Cell-penetrating peptide-adaptors for intracellular cargo delivery

用于细胞内货物递送的细胞穿透肽适配器

• 批准号: 9171796
• 负责人: JONATHAN L MCMURRY
• 金额: $ 40.18万
• 依托单位: KENNESAW STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9171796
• 关键词: Adaptor Signaling Protein; Address; Affinity; Animal Model; Antiviral Agents; Binding; Binding Sites; Biological Assay; Biological Models; Biology; Biomedical Research; Biosensor; Calcium; Calmodulin; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cellular biology; Characteristics; Chimeric Proteins; Clathrin; Complex; Confocal Microscopy; Coupled; Development; Diagnostic; Diagnostics Research; Disease; Dissociation; Doctor of Philosophy; Endocytosis; Endosomes; Environment; Eukaryotic Cell; Future; Genetic Transcription; Goals; HIV; Human; Hydrophobic Interactions; Immunoglobulin G; Institution; Kinetics; Label; Lead; Life; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Mentors; Nucleic Acids; Optics; Penetrance; Penetration; Peptides; Preparation; Property; Proteins; RNA Interference; Recruitment Activity; Research; Research Training; Rest; Speed; Students; System; Technology; Testing; Therapeutic; Therapeutic Uses; Tissues; Toxic effect; Training; Trans-Activators; Transfection; Translational Research; Underrepresented Groups; Work; base; crosslink; cytotoxic; cytotoxicity; design; experience; improved; innovation; interest; macromolecule; member; nanomolar; novel strategies; programs; research study; success; tool; trafficking

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

项目摘要 该提案的目的是进一步开发一种新颖的操纵和标签策略 用于研究，诊断和治疗目的的生活真核细胞的内部。细胞渗透 肽（CPP，也称为蛋白质转导域或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）car 真核细胞。我们已经设计了CPP适应器融合蛋白Tat-Calmodulin（Tat-CAM），该蛋白 由转录和人钙调蛋白的HIV反式激活剂穿透部分的细胞组成。 t CAM结合cam结合位点（CBS），其中含有钙的钙具有纳摩尔亲和力，但 在缺席的情况下可以忽略。因为哺乳动物细胞通常保持钙的静息浓度较低，所以 一旦在细胞内，Cargos就可以从CPP适应器中解散。当前的CPP策略依赖于共价 交联或非特异性疏水相互作用，无法逃脱内体，只提供很小的 货物分子的百分比。我们的CPP适应器代表了细胞渗透的重大进展 由于货物的解离，技术，可以更多地准备对内部的操纵 细胞环境。 我们将使用我们的CPP适应器和类似的结构来研究CPP的基本机械细节 通过细胞的渗透和运输，例如渗透模式，动力学和毒性，我们还将 利用它们提供货物分子，以证明我们的系统效用及其优势 转染，即效率，速度和可调性，并解决KSU和 其他机构。 在这些努力中的成功将验证我们的策略是提供各种各样的适应性工具 大分子，有可能改善癌症治疗剂，抗病毒药和蛋白质的递送以改善 多种疾病以及创建多功能研究和诊断工具

项目成果

Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem.

• DOI: 10.1515/bmc-2017-0023
• 发表时间: 2017-09-26
• 期刊: Biomolecular concepts
• 作者: LeCher JC;Nowak SJ;McMurry JL
• 通讯作者: McMurry JL

SUMO targeting of a stress-tolerant Ulp1 SUMO protease.

• DOI: 10.1371/journal.pone.0191391
• 发表时间: 2018
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Peek J;Harvey C;Gray D;Rosenberg D;Kolla L;Levy-Myers R;Yin R;McMurry JL;Kerscher O
• 通讯作者: Kerscher O