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

Gelman, Anna ; Quintino, Luis LU orcid ; Nordberg, Manja ; Nyeng, Pia ; Brudek, Tomasz ; Nielsen, Leif Kofoed and Hansen, Christian LU (2026) In Biology Methods and Protocols 11(1).
Abstract

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... (More)

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.

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
cell-based assay, chaperone activity, DNAJB6, FRET
in
Biology Methods and Protocols
volume
11
issue
1
article number
bpag005
publisher
Oxford University Press
external identifiers
  • scopus:105030687431
ISSN
2396-8923
DOI
10.1093/biomethods/bpag005
language
English
LU publication?
yes
additional info
Publisher Copyright: © The Author(s) 2026. Published by Oxford University Press.
id
0ffcf565-689e-463d-bfae-303a6b2de2a3
date added to LUP
2026-04-09 16:30:18
date last changed
2026-04-09 16:31:35
@article{0ffcf565-689e-463d-bfae-303a6b2de2a3,
  abstract     = {{<p>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.</p>}},
  author       = {{Gelman, Anna and Quintino, Luis and Nordberg, Manja and Nyeng, Pia and Brudek, Tomasz and Nielsen, Leif Kofoed and Hansen, Christian}},
  issn         = {{2396-8923}},
  keywords     = {{cell-based assay; chaperone activity; DNAJB6; FRET}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Oxford University Press}},
  series       = {{Biology Methods and Protocols}},
  title        = {{Novel cell-based assay enables FRET-based measurements of the dimerization activity of the chaperone DNAJB6}},
  url          = {{http://dx.doi.org/10.1093/biomethods/bpag005}},
  doi          = {{10.1093/biomethods/bpag005}},
  volume       = {{11}},
  year         = {{2026}},
}