RUI: Understanding the Biosynthesis and Function of Headgroup Acylated Glycerophospholipids 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: 1152463
• 负责人: Teresa Garrett
• 金额: $ 21.4万
• 依托单位: Vassar College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1152463&HistoricalAwards=false
• 关键词: RUI; Understanding; Biosynthesis; Function; Headgroup

Intellectual MeritLipids form the semi-permeable bi-layer membrane that surrounds all cells and organelles, compartmentalizing important cellular reactions and metabolites. There is a large diversity of lipids required to form a functional membrane. Glycerophospholipids (GPLs), which contain a glycerol phosphate backbone with two fatty acyl chains esterified to the glycerol and an alcohol added to the phosphate, compromise a significant portion of the membrane lipids. In addition to the major GPLs, E. coli also contains a variety of low abundance lipids whose function is not well understood. One such class of low abundance lipids contains an additional acyl chain added to the headgroup. Headgroup-acylated lipids have been previously shown to impact essential cell functions such as cell division. This research program will characterize two enzymes that impact headgroup-acylated GPL levels in E. coli in order to further shed light on the biological function of headgroup-acylated GPLs. The E. coli enzyme PldB catalyzes the formation of the headgroup-acylated GPL, acyl phosphatidylglycerol (acyl PG) by transferring an acyl chain from a monoacylated GPL (lyso GPL) to the headgroup of PG. The Arabidopsis thaliana enzyme At1g78690p, which acylates lyso GPLs to form di-acylated GPLs in vitro, interestingly, leads to the accumulation of the headgroup-acylated GPL, acyl PG, when over-expressed in E. coli. This A. thaliana protein is a useful tool for probing the interaction between lyso GPL and headgroup-acylated GPL metabolism. In order to illuminate how lyso GPL and headgroup-acylated GPL metabolism interact the substrate specificity of PldB and At1g78690p will be determined. A variety of acyl donors and acyl acceptors will be tested to determine the substrate preference for each enzyme. The enzymatic properties of PldB and At1g78690p will also be investigated. Amino acids critical for catalysis and substrate preference will be identified by testing the activity of mutated PldB and At1g78690p with different acyl donors and acceptors. The biological role of headgroup-acylated GPLs will also be investigated by genetically manipulating E. coli lipid biochemical pathways. Enzymes that alter the levels of headgroup-acylated GPLs will be introduced into wild-type and mutant E. coli strains. For each strain, alterations to the cellular lipid composition will be assessed using liquid chromatography electrospray ionization mass spectrometry. In addition, the growth rate, antibiotic sensitivity and temperature sensitivity of the strains will be tested to determine the phenotypic impacts of alterations of headgroup-acylated GPL levels on cells in a variety of genetic backgrounds. This work will provide the basis to understand the function of these lipids and help, in future work, explain the complexity of lipids found in all cells.Broader ImpactThis research program will impact the field of lipid biochemistry by helping to explain the interaction between lyso GPL and headgroup-acylated GPL metabolism. In addition, undergraduates at Vassar College will have opportunities to engage in substantial research in lipid biochemistry. Every aspect of this research will directly involve undergraduate student researchers. Students will prepare lipid substrates and protein extracts, perform the in vitro enzyme assays, purify the enzymes to homogeneity, and construct the E. coli. They will present their work at scientific conferences as poster and oral presentations and publish in peer-reviewed journals. Particular attention will be given to involve of students at all levels, including freshman and students from under-represented groups, in research leading to increased retention of these students in STEM fields.This project is jointly supported by the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.

脂质形成半透性双层膜，包围所有细胞和细胞器，分隔重要的细胞反应和代谢物。形成功能性细胞膜需要多种脂质。甘油磷脂（GPLs）含有一个磷酸甘油骨架和两个酯化成甘油的脂肪酰基链，并在磷酸基上添加了一个醇，它损害了膜脂的很大一部分。除了主要的gpl外，大肠杆菌还含有多种低丰度的脂质，其功能尚不清楚。一类这样的低丰度脂类含有附加的酰基链添加到头基上。头基团酰化脂质先前已被证明影响基本细胞功能，如细胞分裂。本研究计划将描述影响大肠杆菌头基酰化GPL水平的两种酶，以进一步阐明头基酰化GPL的生物学功能。大肠杆菌酶PldB通过将一个酰基链从单酰基GPL （lyso GPL）转移到PG的头基，催化头基酰化GPL，酰基磷脂酰甘油（acyl PG）的形成。拟南芥酶At1g78690p在体外使lyso GPL酰化形成二酰化GPL，有趣的是，当在大肠杆菌中过表达时，导致头基酰化GPL，酰基PG的积累。该拟南芥蛋白是探测lyso GPL与头群酰化GPL代谢之间相互作用的有用工具。为了阐明lyso GPL和头群酰化GPL代谢如何相互作用，我们将确定PldB和At1g78690p的底物特异性。将测试各种酰基供体和酰基受体，以确定每种酶的底物偏好。我们还将研究PldB和At1g78690p的酶学性质。通过检测突变的PldB和At1g78690p与不同的酰基供体和受体的活性，将确定催化和底物偏好的关键氨基酸。头基酰化GPLs的生物学作用也将通过基因操纵大肠杆菌脂质生化途径进行研究。改变头基酰化gpl水平的酶将被引入野生型和突变型大肠杆菌菌株。对于每个菌株，细胞脂质组成的变化将使用液相色谱-电喷雾电离质谱法进行评估。此外，还将测试菌株的生长速度、抗生素敏感性和温度敏感性，以确定不同遗传背景下头群酰化GPL水平改变对细胞的表型影响。这项工作将为理解这些脂质的功能提供基础，并有助于在未来的工作中解释所有细胞中发现的脂质的复杂性。更广泛的影响本研究项目将通过帮助解释lyso GPL和头基团酰化GPL代谢之间的相互作用来影响脂质生物化学领域。此外，瓦萨学院的本科生将有机会从事脂质生物化学方面的实质性研究。本研究的每个方面都将直接涉及本科生研究人员。学生将准备脂质底物和蛋白质提取物，进行体外酶分析，纯化酶至均匀性，并构建大肠杆菌。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。将特别关注让所有层次的学生，包括新生和来自代表性不足群体的学生参与研究，从而提高这些学生在STEM领域的留用率。本项目由分子与细胞生物科学部生物分子动力学、结构与功能研究小组和化学部生命过程化学研究小组共同支持。