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.

项目概要/摘要 细胞外囊泡 (EV) 包含膜封闭区室的异质库，分泌到 细胞的细胞外环境。真核细胞释放多种可分类的 EV 亚群 根据其膜来源大致分为两类。微泡是通过直接形成的 EV 从质膜向外出芽。外泌体是源自内吞途径的 EV。 多囊泡体（MVB）与质膜融合后，腔内囊泡（ILV） 作为外泌体输出到细胞外空间。外泌体因其管腔内的作用而引起了广泛的兴趣 内容物（例如 miRNA、yRNA 和 tRNA）与其祖细胞不同。 越来越多的证据表明 EV 介导的 miRNA 的细胞间传播 在癌症生物学的各个方面发挥着重要作用。特别是，多项研究表明， miR-122 通过乳腺癌来源的 EV 进行细胞间传播，通过以下方式促进乳腺癌转移： 重新编程体内转移前生态位的葡萄糖代谢。与这条实验线一致 证据，我们的实验室之前证明，无论是在细胞还是无细胞反应中，狼疮 La 抗原 (La) 介导 miR-122 选择性分选并富集到源自转移性乳腺癌的外泌体中 细胞系。然而，La:miR-122 核糖核蛋白 (RNP) 的分子机制 复合物本身是否选择性地整合到乳腺癌来源的外泌体中仍然未知。 在这个拟议的研究项目中，Jordan Ngo 试图阐明 La:miR-122 RNP 被选择性分类到 ILV 中。在具体目标#1中，约旦将评估以下措施的功效： 候选内体受体蛋白组（通过无偏邻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。在具体目标 #2 中，乔丹将 确定 (i) 其在外泌体内非常规分泌和 (ii) 与 miR-122。在具体目标#3中，乔丹将建立一个无细胞反应，概括了选择性分选 La 进入 ILV，从而可以进一步对这种高保真分选机制进行生化剖析。 完成拟议的研究将为特定分子机制提供新的见解 细胞质成分被有效地、选择性地包装到外泌体中，并且通过扩展，识别 转移性乳腺癌的假定治疗靶点，并为将外泌体工程化为有效的药物提供信息 治疗化合物和核酸的递送载体。 在乔丹 NRSA 赞助的培训结束时，乔丹将拥有严格的智力基础， 科学独立性和独特广泛的技术工具包将使他成为杰出的候选人 未来的独立调查员职位。