A Sensitive and Versatile Cell-Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional...

Area of Science:

• Cell Biology
• Molecular Biology
• Neuroscience

Background:

• Conventional protein secretion via ER-Golgi is well-understood, but alternative routes, termed unconventional protein secretion (UcPS), are crucial for many secreted factors.
• Mechanisms of UcPS remain poorly understood, involving direct plasma membrane transport or redirection of intracellular compartments.
• Studying UcPS requires standardized tools to investigate cargo recognition, selection, and transport, especially given cell-type and stress-dependent variations.

Purpose of the Study:

• To develop a reliable cell-based assay for investigating both conventional and unconventional protein secretion.
• To identify intracellular compartments involved in UcPS cargo trafficking.
• To investigate the role of UcPS in disease-associated protein variants and screen for Alzheimer's disease risk factors.

Main Methods:

• Combined split NanoLuc Binary Technology with the Retention Using Selective Hooks (RUSH) system to create a sensitive assay.
• Utilized the assay to identify compartments in UcPS cargo trafficking.
• Applied the assay to study disease-associated mutants (Tau, SOD1) and screen for Alzheimer's disease risk factors.

Main Results:

• Developed a robust and sensitive cell-based assay for studying conventional and unconventional protein secretion.
• Demonstrated altered UcPS in disease-associated mutants of Tau and superoxide dismutase-1 (SOD1).
• Revealed a functional link between amyloid-beta production and Tau UcPS in Alzheimer's disease screening.

Conclusions:

• The developed assay is a powerful tool for studying UcPS mechanisms in physiological and pathological conditions.
• UcPS plays a role in the secretion of disease-associated protein variants.
• This work provides new insights into Alzheimer's disease pathogenesis by linking amyloid-beta to Tau UcPS.