Mechanism of Lupus La:miR-122 ribonucleoprotein sorting into breast cancer-derived exosomes

狼疮 La:miR-122 核糖核蛋白分选至乳腺癌来源的外泌体的机制

• 批准号: 10751552
• 负责人: Jordan Matthew Ngo
• 金额: $ 4.42万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-18 至 2025-07-17
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751552
• 关键词: Antigens; Binding; Biochemical; Biogenesis; Biological Assay; Breast Cancer Cell; Breast Cancer cell line; Breast cancer metastasis; Cancer Biology; Categories; Cell membrane; Cell secretion; Cells; Classification; Complex; Cytoplasm; Data; Disease Progression; Dissection; Electrophoretic Mobility Shift Assay; Endosomes; Eukaryotic Cell; Event; Extracellular Space; Fluorescence Microscopy; Foundations; Fractionation; Future; Genetic; Goals; Imaging Techniques; In Vitro; Integral Membrane Protein; Jordan; Label; Lipids; Lupus; MDA MB 231; Malignant Neoplasms; Mediating; Membrane; Metastatic breast cancer; MicroRNAs; Molecular; Multivesicular Body; National Research Service Awards; Neoplasm Metastasis; Nucleic Acids; Pathway interactions; Pharmaceutical Preparations; Play; Positioning Attribute; Process; Proteins; Proteomics; RNA-Protein Interaction; Reaction; Recombinants; Research; Research Personnel; Research Project Grants; Resolution; Ribonucleoproteins; Role; Small RNA; Sorting; Tertiary Protein Structure; Therapeutic; Training; Transfer RNA; Vesicle; candidate identification; cellular imaging; delivery vehicle; density; diagnostic tool; efficacy evaluation; engineered exosomes; exosome; extracellular; extracellular vesicles; glucose metabolism; in vivo; insight; interest; late endosome; malignant breast neoplasm; microvesicles; neoplastic cell; novel; nucleic acid delivery; overexpression; receptor; reconstitution; stem cells; therapeutic target; tumor progression

PROJECT SUMMARY / ABSTRACT Extracellular vesicles (EVs) comprise a heterogeneous pool of membrane-enclosed compartments secreted to the extracellular milieu of cells. Eukaryotic cells release a variety of EVs subpopulations that can be classified broadly into two categories on the basis of their membrane of origin. Microvesicles are EVs that form by direct outwards budding from the plasma membrane. Exosomes are EVs that originate from the endocytic pathway. Upon fusion of a multivesicular body (MVB) with the plasma membrane, the intraluminal vesicles (ILVs) are exported to the extracellular space as exosomes. Exosomes have elicited broad interest as their intraluminal contents (e.g. miRNAs, yRNAs, and tRNAs) are distinct from their progenitor cells. An accumulating repertoire of evidence has suggested that EV-mediated intercellular propagation of miRNAs plays important roles in various aspects of cancer biology. In particular, several studies have suggested that the intercellular propagation of miR-122 through breast cancer-derived EVs promotes breast cancer metastasis by reprogramming glucose metabolism in the pre-metastatic niche in vivo. Consistent with this line of experimental evidence, our lab demonstrated previously, both in cells and a cell-free reaction, that the Lupus La antigen (La) mediates the selective sorting and enrichment of miR-122 into exosomes derived from a metastatic breast cancer cell line. However, the molecular mechanism(s) by which the La:miR-122 ribonucleoprotein (RNP) complex itself is selectively incorporated into breast cancer-derived exosomes remains unknown. In this proposed research project, Jordan Ngo seeks to elucidate the molecular mechanism(s) by which the La:miR-122 RNP is selectively sorted into ILVs. In Specific Aim #1, Jordan will assess the efficacy by which a panel of candidate endosomal receptor proteins (identified by unbiased proximity labeling proteomics) are able to invoke the capture of cytoplasmic La into ILVs prior to exosome secretion. In Specific Aim #2, Jordan will identify the domains of La required for (i) its unconventional secretion within exosomes and (ii) its interaction with miR-122. In Specific Aim #3, Jordan will establish a cell-free reaction that recapitulates the selective sorting of La into ILVs, allowing further biochemical dissection of this high-fidelity sorting mechanism. Completion of the proposed research will provide novel insights into the molecular mechanisms by which specific cytoplasmic constituents are efficiently and selectively packaged into exosomes, and by extension, identify putative therapeutic targets for metastatic breast cancer and inform efforts to engineer exosomes into efficacious delivery vehicles for therapeutic compounds and nucleic acids. At the conclusion of Jordan’s NRSA-sponsored training, Jordan will have a rigorous intellectual foundation, scientific independence, and a uniquely broad technical toolkit that will make him an outstanding candidate for an independent investigator position in the future.

项目摘要 /摘要 细胞外蔬菜（EV）包括一个分泌的膜封闭室的异质池 细胞外环境。真核细胞释放可以分类的各种电动汽车亚群 根据其原籍膜，大致分为两个类别。微泡是通过直接形成的EV 从质膜向外萌芽。外泌体是源自内吞途径的EV。 在将多个体（MVB）与质膜融合后，内部蔬菜（ILV）为 作为外泌体导出到细胞外空间。外泌体引起了广泛的兴趣作为其内部 含量（例如miRNA，YRNA和TRNA）与其祖细胞不同。 已经提出了积累的证据库存，即EV介导的miRNA的细胞间传播 在癌症生物学的各个方面都起着重要作用。特别是，一些研究表明 miR-122通过乳腺癌衍生的电动汽车的细胞间传播促进乳腺癌转移 重新编程在体内的葡萄糖代谢中。与这条实验线一致 我们的实验室以前在细胞和无细胞反应中证明了证据表明狼疮抗原（LA） 将miR-122的选择性分选和富集介导源自转移性乳腺癌的外泌体 细胞系。但是，LA：miR-122核糖核蛋白（RNP）的分子机制 配合物本身被选择性地纳入乳腺癌衍生的外泌体中，仍然未知。 在这个拟议的研究项目中，约旦非政府组织试图阐明分子机制 LA：MiR-122 RNP选择性地分类为ILV。在特定的目标＃1中，约旦将评估a的效率 候选内体受体蛋白的面板（通过无偏接近标记蛋白质组学鉴定）能够 在外泌体分泌之前，将细胞质LA捕获到ILV中。在特定的目标＃2中，约旦将 确定（i）其在外泌体内非常规分泌所需的洛杉矶域，以及（ii）与之相互作用 mir-122。在特定的目标＃3中，约旦将建立无细胞的反应，以概括性排序 LA进入ILV，允许对这种高保真分类机制的进一步生化解剖。 拟议研究的完成将提供对特定特定机制的新见解 细胞质构成有效，有选择地包装到外泌体中，并通过延伸确定 推定的用于转移性乳腺癌的治疗靶标，并为将外泌体的努力提供为高效的努力 用于治疗化合物和核酸的输送车。 约旦的NRSA赞助培训结束时，约旦将拥有严格的知识基础， 科学独立性和独特的广泛技术工具包，将使他成为杰出的候选人 未来的独立研究者职位。