Quantification of the Interactions Between BCL-2 Proteins by Fluorescence Correlation Spectroscopy

Murad, Fabronia; García‐Sáez, Ana J.

doi:10.1007/978-1-4939-8861-7_20

Cited by 3 publications

(1 citation statement)

References 13 publications

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“…Ensemble measurements, which do not distinguish between the soluble and membrane forms of the proteins, can thus only return an apparent affinity, which depends on the amount and type of lipids present in the system. Interactions between different pairs of Bcl-2 family proteins embedded in the membrane of giant unilamellar vesicles have also been detected using linear scan cross-correlation spectroscopy [ 25 , 26 , 27 ]. Yet these experiments cannot distinguish between the different conformations or stoichiometries these proteins are known to adopt in lipid membranes [ 28 , 29 ].…”

Section: Introductionmentioning

confidence: 99%

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane

Rose

Kurylowicz

Mahmood

et al. 2021

IJMS

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The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6/m2 to ≃0.1/m2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.

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Section: Introductionmentioning

confidence: 99%

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane

Rose

Kurylowicz

Mahmood

et al. 2021

IJMS

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A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells

Subas Satish,

Iyer,

Shi

et al. 2024

Cell Death Differ

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BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.

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Bcl-xL inhibits tBid and Bax via distinct mechanisms

Murad

García‐Sáez

2021

Faraday Discuss.

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Quantification of the Interactions Between BCL-2 Proteins by Fluorescence Correlation Spectroscopy

Cited by 3 publications

References 13 publications

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane

A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells

Bcl-xL inhibits tBid and Bax via distinct mechanisms

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