in the cell as well as how levels of headgroup-acylated GPL are regulated in the bacterium; and the impact of changing levels of these lipids on how much the membrane can bend. This work will lay a basis for understanding the function of these minor, yet important, lipids and help, in future work, to explain the complexity of lipids in all cells. A third objective of this project is directly to engage underrepresented students in original research. An innovative program called Diving into Research brings six admitted Vassar College students to campus before the beginning of their freshman year to participate for 4 to 5 weeks in Vassar's 10-week Undergraduate Research Summer Institute. By establishing strong connections with peer and faculty research mentors early on, students from underrepresented groups, including low socioeconomic and first-generation college goers, are placed on a path to success in STEM (Science, Technology, Engineering and Mathematics)careers.Throughout this project, undergraduate students will be involved in original research on the structure, function and biosynthesis of a class of understudied lipids called headgroup-acylated glycerophospholipids (GPLs) in the bacterium Escherichia coli. The biosynthesis of lipids, including low abundance lipids such as headgroup-acylated GPLs, affects essential cell functions such as cell division and maintaining cell membrane integrity. Determining the substrate specificity and enzymatic mechanism of two enzymes, PldB and At1g78690, known to impact headgroup-acylated GPL levels in E. coli, and the careful structural determination of these enzymes in vitro products, will provide valuable insights into the basis of each enzyme's unique catalytic mechanisms. Students involved in this project will prepare lipid substrates and protein extracts, perform in vitro enzyme assays, purify enzymes and construct the necessary E. coli strains. In addition, they will employ modern techniques of nuclear magnetic resonance and mass spectrometry to characterize lipid structure. Understanding the impact of altering headgroup-acylated GPL levels in vivo will yield insights into how headgroup-acylated GPLs affect cell division and the maintenance of membrane integrity. Experiments designed to understand how cells regulate headgroup-acylated GPL levels and how those lipids impact membrane curvature will expose undergraduate researchers to techniques in genetics and cell biology, including mutagenesis and confocal microscopy. This work will yield insight into the biosynthesis and function of this under-investigated class of lipids related to their emerging roles in important cellular processes. This research project, which will influence the field of lipid biochemistry by helping to explain the complexity of lipids in all cells, will positively affect participating undergraduates at Vassar College by expanding their opportunity to engage in substantial research in lipid biochemistry. Every aspect of this research will directly involve undergraduate student researchers. They will present their work at scientific conferences as poster and oral presentations and publish in peer-reviewed journals, setting these students on a path to successful careers in STEM-related fields.

该项目提出了一个综合研究和教育计划，吸引本科生对大肠杆菌中一类被称为头基酰化甘油磷脂（GPLs）的脂类的结构、功能和生物合成进行原创性研究。脂质是活细胞的关键部分，它们与其他大分子（如蛋白质）一起形成包围所有细胞的膜。脂质支持基本的细胞功能，如细胞分裂和维持包围和划分细胞的膜。两个主要的研究目标是：了解影响细胞中头群酰化GPL水平的两种蛋白质的功能特性，以及头群酰化GPL在细菌中的水平是如何调节的；以及这些脂质水平的变化对细胞膜弯曲程度的影响。这项工作将为理解这些次要但重要的脂质的功能奠定基础，并有助于在未来的工作中解释所有细胞中脂质的复杂性。该项目的第三个目标是直接让代表性不足的学生参与原创研究。一个名为“潜入研究”（dive into Research）的创新项目让六名被瓦萨学院（Vassar College）录取的学生在大一开始前来到校园，参加为期10周的本科生暑期研究学院（Undergraduate Research Summer Institute），为期四到五周。通过早期与同行和教师研究导师建立牢固的联系，来自弱势群体的学生，包括社会经济地位低的学生和第一代大学生，可以走上STEM（科学、技术、工程和数学）职业生涯的成功之路。在整个项目中，本科生将参与对大肠杆菌中一类被称为头基酰化甘油磷脂（GPLs）的脂类的结构、功能和生物合成的原始研究。脂类的生物合成，包括低丰度的脂类，如头基团酰化的gpl，影响基本的细胞功能，如细胞分裂和维持细胞膜完整性。确定已知影响大肠杆菌头基酰化GPL水平的两种酶PldB和At1g78690的底物特异性和酶促机制，并仔细测定这些酶的体外产物的结构，将为每种酶独特的催化机制的基础提供有价值的见解。参与该项目的学生将制备脂质底物和蛋白质提取物，进行体外酶分析，纯化酶并构建必要的大肠杆菌菌株。此外，他们将采用核磁共振和质谱的现代技术来表征脂质结构。了解改变头群酰化GPL水平在体内的影响，将有助于深入了解头群酰化GPL如何影响细胞分裂和维持膜完整性。旨在了解细胞如何调节头群酰化GPL水平以及这些脂质如何影响膜曲率的实验将使本科研究人员接触到遗传学和细胞生物学技术，包括诱变和共聚焦显微镜。这项工作将深入了解这类未被研究的脂类的生物合成和功能，以及它们在重要细胞过程中的新兴作用。该研究项目将通过帮助解释所有细胞中脂质的复杂性来影响脂质生物化学领域，并将通过扩大参与脂质生物化学实质性研究的机会，对瓦萨学院参与的本科生产生积极影响。本研究的每个方面都将直接涉及本科生研究人员。他们将在科学会议上以海报和口头报告的形式展示他们的工作，并在同行评审的期刊上发表，为这些学生在stem相关领域的成功职业生涯铺平道路。