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.

摘要 本项目的总体目标是开发基于硼酸肽适配体的荧光探针， 蛋白质（BapaFP），用于检测邻位含二醇的分子，包括各种碳水化合物， 碳水化合物衍生物。特别是，这个为期三年的项目是设计BapaFP用于活细胞成像， 核苷酸糖是多糖酶促形成的信号分子和供体， 糖蛋白、糖脂和糖基化次级代谢产物。 这项新技术的特点是一个基因编码的硼酸部分，以诱导初始相互作用， 含邻位二醇的分子，增强亲和力和特异性的延伸肽适体，和 循环排列的红色荧光蛋白（cpRFP）结构域用于荧光读数。这项工作具有很大的 有望填补技术空白，使荧光成像和跟踪各种碳水化合物， 活细胞中的碳水化合物衍生物。 我们将努力实现以下具体目标： 1.设计BapaFP以选择性地检测核苷酸糖。 2.在活的哺乳动物细胞中表征BapaFP。 该项目将导致新的研究工具，用于成像活哺乳动物细胞中的核苷酸糖， 亚细胞结构域由于核苷酸糖将代谢与信号传导（例如，翻译后 修饰（PTM）、表观遗传学、胞吐信号传导和表面相互作用）和生物学结果， 这些新工具将促进与聚糖介导的信号传导和疾病相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。由于BapaFP技术简单，成本效益高， 可扩展的，并可以采用任何实验室与基本的分子和细胞生物学知识，我们希望 BapaFP广泛应用于碳水化合物和碳水化合物的跟踪、鉴定和分析 衍生物.