A Novel Plate Reader-Based Protocol for Measurement of DNAJB6 Dimerization Activity

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Neurodegenerative diseases like Parkinson's, Huntington's, and Alzheimer's are characterized by misfolded protein accumulation.
• Protein homeostasis, crucial for neuronal health, is often compromised in these disorders.
• The chaperone DNAJB6 plays a vital role in preventing protein aggregation and is expressed in neurons.

Purpose of the Study:

• To develop a high-throughput screening (HTS) assay for DNAJB6 activity.
• To identify compounds that modulate DNAJB6 function for potential therapeutic applications.
• To facilitate drug discovery for neurodegenerative diseases by targeting protein homeostasis.

Main Methods:

• Utilized genetically engineered HEK293 cells stably expressing DNAJB6 fused to CFP or YFP.
• Employed a plate reader format to measure fluorescence resonance energy transfer (FRET) signals.
• Assessed DNAJB6 dimerization as a proxy for its activity.

Main Results:

• Established a feasible HTS assay for DNAJB6 activity in a plate reader format.
• Demonstrated that DNAJB6 dimerization, measured by FRET, correlates with its chaperone function.
• The assay is suitable for screening compound libraries to find modulators of DNAJB6 activity.

Conclusions:

• The developed FRET-based assay provides a robust platform for HTS of DNAJB6 activity.
• This assay can accelerate the discovery of novel therapeutics targeting protein misfolding in neurodegenerative diseases.
• The protocol serves as a foundation for identifying compounds that enhance DNAJB6-mediated neuroprotection.