Novel cell-based assay enables FRET-based measurements of the dimerization activity of the chaperone DNAJB6.

The chaperone DNAJB6 inhibits aggregation of several amyloid proteins, such as α-synuclein and huntingtin, which are involved in neurodegenerative diseases. Here, we designed a cell-based assay to measure DNAJB6 dimerization in HEK293 cells by fluorescent resonance energy transfer (FRET), as previous studies suggest that the activity of DNAJB6 is dependent on dimerization. The HEK293 cells were engineered to stably express DNAJB6 coupled to cyan fluorescent protein and yellow fluorescent protein, respectively. A platereader format was used to analyze the FRET signal from dimerization. Stimulation with Tunicamycin, a positive control that induces protein misfolding, or with α-synuclein preformed fibrils, increased the FRET signal significantly, in these cells. This increase in FRET signal reflects enhanced dimerization activity, which is suggested to correlate with chaperone activity. To our knowledge, this study is the first to measure a DNAJ protein dimerization activity using a FRET-based approach. These cells could serve as an experimental platform to screen for compounds that modulate DNAJB6 dimerization activity, which in the future may aid in identifying potential therapeutic targets.