Denus Morgane, Filaquier Aurore, Fargues William, Néel Eloïse, Stewart Sarah E, Colladant Maëlle, Curel Thomas, Mezghrani Alexandre, Marin Philippe, Claeysen Sylvie, Rubinsztein David C, Parmentier Marie-Laure, Villeneuve Julien
Institute of Functional Genomics (IGF), University of Montpellier, CNRS, INSERM, Montpellier, France.
Department of Biochemistry and Chemistry, School of Agriculture, Biomedicine and Environment, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
Traffic. 2025 Apr;26(4-6):e70009. doi: 10.1111/tra.70009.
In addition to the conventional endoplasmic reticulum (ER)-Golgi secretory pathway, alternative routes are increasingly recognized for their critical roles in exporting a growing number of secreted factors. These alternative processes, collectively referred to as unconventional protein secretion (UcPS), challenge traditional views of protein and membrane trafficking. Unlike the well-characterized molecular machinery of the conventional secretory pathway, the mechanisms underlying UcPS remain poorly understood. Various UcPS pathways may involve direct transport of cytosolic proteins across the plasma membrane or the incorporation of cargo proteins into intracellular compartments redirected for secretion. Identifying the specific chaperones, transporters and fusion machinery involved in UcPS cargo recognition, selection and transport is crucial to decipher how cargo proteins are selectively or synergistically directed through multiple secretory routes. These processes can vary depending on cell type and in response to particular stress conditions or cellular demands, underscoring the need for standardized tools and methods to study UcPS. Here, we combine the sensitivity of split NanoLuc Binary Technology with the versatility of the Retention Using Selective Hooks (RUSH) system to develop a straightforward and reliable cell-based assay for investigating both conventional and unconventional protein secretion. This system allows for the identification of intracellular compartments involved in UcPS cargo trafficking. Additionally, its sensitivity enabled us to demonstrate that disease-associated mutants or variants of Tau and superoxide dismutase-1 (SOD1) show altered secretion via UcPS. Finally, we leveraged this assay to screen for Alzheimer's disease risk factors, revealing a functional link between amyloid-beta production and Tau UcPS. This robust assay provides a powerful tool for increasing our knowledge of protein secretion mechanisms in physiological and pathological contexts.
除了传统的内质网（ER）-高尔基体分泌途径外，替代途径在输出越来越多的分泌因子中所起的关键作用也日益得到认可。这些替代过程统称为非常规蛋白质分泌（UcPS），对蛋白质和膜运输的传统观点提出了挑战。与传统分泌途径中特征明确的分子机制不同，UcPS的潜在机制仍知之甚少。各种UcPS途径可能涉及胞质蛋白直接穿过质膜运输，或将货物蛋白整合到重定向用于分泌的细胞内区室中。确定参与UcPS货物识别、选择和运输的特定分子伴侣、转运蛋白和融合机制，对于解读货物蛋白如何通过多种分泌途径被选择性或协同引导至关重要。这些过程可能因细胞类型而异，并响应特定的应激条件或细胞需求，这突出了研究UcPS需要标准化工具和方法。在这里，我们将分裂型纳米荧光素酶二元技术的敏感性与选择性挂钩保留（RUSH）系统的多功能性相结合，开发了一种简单可靠的基于细胞的检测方法，用于研究传统和非常规蛋白质分泌。该系统能够识别参与UcPS货物运输的细胞内区室。此外，其敏感性使我们能够证明，与疾病相关的Tau和超氧化物歧化酶-1（SOD1）突变体或变体通过UcPS显示出分泌改变。最后，我们利用该检测方法筛选阿尔茨海默病风险因素，揭示了淀粉样β蛋白产生与Tau UcPS之间的功能联系。这种强大的检测方法为增进我们对生理和病理背景下蛋白质分泌机制的了解提供了有力工具。
