BapaFPs: Genetically encoded fluorescent indicators to image live-cell nucleotide sugars

BapaFP：基因编码的荧光指示剂，用于对活细胞核苷酸糖进行成像

• 批准号: 9750256
• 负责人: Huiwang Ai
• 金额: $ 47.43万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-24 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750256
• 关键词: Adopted; Adoption; Affinity; Amino Acids; Antigens; Apoptosis; Bacterial Infections; Baculoviruses; Binding; Biological; Biology; Biosensor; Boronic Acids; Carbohydrates; Cell Communication; Cell Compartmentation; Cell Line; Cells; Cellular biology; Corneal dystrophy; Detection; Development; Diabetes Mellitus; Disease; Engineering; Enzymes; Epigenetic Process; Fluorescence; Generations; Genetic Code; Glycolipids; Glycols; Glycoproteins; Graph; Image; Imaging Device; Inclusion Bodies; Infection; Inflammation; Knowledge; Laboratories; Link; Liver Fibrosis; Malignant Neoplasms; Mammalian Cell; Mediating; Metabolic; Metabolism; Methods; Modification; Molecular Biology; Mutagenesis; Myopathy; Nucleotide Sugars Metabolism Pathway; Outcome; Pattern; Peptide aptamers; Play; Polysaccharides; Post-Translational Protein Processing; Proteins; Purinoceptor; Research; Role; Sialic Acids; Signal Transduction; Signaling Molecule; Specificity; Structure; Surface; System; Technology; Tertiary Protein Structure; Uridine Diphosphate Sugars; Viral Vector; Virus Diseases; Work; base; carbohydrate metabolism; cost effective; extracellular; fluorescence imaging; glycosylation; glycosyltransferase; high throughput screening; imaging capabilities; imaging probe; inhibitor/antagonist; innovation; live cell imaging; macula; mutant; new technology; novel; nutrition; public health relevance; ratiometric; red fluorescent protein; response; sugar nucleotide; technology development; tool; vector

ABSTRACT The overall objective of this project is to develop boronic acid-containing peptide aptamer-based fluorescent proteins (BapaFPs) for the detection of vicinal diol-containing molecules, including various carbohydrates and carbohydrate derivatives. In particular, this three-year project is to engineer BapaFPs for live-cell imaging of nucleotide sugars that are signaling molecules and donors for the enzymatic formation of polysaccharides, glycoproteins, glycolipids, and glycosylated secondary metabolite. This new technology features a genetically encoded boronic acid moiety to induce initial interactions with vicinal diol-containing molecules, (an) extended peptide aptamer(s) to enhance affinity and specificity, and a circularly permuted red fluorescent protein (cpRFP) domain for fluorescence readouts. This work holds great promise to fill the technical gap to enable fluorescence imaging and tracking of various carbohydrates and carbohydrate derivatives in live cells. We will pursue the following specific aims: 1. Engineer BapaFPs to selectively detect nucleotide sugars. 2. Characterize BapaFPs in live mammalian cells. This project will lead to new research tools for imaging nucleotide sugars in live mammalian cells and subcellular domains. Since nucleotide sugars connect metabolism to signaling (e.g. posttranslational modifications (PTMs), epigenetics, exocytotic signaling, and surface interactions) and biological outcomes, these new tools will catalyze a large array of biological studies related to glycan-mediated signaling and disease. In addition, these new biosensors will accelerate the discovery of selective inhibitors for glycosyltransferases and glycan modification enzymes. Because the BapaFP technology is straightforward, cost-effective, and scalable, and can be adopted by any laboratory with basic molecular and cell biology knowledge, we expect the broad application of BapaFPs for the tracking, identification, and analysis of carbohydrates and carbohydrate derivatives.

抽象的 该项目的总体目的是开发含硼酸的肽适体的荧光 蛋白质（BAPAFPS）用于检测含有二醇的分子，包括各种碳水化合物和 碳水化合物衍生物。特别是，这个为期三年的项目是为BAPAFPS设计 核苷酸糖是信号分子和供体的酶形成多糖的酶形成，即 糖蛋白，糖脂和糖基化的二级代谢产物。 这项新技术具有遗传编码的硼酸部分，以诱导与 含二醇的分子，（AN）扩展的肽适体以增强亲和力和特异性，A 用于荧光读数的循环置换的红色荧光蛋白（CPRFP）结构域。这项工作很棒 承诺填补技术空白，以实现各种碳水化合物的荧光成像和跟踪 活细胞中的碳水化合物衍生物。 我们将追求以下具体目标： 1。工程师BAPAFPS选择性检测核苷酸糖。 2。表征活哺乳动物细胞中的BAPAFP。 该项目将为活哺乳动物细胞和 亚细胞域。由于核苷酸糖将代谢连接到信号传导（例如翻译后 修饰（PTMS），表观遗传学，胞外信号传导和表面相互作用）和生物学结果， 这些新工具将催化与聚糖介导的信号传导和疾病有关的大量生物学研究。 此外，这些新的生物传感器将加速发现糖基转移酶的选择性抑制剂 和聚糖修饰酶。因为BAPAFP技术是简单的，具有成本效益的，并且 可扩展，任何具有基本分子和细胞生物学知识的实验室都可以采用，我们期望 BAPAFP的广泛应用用于碳水化合物和碳水化合物的跟踪，识别和分析 衍生物。