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 蛋白质分泌在开发和维持多细胞生物中起着核心作用。专业单元格 组织类型通过调节或本构分泌分泌蛋白质。被调节的分泌发生 到引起信号分子释放的细胞外刺激，而本构分泌有助于 提供组织完整性的细胞外基质组件的沉积。即使这些过程 对于人类健康非常重要，该特征确定蛋白质是否被分泌 受调节或收缩的分泌仍然未知。细胞内蛋白质分布的中心调节剂 是反式高尔基网络（TGN），将分泌蛋白分类为特定的水泡载体 将它们靶向细胞内存储颗粒（调节分泌）或细胞表面（组成型分泌）。 识别识别溶酶体M6P标签的甘露糖-6-磷酸受体（M6P-R）的鉴定导致 特定分类受体还分类分泌蛋白的想法。但是，保守的识别信号 或货物受体仍然未知。这些分子如何识别和分类以靶向 正确的目的地？ 将大分子浓缩到生物分子冷凝水中的概念 （LLP）彻底改变了现代细胞生物学。人类细胞使用此原理组织生化 在空间上没有膜。我们最近的研究提高了种族隔离的新颖可能性 TGN Lumen中的分泌蛋白遵循了这一概念。我们的工作表明纯化的染色体蛋白 （CGS）或CAB45在TGN环境中进行液态液相分离（LLP）。两种蛋白质都是 建议与分泌的蛋白质（客户）共同聚集，以促进其分类和包装。我们显示 CG或CAB45液体而不是固体聚集体对于客户分类和包装至关重要。尽管如此， LLPS依赖性客户包装的基本机制仍然难以捉摸。因此，我们的长期 目标是了解调节（通过CGS）和 本构（由CAB45）分泌。 我们的建议旨在确定在重组系统中依赖LLP的机制 重新创建TGN Lumen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的腔内小叶相关。我们将使用调节的细胞培养模型（p12 细胞）或本构（皮肤成纤维细胞）分泌以验证我们的体外导致活细胞的结果。我们的概念会 确定凝结物形成的分子要求，客户识别机制和 调节和本构分泌中的囊泡形成。这些结果将提供基本 了解细胞生物学中令人兴奋的新范式并影响病理学的研究 由蛋白质分泌有缺陷引起的，例如精神疾病或癌症。