Dataset Information

Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons.

ABSTRACT: B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic member of the Bcl2 family of proteins, which supports neurite outgrowth and neurotransmission by improving mitochondrial function. During excitotoxic stimulation, however, Bcl-xL undergoes post-translational cleavage to ?N-Bcl-xL, and accumulation of ?N-Bcl-xL causes mitochondrial dysfunction and neuronal death. In this study, we hypothesized that the generation of reactive oxygen species (ROS) during excitotoxicity leads to formation of ?N-Bcl-xL. We further proposed that the application of an antioxidant with neuroprotective properties such as ?-tocotrienol (TCT) will prevent ?N-Bcl-xL-induced mitochondrial dysfunction via its antioxidant properties. Primary hippocampal neurons were treated with ?-TCT, glutamate, or a combination of both. Glutamate challenge significantly increased cytosolic and mitochondrial ROS and ?N-Bcl-xL levels. ?N-Bcl-xL accumulation was accompanied by intracellular ATP depletion, loss of mitochondrial membrane potential, and cell death. ?-TCT prevented loss of mitochondrial membrane potential in hippocampal neurons overexpressing ?N-Bcl-xL, suggesting that ?N-Bcl-xL caused the loss of mitochondrial function under excitotoxic conditions. Our data suggest that production of ROS is an important cause of ?N-Bcl-xL formation and that preventing ROS production may be an effective strategy to prevent ?N-Bcl-xL-mediated mitochondrial dysfunction and thus promote neuronal survival.

SUBMITTER: Park HA

PROVIDER: S-EPMC6982044 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons.

Park Han-A HA Mnatsakanyan Nelli N Broman Katheryn K Davis Abigail U AU May Jordan J Licznerski Pawel P Crowe-White Kristi M KM Lackey Kimberly H KH Jonas Elizabeth A EA

International journal of molecular sciences 20191228 1

B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic member of the Bcl2 family of proteins, which supports neurite outgrowth and neurotransmission by improving mitochondrial function. During excitotoxic stimulation, however, Bcl-xL undergoes post-translational cleavage to ∆N-Bcl-xL, and accumulation of ∆N-Bcl-xL causes mitochondrial dysfunction and neuronal death. In this study, we hypothesized that the generation of reactive oxygen species (ROS) during excitotoxicity leads to formation of ...[more]

PMID: 31905614

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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.

Project description:BackgroundNatural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s).Methodology/principal findingsEndothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase.Conclusion/significanceApoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.

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Dataset Information

Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons.

Publications

Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons.

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