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Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

ABSTRACT: The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices ?1/?6 and the ?1-?2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.

SUBMITTER: Uchime O

PROVIDER: S-EPMC4697190 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

Uchime Onyinyechukwu O Dai Zhou Z Biris Nikolaos N Lee David D Sidhu Sachdev S SS Li Sheng S Lai Jonathan R JR Gavathiotis Evripidis E

The Journal of biological chemistry 20151112 1

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how t ...[more]

PMID: 26565029

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Project description:Background:Acute kidney injury (AKI) caused by bee stings is common, with characteristics of acute onset, severe illness, and high mortality. Melittin, a major component of bee venom, has been considered to play a key role in bee sting related AKI. This study aims to illustrate whether melittin could lead to apoptosis of renal tubular epithelial cells (RTECs) and to investigate its mechanism. Methods:In vivo, 45 mice were randomly divided into the melittin group (n=30, injected with melittin into the tail vein according to the total dose of 4.0 ug/g weight) and the control group (n=15, injected with the same volume of saline into the tail vein). In vitro, human RTECs (HK-2) were cultured and treated with melittin (2ug/ml or 4ug/ml) and TNF-α (10ng/ml). Biochemical analysis, HE stains, and electron microscope were performed to evaluate renal function and pathological changes. TUNEL stains and flow cytometry were performed to detect apoptosis. Real-time PCR was performed to detect mRNA levels of Bax, Bcl-2, and TNF-α. Simple western assay and immunohistochemical (IH) and immunofluorescent (IF) stains were performed for protein detection. Results:Melittin successfully induced AKI in mice. Compared with the control group, obvious injury and apoptosis of RTECs were observed in the melittin group; the mRNA and protein expressions of Bax were significantly increased, while the expression of Bcl-2 was significantly decreased. The serum TNF-αlevel in melittin group was significantly higher than that in control group. In vitro, the results confirmed that melittin can cause HK-2 cells apoptosis. The trends of expression of Bax and Bcl-2 were consistent with the results in vivo. The levels of TNF-α mRNA and protein by PCR and Western blot were significantly higher in melittin group than those in control group. Conclusion:Melittin can lead to the apoptosis of RTECs, which may be mediated by upregulating the expression of Bax/Bcl-2 and activating the TNF-α signaling pathway.

Dataset Information

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

Publications

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

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