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 这项提案的目标是进一步开发一种新的策略，用于操纵和标记 用于研究、诊断和治疗目的的活真核细胞内部。细胞穿透 多肽（CPP，也称为蛋白转导结构域或PTD）提供了诱人的前景， 通过质膜外源递送用户定义的蛋白质（和其它生物分子）货物 真核细胞。我们设计了一种CPP接头融合蛋白TAT-CaM（TAT-CaM）， 由来自HIV转录反式激活因子和人钙调蛋白的细胞穿透部分组成。达特- 钙调素结合钙调素结合位点（CBS）含有货物与纳摩尔亲和力的存在下，钙，但 在其缺席的情况下可以忽略。因为哺乳动物细胞通常保持低的静息钙浓度， 一旦进入细胞，货物就从CPP-衔接子解离。当前的CPP策略依赖于共价键 交联或非特异性疏水相互作用，不能逃脱内体，仅递送非常小的内体。 货物分子的百分比。我们的CPP适配器代表了细胞渗透的重大进展 技术，因为货物的解离，允许更容易地操纵内部 细胞的环境。 我们将使用我们的CPP适配器和类似的结构来研究CPP的基本机制细节 渗透和运输通过细胞，如模式的渗透，动力学和毒性，我们还将 利用它们来运送货物分子，这将证明我们系统的实用性及其优势， 转染，即效率，速度和可调性，并解决生物医学研究问题，在KSU和 其他机构。 这些努力的成功将证明，我们的战略是一个适应性强的工具，可以提供各种各样的 大分子，潜在地改善癌症治疗剂、抗病毒药和蛋白质的递送，以改善癌症的治疗效果。 以及创造多功能的研究和诊断工具

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.

SUMO靶向耐应力的ULP1 SUMO蛋白酶。

• 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