Mechanism of secretory cargo sorting at the trans-Golgi Network (TGN)

跨高尔基体网络（TGN）的分泌性货物分类机制

• 批准号: 10623825
• 负责人: Julia von Blume
• 金额: $ 32.79万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623825
• 关键词: Adrenal Glands; Biochemical Process; Blood Circulation; Breathing; Cell Culture Techniques; Cell surface; Cells; Cellular biology; Chromogranins; Client; Cytoplasmic Granules; Deposition; Destinations; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Goals; Health; Human; IGF Type 2 Receptor; In Vitro; Liquid substance; Malignant Neoplasms; Mechanics; Membrane; Mental disorders; Modeling; Modernization; Molecular; Muscle; Neuroendocrine Cell; Organism; Pathology; Phase; Physical condensation; Play; Process; Protein Secretion; Proteins; Research; Role; Signal Transduction; Signaling Molecule; Skin; Sorting; Stimulus; Stress; System; Tissues; Work; carbohydrate metabolism; cell type; extracellular; macromolecule; membrane model; novel; protein distribution; receptor; reconstitution; response; secretory protein; segregation; trans-Golgi Network

Project summary Protein secretion plays a central role in developing and maintaining multicellular organisms. Specialized cell types in tissues secrete proteins by regulated or constitutive secretion. Regulated secretion occurs in response to an extracellular stimulus that elicits the release of signaling molecules, while constitutive secretion facilitates the deposition of extracellular matrix components that provide tissue integrity. Even though these processes are highly significant for human health, features that determine whether a protein is secreted by regulated or constrictive secretion remain unknown. A central regulator of intracellular protein distribution is the trans-Golgi Network (TGN), which sorts and packages secretory proteins into specific vesicular carriers targeting them to intracellular storage granules (regulated secretion) or the cell surface (constitutive secretion). The identification of the mannose-6-phosphate receptor (M6P-R) that recognizes M6P tags of lysosomal led to the idea that specific sorting receptors also sort secretory proteins. However, conserved recognition signals or cargo receptors remain unknown. How are these molecules recognized and sorted for targeting the correct destination? The concept of concentrating macromolecules into biomolecular condensates by liquid-liquid phase separation (LLPS) has revolutionized modern cell biology. Human cells use this principle to organize biochemical processes spatially without a membrane. Our recent research raises the novel possibility that the segregation of secretory proteins in the TGN lumen follows this concept. Our work has shown that purified chromogranins (CGs) or Cab45 undergo liquid-liquid phase separation (LLPS) in the milieu of TGN. Both proteins have been suggested to co-aggregate with secreted proteins (clients) to facilitate their sorting and packaging. We show that CG or Cab45 liquids, not solid aggregates, are essential for client sorting and packaging. Nonetheless, the underlying mechanisms of LLPS-dependent client packaging remain elusive. Therefore, our long-term goal is to understand the molecular basis of LLPS-dependent cargo sorting for regulated (by CGs) and constitutive (by Cab45) secretion. Our proposal aims at identifying the mechanisms of LLPS-dependent sorting in reconstituted systems that recreate the milieu of the TGN lumen. We will include model membranes to examine if and how these condensates associate with the luminal leaflet of the TGN. We will use cell culture models of regulated (P12 cells) or constitutive (skin fibroblasts) secretion to validate our in-vitro results in living cells. Our concept will establish the molecular requirements for condensate formation, the mechanisms of client recognition and vesicular formation in regulated and constitutive secretion. These results will provide a fundamental understanding of an exciting new paradigm in cell biology and impact the research of pathologies caused by defective protein secretion, such as psychiatric disorders or cancer.

项目摘要 蛋白质分泌在多细胞生物的发育和维持中起着核心作用。特化细胞 组织中的类型通过调节性或组成性分泌来分泌蛋白质。调节分泌是为了回应 细胞外刺激，刺激信号分子的释放，而组成性分泌促进 提供组织完整性的细胞外基质成分的沉积。尽管这些过程 对人类健康非常重要，这些特征决定了蛋白质是否由 受调节的或收缩性的分泌仍然是未知的。细胞内蛋白质分布的中枢调节因子 是高尔基体网络（trans-Golgi Network，TGN），它将分泌蛋白分类并包装到特定的囊泡载体中 将它们靶向细胞内储存颗粒（调节分泌）或细胞表面（组成性分泌）。 识别溶酶体M6 P标签的甘露糖-6-磷酸受体（M6 P-R）的鉴定导致 特定的分选受体也能分选分泌蛋白的想法。然而，保守的识别信号 或货物受体仍然未知。这些分子是如何识别和分类的， 目的地正确吗？ 液-液相分离法将大分子浓缩成生物分子凝聚物的概念 LLPS彻底改变了现代细胞生物学。人类细胞利用这一原理来组织生化 在没有膜的情况下进行空间处理。我们最近的研究提出了一种新的可能性， TGN腔中分泌蛋白的表达遵循这一概念。我们的工作表明，纯化的嗜铬粒蛋白 (CGs)或Cab 45在TGN环境中进行液-液相分离（LLPS）。这两种蛋白质都是 建议与分泌蛋白（客户）共聚集，以促进其分选和包装。我们表明 CG或Cab 45液体，而不是固体骨料，是客户分拣和包装的关键。尽管如此， 依赖LLPS的客户端打包的基础机制仍然是难以捉摸的。因此，我们的长期 目的是了解LLPS依赖性货物分选的分子基础，用于调节（通过CG）和 组成型（Cab 45）分泌。 我们的建议旨在确定重组系统中LLPS依赖性分选的机制， 重建TGN腔的环境。我们将包括模型膜，以检查是否以及如何这些 冷凝物与TGN的管腔小叶结合。我们将使用调节的细胞培养模型（P12 细胞）或组成型（皮肤成纤维细胞）分泌来验证我们在活细胞中的体外结果。我们的理念将 建立冷凝物形成的分子要求，客户识别的机制， 在调节和组成性分泌中形成囊泡。这些结果将提供一个基本的 了解细胞生物学中令人兴奋的新范式并影响病理学研究 由蛋白质分泌缺陷引起，如精神疾病或癌症。