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.

项目总结/摘要 细胞外囊泡（EV）包含分泌到细胞外的膜封闭区室的异质池。 细胞的细胞外环境。真核细胞释放出多种EV亚群， 根据它们的起源膜，大致分为两类。微囊泡是通过直接 从质膜向外出芽。外泌体是源自内吞途径的EV。 当多泡体（MVB）与质膜融合时，管腔内囊泡（ILV）被释放。 作为外泌体输出到细胞外空间。外泌体由于其管腔内 在一些实施方案中，细胞内含物（例如miRNA、yRNA和tRNA）与它们的祖细胞不同。 越来越多的证据表明EV介导的miRNAs的细胞间增殖 在癌症生物学的各个方面都发挥着重要作用。特别是，一些研究表明， miR-122通过乳腺癌来源的EV的细胞间传播通过以下方式促进乳腺癌转移： 在体内转移前小生境中重编程葡萄糖代谢。与这条实验线一致 证据，我们的实验室以前证明，无论是在细胞和无细胞反应，狼疮La抗原（La） 介导miR-122选择性分选和富集到来源于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） 复合物本身选择性地掺入乳腺癌来源的外泌体仍然是未知的。 在这个拟议的研究项目中，Jordan Ngo试图阐明 La：miR-122 RNP被选择性地分选到ILV中。在具体目标#1中，Jordan将评估 一组候选内体受体蛋白（通过无偏邻近标记蛋白质组学鉴定）能够 以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 鉴定La的结构域，所述结构域是（i）其在外来体内的非常规分泌和（ii）其与 miR-122在具体目标#3中，Jordan将建立一种无细胞反应，该反应重现了细胞的选择性分选。 La进入ILV，允许进一步的生化解剖这种高保真分选机制。 完成拟议的研究将提供新的见解的分子机制，具体 细胞质成分被有效地和选择性地包装到外泌体中，并通过延伸，鉴定 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 用于治疗化合物和核酸的递送载体。 在约旦的NRSA赞助的培训结束时，约旦将有一个严格的知识基础， 科学的独立性，以及独特的广泛的技术工具包，这将使他成为一个杰出的候选人， 未来的独立调查员。