Nanodelivery of functional proteins to phagosomal membranes

将功能蛋白纳米递送至吞噬体膜

• 批准号: 10365947
• 负责人: DEBORAH J. NELSON
• 金额: $ 70.4万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-09 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10365947
• 关键词: 3-Dimensional; Adoptive Transfer; Alveolar Macrophages; Anabolism; Anions; Autologous; Bacteria; Biological Assay; Cell Compartmentation; Cell membrane; Cells; Complex; Confocal Microscopy; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoplasmic Protein; Dimensions; Disease; Engineering; Epithelial Cells; Fluorescent Dyes; Genetic Engineering; Hemagglutinin; Image; Immune; Inbred CFTR Mice; Infection; Inflammatory Response; Integral Membrane Protein; Ion Channel; Label; Laboratories; Lead; Light; Lung; Measurement; Measures; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Microfluidics; Microscopy; Monitor; Mus; Nano delivery; Nose; Nucleic Acids; Phagocytes; Phenotype; Photons; Population; Probability; Process; Proteins; Reaction; Respiratory System; Screening procedure; Structure; Surface; Surveys; Techniques; Testing; Therapeutic; Tissues; Total Internal Reflection Fluorescent; Universities; Vesicle; Viral; Viral Proteins; Work; X ray spectroscopy; alveolar epithelium; base; cystic fibrosis patients; density; design; experience; experimental study; extracellular vesicles; fighting; inflammatory modulation; interest; mouse model; nano container; nanocarrier; particle; patch clamp; receptor; response; restoration; screening; time use; uptake; vesicular release

Project Summary Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo including soluble cytoplasmic proteins and nucleic acids. They are a heterogeneous group of particles generally classified according to subcellular origin, dimension, and tetraspanin surface expression. Circulating EVs may act as nanocontainers capable of recognizing target cells through membrane receptors and communicating cargo through membrane fusion, a notoriously low probability process. Interaction with recipient cells can lead to their acquisition of EV surface proteins and luminal contents although the mechanisms controlling this exchange remain unclear. Their complex cargo and well as surface markers are highly variable with respect to functional impact and dependent upon the tissue context from which they are released. A central focus of the proposed work is the isolation and identification of functionally competent small EVs (30-150 nm) carrying an ion channel of interest which can then be transferred to a naïve, non-expressing cell. Functionally significant EV-mediated channel translocation from one cell to another would require a high density of channel expression per vesicle during EV biosynthesis in the donor cell and reliable fusion with the plasma membrane and endocytic/phagocytic compartments of recipient cells. In order to facilitate EV membrane fusion we will co-express the viral protein fusogen hemagglutinin subtype 7 (HA). Expression of a genetically engineered blue light-activated Ca2+ channel switch (BACCS) which opens the Ca2+ selective ORAI ion channel in response to light will be used in the design of screening strategies exploring vesicle fusion and channel transfer. The light- sensitive probe will be used as screening tool for functional Ca2+ channel transfer in non-responsive recipient cells via isolated EVs derived from BACCS-ORAI expressing cells. Once we have established optimal conditions for EV-mediated channel transfer, we will leverage our experience in the study of the anion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in murine alveolar macrophages (AMs) and epithelial cells and study the EV-mediated transfer of CFTR to cftr-/- cells in the context of the disease of cystic fibrosis (CF) using adoptive transfer techniques.

项目摘要 细胞外囊泡是一种细胞源性的膜结构，携带跨膜蛋白 和包括可溶性细胞质蛋白和核酸的腔货物。他们是一个 通常根据亚细胞起源、尺寸和 四跨膜蛋白表面表达。循环EV可以充当能够识别 靶细胞通过膜受体和沟通货物通过膜融合， 这是一个非常低概率的过程。与受体细胞的相互作用可以导致它们获得 EV表面蛋白和管腔内容物，尽管控制这种交换的机制仍然存在 不清楚它们的复杂货物和表面标记物在以下方面高度可变： 功能影响，并取决于它们被释放的组织环境。中央 拟议工作的重点是分离和识别功能胜任的小 携带感兴趣的离子通道的EV（30-150 nm），然后可以将其转移到幼稚的， 非表达细胞。功能显著的EV介导的通道从一个细胞易位到 另一个是在EV生物合成过程中需要每个囊泡高密度的通道表达。 供体细胞和可靠的融合与质膜和内吞/吞噬区室 受体细胞为了促进EV膜融合，我们将共表达病毒蛋白融合子 血凝素亚型7（HA）。基因工程蓝光激活钙离子的表达 通道开关（BACCS），其响应于光而打开Ca 2+选择性奥赖离子通道， 用于探索囊泡融合和通道转移的筛选策略的设计。光- 敏感的探针将被用作筛选工具的功能性钙通道转移在非响应 通过衍生自表达BACCS-ORAI的细胞的分离的EV将细胞移植到受体细胞。一旦我们 建立了EV介导的通道转移的最佳条件，我们将利用我们的经验， 囊性纤维化跨膜电导调节因子（CFTR）在大鼠前列腺增生症中的作用 小鼠肺泡巨噬细胞（AM）和上皮细胞，并研究EV介导的CFTR转移 使用过继转移技术在囊性纤维化（CF）疾病的背景下将cftr-/-细胞转移到cftr-/-细胞。

项目成果

Chloride-hydrogen antiporters ClC-3 and ClC-5 drive osteoblast mineralization and regulate fine-structure bone patterning in vitro.

氯化物 - 氢抗抗植物CLC-3和CLC-5驱动成骨细胞矿化，并在体外调节精细结构骨图案。

• DOI: 10.14814/phy2.12607
• 发表时间: 2015-11
• 期刊: Physiological reports
• 影响因子: 2.5
• 作者: Larrouture QC;Nelson DJ;Robinson LJ;Liu L;Tourkova I;Schlesinger PH;Blair HC
• 通讯作者: Blair HC