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Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL.

ABSTRACT: BCL-XL is a dominant inhibitor of apoptosis and a significant anti-cancer drug target. Endogenous BCL-XL is integral to the mitochondrial outer membrane (MOM). BCL-XL reconstituted in detergent-free lipid bilayer nanodiscs is anchored to the nanodisc lipid bilayer membrane by tight association of its C-terminal tail, while the N-terminal head retains the canonical structure determined for water-soluble, tail-truncated BCL-XL, with the surface groove solvent-exposed and available for BH3 ligand binding. To better understand the conformation and dynamics of this key region of BCL-XL we have developed methods for isolating the membrane-embedded C-terminal tail from its N-terminal head and for preparing protein suitable for structural and biochemical studies. Here, we outline the methods for sample preparation and characterization and describe previously unreported structural and dynamics features. We show that the C-terminal tail of BCL-XL forms a transmembrane ?-helix that retains a significant degree of conformational dynamics. We also show that the presence of the intact C-terminus destabilizes the soluble state of the protein, and that the small fraction of soluble recombinant protein produced in Escherichia coli is susceptible to proteolytic degradation of C-terminal residues beyond M218. This finding impacts the numerous previous studies where recombinant soluble BCL-XL was presumed to be full-length. Nevertheless, the majority of recombinant BCL-XL produced in E. coli is insoluble and protected from proteolysis. This protein retains the complete C-terminal tail and can be reconstituted in lipid bilayers in a folded and active state.

SUBMITTER: Yao Y

PROVIDER: S-EPMC4842142 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL.

Yao Yong Y Nisan Danielle D Fujimoto Lynn M LM Antignani Antonella A Barnes Ashley A Tjandra Nico N Youle Richard J RJ Marassi Francesca M FM

Protein expression and purification 20160223

BCL-XL is a dominant inhibitor of apoptosis and a significant anti-cancer drug target. Endogenous BCL-XL is integral to the mitochondrial outer membrane (MOM). BCL-XL reconstituted in detergent-free lipid bilayer nanodiscs is anchored to the nanodisc lipid bilayer membrane by tight association of its C-terminal tail, while the N-terminal head retains the canonical structure determined for water-soluble, tail-truncated BCL-XL, with the surface groove solvent-exposed and available for BH3 ligand b ...[more]

PMID: 26923059

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Project description:ABT-737 is a pharmacological inhibitor of the anti-apoptotic activity of B-cell lymphoma-extra large (Bcl-xL) protein; it promotes apoptosis of cancer cells by occupying the BH3-binding pocket. We have shown previously that ABT-737 lowers cell metabolic efficiency by inhibiting ATP synthase activity. However, we also found that ABT-737 protects rodent brain from ischemic injury in vivo by inhibiting formation of the pro-apoptotic, cleaved form of Bcl-xL, ΔN-Bcl-xL. We now report that a high concentration of ABT-737 (1 μM), or a more selective Bcl-xL inhibitor WEHI-539 (5 μM) enhances glutamate-induced neurotoxicity while a low concentration of ABT-737 (10 nM) or WEHI-539 (10 nM) is neuroprotective. High ABT-737 markedly increased ΔN-Bcl-xL formation, aggravated glutamate-induced death and resulted in the loss of mitochondrial membrane potential and decline in ATP production. Although the usual cause of death by ABT-737 is thought to be related to activation of Bax at the outer mitochondrial membrane due to sequestration of Bcl-xL, we now find that low ABT-737 not only prevents Bax activation, but it also inhibits the decline in mitochondrial potential produced by glutamate toxicity or by direct application of ΔN-Bcl-xL to mitochondria. Loss of mitochondrial inner membrane potential is also prevented by cyclosporine A, implicating the mitochondrial permeability transition pore in death aggravated by ΔN-Bcl-xL. In keeping with this, we find that glutamate/ΔN-Bcl-xL-induced neuronal death is attenuated by depletion of the ATP synthase c-subunit. C-subunit depletion prevented depolarization of mitochondrial membranes in ΔN-Bcl-xL expressing cells and substantially prevented the morphological change in neurites associated with glutamate/ΔN-Bcl-xL insult. Our findings suggest that low ABT-737 or WEHI-539 promotes survival during glutamate toxicity by preventing the effect of ΔN-Bcl-xL on mitochondrial inner membrane depolarization, highlighting ΔN-Bcl-xL as an important therapeutic target in injured brain.

Dataset Information

Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL.

Publications

Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL.

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