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.

项目概要 蛋白质分泌在多细胞生物的发育和维持中起着核心作用。专门细胞 组织中的类型通过调节或组成性分泌来分泌蛋白质。调节分泌发生响应 细胞外刺激引起信号分子的释放，而组成型分泌促进 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 调节或收缩的分泌仍然未知。细胞内蛋白质分布的中央调节器 是跨高尔基体网络 (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（组成型分泌）。 识别溶酶体 M6P 标签的 6-磷酸甘露糖受体 (M6P-R) 的鉴定导致 特定分选受体也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。这些分子如何被识别和分类以靶向 正确的目的地？ 通过液-液相分离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。人体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，即隔离 TGN 管腔中分泌蛋白的分布遵循这一概念。我们的工作表明，纯化的嗜铬粒蛋白 (CG) 或 Cab45 在 TGN 环境中进行液-液相分离 (LLPS)。两种蛋白质均已 建议与分泌蛋白（客户）共聚集，以促进其分类和包装。我们展示 CG 或 Cab45 液体（而不是固体骨料）对于客户分类和包装至关重要。尽管如此， 依赖于 LLPS 的客户端打包的底层机制仍然难以捉摸。因此，我们的长期 目标是了解 LLPS 依赖性货物分选的分子基础（通过 CG）和 组成型（由 Cab45）分泌。 我们的建议旨在确定重构系统中依赖于 LLPS 的排序机制 重建 TGN 流明的环境。我们将包括模型膜来检查这些是否以及如何 冷凝物与 TGN 的管腔小叶相关。我们将使用受监管的细胞培养模型（P12 细胞）或组成型（皮肤成纤维细胞）分泌，以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、客户识别机制和 调节和组成性分泌中的囊泡形成。这些结果将为 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，例如精神疾病或癌症。