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

摘要 本项目的总体目标是开发基于含硼酸肽适体的荧光材料。 用于检测附近含二醇分子的蛋白质(BapaFP)，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活体细胞成像设计BapaFP 核糖类是多糖酶形成的信号分子和供体， 糖蛋白、糖脂和糖化次生代谢物。 这项新技术的特点是一种基因编码的硼酸部分可以诱导与 邻位二元醇分子，用于增强亲和力和特异性的延伸肽适配子(S)，以及 用于荧光读数的循环置换红色荧光蛋白(CpRFP)结构域。这部作品很有感染力 承诺填补技术空白，实现对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.设计BapaFP以选择性地检测核苷酸糖。 2.在哺乳动物活细胞中鉴定BapaFP。 该项目将导致新的研究工具，以成像活的哺乳动物细胞和 亚细胞结构域。由于核苷酸糖将新陈代谢与信号联系起来(例如，翻译后 修饰(PTM)、表观遗传学、胞吐信号和表面相互作用)和生物结果， 这些新工具将催化大量与多糖介导的信号转导和疾病相关的生物学研究。 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。 和葡聚糖修饰酶。因为BapaFP技术简单、经济高效，而且 可扩展的，并且可以被任何具有基本分子和细胞生物学知识的实验室采用，我们期望 BapaFP在碳水化合物和碳水化合物跟踪、鉴定和分析中的广泛应用 衍生品。

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.