Methods to Probe Conformational Activation and Mitochondrial Activity of Proapoptotic BAK

Singh, Geetika; Moldoveanu, Tudor

doi:10.1007/978-1-4939-8861-7_13

Cited by 4 publications

(7 citation statements)

References 42 publications

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“…The binding of activating BH3 ligands disrupts helix α1 contacts to helices α3 and α5 of the C-bundle observed in apo BAK. BH3 ligand-activated BAK exhibits electrostatic contacts between helix α1 and helix α2 of the N-bundle, which is dynamic and susceptible to m-calpain proteolysis in endogenous mitochondrial BAK 19 , 25 , 49 (Fig. 7 inset and Supplementary Movie 1 ).…”

Section: Discussionmentioning

confidence: 99%

Structural basis of BAK activation in mitochondrial apoptosis initiation

et al. 2022

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BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

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Section: Discussionmentioning

confidence: 99%

Structural basis of BAK activation in mitochondrial apoptosis initiation

et al. 2022

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“…In these cells, mitochondrial R127A(s) BAK was partially degraded into two fragments resembling those produced by limited proteolysis with m -calpain, suggesting that an endogenous protease may degrade this mutant. This mutant failed to respond to the thiol-targeting crosslinker BMH (bismaleimidohexane), which can detect oligomerization with activated WT BAK ( Figure 5 E) ( Singh and Moldoveanu, 2019 ). Remarkably, adding WT BID BH3 ± SJ572946 did not change the banding pattern obtained with m -calpain proteolysis, suggesting that this mutant is spontaneously misfolded at the mitochondria and inactive in poration ( Figure 5 E).…”

Section: Resultsmentioning

confidence: 99%

“…Mitochondria were purified from BCL2allKO HCT116 cells constitutively expressing R127A(s) BAK from pMX-BAK-IRES-GFP by differential centrifugation according to published protocols ( Singh and Moldoveanu, 2019 ). In poration assays, mitochondria were incubated for 2 h with WT BID BH3 peptide (1–50 μM) ± SJ572946 (200 μM) then centrifuged for 10 min at 4°C at the maximum speed achieved in a Sorvall HIGHPlate™ 6000 swinging-bucket rotor of a Sorvall Legend XTR tabletop centrifuge (Thermo Fisher).…”

Section: Methodsmentioning

confidence: 99%

Small molecule SJ572946 activates BAK to initiate apoptosis

Sekar¹,

Singh²,

Qin³

et al. 2022

iScience

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“…[86,105] The entire pool of effectors in each cell need not be found in mode 2, and a mixture of dormant and mode 2 effectors is usually observed as detected by proteolytic sensitivity which can distinguish between the two effector conformations. [86,106] Exactly how the conversion of robust apoptosis, which lead to the idea that the effectors are activated by the mitochondrial outer membrane. [35,107] It is documented that BAK and BAX are activated by sphingosine-1-phosphate and hexadecenal which cooperate with tBID, respectively, [108,109] although the role of these lipids has not been reassessed in the AKO cells.…”

Section: Bak and Bax Are Directly Activated By Bh3-only Initiators Au...mentioning

confidence: 99%

“…[ 86,105 ] The entire pool of effectors in each cell need not be found in mode 2, and a mixture of dormant and mode 2 effectors is usually observed as detected by proteolytic sensitivity which can distinguish between the two effector conformations. [ 86,106 ] Exactly how the conversion of the effectors to mode 2 conformation occurs is unclear but the effectors must present an open conformation with the BH3 region exposed for sequestration by the pro‐survival BCL‐2 proteins. Derepression of pro‐survival BCL‐2 proteins with inhibitors known as BH3 mimetics can induce robust apoptosis when effectors are present in mode 2.…”

Section: Introductionmentioning

confidence: 99%

Apoptotic mitochondrial poration by a growing list of pore‐forming BCL‐2 family proteins

Moldoveanu

2023

BioEssays

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The pore‐forming BCL‐2 family proteins are effectors of mitochondrial poration in apoptosis initiation. Two atypical effectors—BOK and truncated BID (tBID)—join the canonical effectors BAK and BAX. Gene knockout revealed developmental phenotypes in the absence the effectors, supporting their roles in vivo. During apoptosis effectors are activated and change shape from dormant monomers to dynamic oligomers that associate with and permeabilize mitochondria. BID is activated by proteolysis, BOK accumulates on inhibition of its degradation by the E3 ligase gp78, while BAK and BAX undergo direct activation by BH3‐only initiators, autoactivation, and crossactivation. Except tBID, effector oligomers on the mitochondria appear as arcs and rings in super‐resolution microscopy images. The BH3‐in‐groove dimers of BAK and BAX, the tBID monomers, and uncharacterized BOK species are the putative building blocks of apoptotic pores. Effectors interact with lipids and bilayers but the mechanism of membrane poration remains elusive. I discuss effector‐mediated mitochondrial poration.

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Methods to Probe Conformational Activation and Mitochondrial Activity of Proapoptotic BAK

Cited by 4 publications

References 42 publications

Structural basis of BAK activation in mitochondrial apoptosis initiation

Structural basis of BAK activation in mitochondrial apoptosis initiation

Small molecule SJ572946 activates BAK to initiate apoptosis

Apoptotic mitochondrial poration by a growing list of pore‐forming BCL‐2 family proteins

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