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

项目摘要/摘要 细胞外小泡(EVS)由不同种类的膜封闭隔室组成，分泌到 细胞的胞外环境。真核细胞释放各种EVS亚群，这些亚群可以分类 根据它们的原产膜大致分为两类。微囊是通过直接形成的电动汽车 从质膜向外萌发。外切体是起源于内吞途径的EV。 当多囊泡体(MVB)与质膜融合时，腔内小泡(ILV)是 以胞外体的形式输出到细胞外空间。外切体由于其腔内结构而引起了人们的广泛兴趣。 内容物(例如miRNAs、yRNAs和tRNAs)与它们的祖细胞不同。 一系列积累的证据表明，EV介导的miRNAs的细胞间繁殖 在癌症生物学的各个方面发挥着重要的作用。特别是，几项研究表明， MiR-122通过乳腺癌EVS的细胞间增殖通过以下途径促进乳腺癌转移 在体内对转移前的生态位的葡萄糖代谢进行重新编程。与这条实验路线相一致 有证据表明，我们的实验室以前在细胞和无细胞反应中都证明，狼疮La抗原(La) 介导miR-122在转移性乳腺癌来源的外体中的选择性分选和浓缩 细胞系。然而，La：miR-122核糖核蛋白的分子机制(S) 复合体本身被选择性地结合到乳腺癌衍生的外切体中仍不清楚。 在这项拟议的研究项目中，乔丹·恩戈试图阐明(S) LA：MIR-122 RNP被选择性地分类为ILV。在具体目标1中，乔丹将评估一项 候选内体受体蛋白小组(通过无偏的邻近标记蛋白质组学鉴定)能够 在胞外体分泌之前将胞质La捕获到ILV中。在第二个具体目标中，乔丹将 确定La在外体中的非常规分泌和(Ii)与其相互作用所需的结构域 和平号-122。在具体目标#3中，乔丹将建立一种无细胞反应，概括了选择性分选 使这种高保真分选机制得到进一步的生化剖析。 这项拟议的研究的完成将为研究特定的分子机制提供新的见解 细胞质成分被有效和选择性地包装到外体中，并延伸到识别 转移性乳腺癌的假定治疗靶点，并为将外显子改造为有效的努力提供信息 治疗性化合物和核酸的运输工具。 在约旦NRSA赞助的训练结束后，乔丹将拥有严格的智力基础， 科学独立性和独特的广泛的技术工具包，这将使他成为杰出的候选人 未来的独立调查员职位。

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.