N-terminal acylation of Lipoproteins in Firmicutes

厚壁菌门中脂蛋白的 N 末端酰化

• 批准号: 10394728
• 负责人: Timothy C. Meredith
• 金额: $ 31.4万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394728
• 关键词: Accounting; Acylation; Affect; Affinity; Amides; Bacteria; Bacterial Genome; Bacterial Infections; Bacterial Physiology; Bacterial Proteins; Binding; Biological; Biological Assay; Biology; Cell Adhesion; Cell membrane; Complex; Copper; Cysteine; Data; Databases; Detection; Deuterium; Diglycerides; Elements; Enterococcus faecalis; Environment; Environmental Monitoring; Enzymes; Firmicutes; Genes; Genetic Transcription; Glycerol; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Immune response; Immune system; In Vitro; Infection; Integral Membrane Protein; Ions; Label; Lipoprotein Binding; Lipoproteins; Mass Spectrum Analysis; Measures; Mediating; Membrane; Methods; Modification; Monitor; Multiprotein Complexes; Mutase; N acylation; N-terminal; Names; Natural Immunity; Nutrient; Operon; Organism; Pathway interactions; Pattern; Phenotype; Phospholipids; Physiology; Plasmids; Play; Process; Proteins; Receptor Signaling; Recombinants; Report (document); Reporter; Research; Resistance; Role; Signal Transduction; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staphylococcus aureus; Stress; Structure; Surface; System; TLR1 gene; TLR2 gene; TLR6 gene; Testing; Time; Toll-like receptors; Variant; Virulence; Virulence Factors; amino group; antimicrobial drug; bacterial fitness; beta-Galactosidase; cell envelope; esterase; fluorophore; genome-wide; globular protein; insight; novel; paralogous gene; pressure; prevent; promoter; reconstitution; response; theories; uptake; whole genome

Project Summary The long term goal of the application is to understand why and how lipoproteins from Firmicutes undergo modifications. Lipoproteins are membrane associated globular proteins anchored to the bacterial membrane surface through a lipidated N-terminal cysteine residue. They are ubiquitous cell envelope structures found in both gram positive and negative bacteria, accounting for 1-5% of all genes in a typical bacterial genome. Lipoproteins play roles in nearly every aspect of bacterial cell envelope physiology, from nutrient acquisition to mediating cellular contacts. Lipoproteins are also important during the detection and mounting of initial immune responses to clear bacteria infections. Due to their multiple essential cellular roles, abundance, universal distribution, and their unique and highly conserved structure, innate immunity detects the presence of bacteria through binding lipoproteins using Toll-like receptor 2 (TLR-2) complexes. TLR2 binds lipoproteins through forming heterodimers, using either TLR-1 or TLR-6 depending on the state of N-acylation. As such, understanding how and why certain bacteria utilize lipoprotein structural variations is pertinent to both fundamental bacterial physiology and infection biology. In particular, this application studies the role of N-acylation in Enterococcus faecalis. There is emerging evidence that lipoproteins are differentially N-acylated amongst bacteria in the Firmicutes phylum. A class of integral membrane N-acylating proteins named Lit was identified in E. faecalis that makes lyso- form lipoproteins in this organism. The lyso- form of lipoprotein has a unique acyl chain distribution pattern. Whereas the diacylglyceryl form has both acyl chains on the glyceryl residue, and the triacyl form has these two acyl chains plus a third N-terminal acyl chain connected through an amide bond to the α-amino cysteine group, the lyso-form has a single acyl chain on both the N-terminus and glyceryl unit. The lyso- structure suggests an acyl chain is removed from the diacylglyceryl unit and transferred to the N-terminus to form the lyso- structure or that there are lipoprotein esterases working concert with Lit. The application aims to reconstitute Lit activity by recombinant expression so as to test the proposed intramolecular transferase using specifically labeled diacylglyceryl lipoprotein substrates. The products will be characterized by MALDI mass spectrometry to determine acyl chain donor origin. A second aim is to test environmental stress condition in order to gain insight into the biological role for N-acylation using the E. faecalis/Δlit isogenic paired strain set. The third aim is to probe how Lit affects TLR-2 signaling, as preliminary data has suggested lyso-form lipoproteins bind in more complex ways to TLR2 than their triacyl- and diacyl-glycerol congeners. The final aim is to develop functional assays to uncover other lipoprotein N-terminal modifying enzymes.

项目摘要 该应用程序的长期目标是了解为什么来自Fircicutes的脂蛋白 进行修改。脂蛋白是固定在细菌上的膜相关的球状蛋白 膜表面通过脂化的N末端半胱氨酸居住。它们是无处不在的细胞信封 在革兰氏阳性和阴性细菌中发现的结构，占典型基因的1-5％ 细菌基因组。脂蛋白在细菌细胞包膜生理的几乎每个方面都起着作用， 营养获取以介导细胞接触。脂蛋白在检测过程中也很重要， 对清除细菌感染的初始免疫反应的安装。由于其多个必需的细胞角色， 抽象，普遍分布及其独特且高度组成的结构，先天免疫学检测 使用Toll样受体2（TLR-2）复合物通过结合脂蛋白通过结合脂蛋白存在细菌。 TLR2结合 脂蛋白通过形成异二聚体，使用TLR-1或TLR-6取决于N-酰化状态。 因此，了解某些细菌如何利用脂蛋白结构变异的方式以及为什么与两者有关 基本细菌生理和感染生物学。 特别是，该应用研究了N-酰化在粪肠球菌中的作用。有新兴 证据表明，脂蛋白在Firmicutes Phylum中细菌中N-酰基不同。一类 在E. faecalis中鉴定出了综合的膜N-酰化蛋白，该蛋白在粪肠球菌中鉴定 该生物中的脂蛋白。脂蛋白的溶作型形式具有独特的酰基链分布模式。 二酰基糖基形式在糖基居住地上均具有酰基链，而三酰基形式具有这些 两个通过酰胺键连接到α-氨基半胱氨酸的两个N末端酰基链的两个N端酰基链 组，Lyso形式在N末端和糖基单元上具有单个酰基链。抒情结构 建议从二酰基糖基单元中除去酰基链并转移到N末端以形成 溶血结构或有LIT的脂蛋白酯酶工作音乐会。该申请旨在 通过重组表达来重构点亮活性，以便使用拟议的分子内转移酶使用 特异性标记的二酰基糖脂蛋白底物。产品将以Maldi质量为特征 确定酰基链供体起源的光谱法。第二个目的是测试环境压力条件 为了使用大肠杆菌/ΔLIT等源性配对应变集，以深入了解N酰基的生物学作用。 第三个目的是探测LIT如何影响TLR-2信号传导，因为初步数据表明了溶解形式 脂蛋白与TLR2更复杂的脂蛋白比其三酰基和二酰基甘油同源物结合。最终目标 是开发功能测定，以发现其他脂蛋白N末端修饰酶。