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

Progressive neurodegeneration linked to the accumulation of misfolded proteins is a hallmark of several neurodegenerative disorders, including Parkinson's disease, Huntington's disease, and Alzheimer's disease. Dysfunction in the protein homeostasis machinery correlates with pathology. The chaperone protein DNAJB6 is expressed in neurons and oligodendrocytes and has been shown to play a key role in preventing amyloid aggregation by binding to amyloidogenic proteins and facilitating their refolding or degradation, in cooperation with other chaperones. Here, we describe a simple and feasible assay that enables high-throughput screening for DNAJB6 activity in a plate reader format. We use genetically engineered HEK293 cells that stably express DNAJB6 fused to either CFP or YFP. These cells can be plated into multi-well plates, and the fluorescence resonance energy transfer (FRET) signal can be measured for analysis of DNAJB6 dimerization, which is linked to DNAJB6 activity. The protocol can be used for drug screening and to identify compounds that increase DNAJB6 dimerization, and can serve as a starting point for finding new medicines that act through modulating DNAJB6 activity. Key features • The protocol requires a plate reader capable of FRET analysis and bandwidth adjustment for CFP/YFP separation. It was developed using a CLARIOstar plate reader. • The protocol requires access to the authors' FRET DNAJB6 cell line or equivalent cells with stable expression of CFP/YFP-DNAJB6. • The assay measures DNAJB6 dimerization and can potentially be adapted to other proteins whose functional state is linked to dimerization activity. • The protocol is useful for compound screening purposes and requires pre-existing knowledge of basic cell culture.