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

项目总结 蛋白质分泌在多细胞生物体的发育和维持中起着核心作用。特化细胞 组织中的各种类型通过调节或结构性分泌来分泌蛋白质。调节分泌发生在反应中 细胞外刺激引起信号分子的释放，而结构性分泌促进 提供组织完整性的细胞外基质成分的沉积。即使这些过程 对人类健康非常重要，这些特征决定了蛋白质是否由 调节或收缩的分泌物仍不清楚。细胞内蛋白质分布的中枢调节因子 是跨高尔基体网络(TGN)，它将分泌蛋白质分类并包装成特定的囊泡载体 靶向细胞内存储颗粒(调节分泌)或细胞表面(结构性分泌)。 识别溶酶体M6P标签的甘露糖-6-磷酸受体(M6P-R)的鉴定导致 这种观点认为，特定的分拣受体也会对分泌蛋白质进行分拣。然而，保守的识别信号 或者，货物受体仍然未知。这些分子是如何识别和分类的，以靶向 正确的目的地？ 液液相分离将大分子浓缩成生物分子凝聚物的概念 (LLP)已经彻底改变了现代细胞生物学。人类细胞使用这一原理来组织生化 在没有膜的情况下在空间上进行。我们最近的研究提出了一种新的可能性：种族隔离 TGN管腔中的分泌蛋白质遵循这一概念。我们的工作表明，纯化的嗜铬颗粒 (CGS)或Cab45在TGN环境中进行液-液相分离(LLP)。这两种蛋白质都是 建议与分泌蛋白(客户)共同聚集，以方便其分类和包装。我们展示了 CG或Cab45液体，而不是固体聚集体，是客户分拣和包装所必需的。尽管如此， 依赖有限合伙人的客户打包的基本机制仍然难以捉摸。因此，我们的长期 目标是了解依赖于LLP的货物分拣的分子基础(由CGs监管)和 构成(由Cab45)分泌物。 我们的建议旨在确定重组系统中依赖于LLP的分类机制 重建TGN管腔的环境。我们将包括模型膜，以检查是否以及如何 凝结物与TGN的管腔小叶伴生。我们将使用调控的细胞培养模型(P12 细胞)或构成(皮肤成纤维细胞)分泌物，以验证我们在活细胞中的体外结果。我们的理念将 建立凝析油形成的分子要求、客户识别机制和 调节分泌物和结构性分泌物中的囊泡形成。这些结果将为我们提供一个基本的 对细胞生物学中一个令人兴奋的新范式的理解和对病理学研究的影响 由蛋白质分泌缺陷引起的，如精神疾病或癌症。

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.