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 - Funding Information:
This work was supported by NIH grants R01 CA259110 (J.E.C.) and R01 CA206005 (J.E.C.), the JJR Foundation , the William A. Spivak Fund , the Fridolin Charitable Trust , an American Cancer Society Research Scholar Award , a Leukemia & Lymphoma Society Career Development Award , and an Irma T. Hirschl/Monique Weill-Caulier Trust Research Award . This work was also supported in part by two research grants ( 5FY1174 and 1FY13416 ) from the March of Dimes Foundation , a Collaborative Pilot Award from the Melanoma Research Alliance , a Developmental Research Pilot Project Program within the Department of Oncological Sciences at the Icahn School of Medicine at Mount Sinai, and the Tisch Cancer Institute Cancer Center support grant ( P30 CA196521 ). The authors wish to thank Drs. Thibaud T. Renault and Mark P.A. Luna-Vargas for their assistance in the development of the assays used within this work; Dr. Evripidis Gavathiotis for providing the BAX α9 , BAX ℓ1–2 , and BAX K21E plasmids; and Dr. James LaBelle for his aid and expertise with BH3 peptides.
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 -