TY - JOUR
T1 - A kinetic fluorescence polarization ligand assay for monitoring BAX early activation
AU - Gelles, Jesse D.
AU - Mohammed, Jarvier N.
AU - Chen, Yiyang
AU - Sebastian, Tara M.
AU - Chipuk, Jerry Edward
N1 - Publisher Copyright:
© 2022 The Author(s)
PY - 2022/3/28
Y1 - 2022/3/28
N2 - Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of BAX are commonly limited to methodologies measuring endpoint phenotypes and do not assess activation of monomeric BAX. Here, we present FLAMBE, a fluorescence polarization ligand assay for monitoring BAX early activation, that measures activation-induced release of a peptide probe in real time. Using complementary parallel and tandem biochemical techniques, we validate, corroborate, and apply FLAMBE to a contemporary repertoire of BAX modulators, characterizing their contributions within the early steps of BAX activation. Additionally, we use FLAMBE to reveal that historically “dead” BAX mutants remain responsive to activation as quasi-functional monomers. We also identify data metrics for comparative analyses and demonstrate that FLAMBE data align with downstream functional observations. Collectively, FLAMBE advances our understanding of BAX activation and fills a methodological void for studying BAX with broad applications in cell biology and therapeutic development.
AB - Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of BAX are commonly limited to methodologies measuring endpoint phenotypes and do not assess activation of monomeric BAX. Here, we present FLAMBE, a fluorescence polarization ligand assay for monitoring BAX early activation, that measures activation-induced release of a peptide probe in real time. Using complementary parallel and tandem biochemical techniques, we validate, corroborate, and apply FLAMBE to a contemporary repertoire of BAX modulators, characterizing their contributions within the early steps of BAX activation. Additionally, we use FLAMBE to reveal that historically “dead” BAX mutants remain responsive to activation as quasi-functional monomers. We also identify data metrics for comparative analyses and demonstrate that FLAMBE data align with downstream functional observations. Collectively, FLAMBE advances our understanding of BAX activation and fills a methodological void for studying BAX with broad applications in cell biology and therapeutic development.
KW - BAX
KW - BCL-2 family
KW - apoptosis
KW - fluorescence polarization
KW - high throughput
KW - kinetic assay
UR - http://www.scopus.com/inward/record.url?scp=85126550942&partnerID=8YFLogxK
U2 - 10.1016/j.crmeth.2022.100174
DO - 10.1016/j.crmeth.2022.100174
M3 - Article
AN - SCOPUS:85126550942
SN - 2667-2375
VL - 2
JO - Cell Reports Methods
JF - Cell Reports Methods
IS - 3
M1 - 100174
ER -