Cell-penetrating peptide-adaptors for intracellular cargo delivery

项目总结 这项提议的目标是进一步开发一种新的战略，用于操纵和标记 用于研究、诊断和治疗目的的活的真核细胞的内部。细胞穿透性 多肽(CPPs，也称为蛋白质转导结构域，或PTD)提供了诱人的前景 通过质膜外源输送用户定义的蛋白质(和其他生物分子)货物 真核细胞。我们设计了一种CPP-接头融合蛋白TAT-钙调蛋白(TAT-CaM)，它可以 由HIV转录反式激活因子和人钙调素的细胞穿透部分组成。达特- 在有钙存在的情况下，CaM与含有CaM结合位点(CBS)的货物具有纳摩尔亲和力 在它不存在的情况下，可以忽略不计。因为哺乳动物细胞通常保持较低的静息钙浓度， 一旦进入细胞，货物就会从CPP适配器上解离。当前的CPP战略依赖于共价 交联性或非特异性疏水相互作用，不能逃脱内小体，只提供非常小的 货物分子的百分比。我们的CPP适配器代表着细胞渗透率的显著进步 技术因为货物的解离，让内部的操控更加便捷 细胞的环境。 我们将使用我们的CPP适配器和类似的构造来研究CPP的基本机制细节 通过细胞的渗透和运输，如渗透模式、动力学和毒性，我们还将 利用它们来运送货物分子，以展示我们系统的实用性及其相对于 转基因，即效率、速度和可调性，并在科罗拉多州立大学和 其他机构。 这些努力的成功将验证我们的战略是一种适应性强的工具，可以交付广泛的 大分子，潜在地改善癌症治疗药物、抗病毒药物和蛋白质的输送以改善 各种疾病，以及创建通用的研究和诊断工具

PROJECT SUMMARY The goal of this proposal is to further the development a novel strategy for the manipulation and labeling of the interior of living eukaryotic cells for research, diagnostic and therapeutic purposes. Cell-penetrating peptides (CPPs, also called protein transduction domains, or PTDs) offer the tantalizing prospect of exogenously delivering user-defined protein (and other biomolecular) cargos through the plasma membranes of eukaryotic cells. We have designed a CPP-adaptor fusion protein, TAT-Calmodulin (TAT-CaM), that consists of the cell penetrating moiety from HIV transactivator of transcription and human calmodulin. TAT- CaM binds CaM binding-site (CBS) containing cargos with nanomolar affinity in the presence of calcium but negligibly in its absence. Because mammalian cells typically maintain low resting concentrations of calcium, cargos dissociate from the CPP-adaptor once inside the cell. Current CPP strategies rely on covalent crosslinking or nonspecific hydrophobic interactions, fail to escape endosomes and only deliver a very small percentage of cargo molecules. Our CPP-adaptors represent a significant advance in cell-penetration technology because of the dissociation of cargo, allowing more ready manipulation of the internal environments of cells. We will use our CPP-adaptor and similar constructs to investigate the basic mechanistic details of CPP penetration of and trafficking through cells such as mode of penetrance, kinetics, and toxicity We will also utilize them to deliver cargo molecules that will demonstrate the utility of our system and its advantages over transfection, namely efficiency, speed and tunability and address biomedical research questions at KSU and other institutions. Success in these endeavors will validate that our strategy is an adaptable tool for delivery of a wide array of macromolecules, potentially improving the delivery of cancer therapeutics, antivirals, and proteins to ameliorate a variety of disorders as well as creating versatile research and diagnostic tools