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Engineering tocopherol selectivity in ?-TTP: a combined in vitro/in silico study.

ABSTRACT: We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of [Formula: see text]-tocopherol transfer protein ([Formula: see text]-TTP) towards [Formula: see text]-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for [Formula: see text]-tocopherol to [Formula: see text]-TTP 8.26[Formula: see text]2.13 kcal mol[Formula: see text] lower than that of [Formula: see text]-tocopherol. Our calculations show that [Formula: see text]-tocopherol binds to [Formula: see text]-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of [Formula: see text]-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to [Formula: see text]-tocopherol, with striking structural similarities to the wild-type-[Formula: see text]-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of [Formula: see text]-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover, the identification of a [Formula: see text]-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for [Formula: see text]-tocopherol selection in omnivorous animals.

SUBMITTER: Helbling RE

PROVIDER: S-EPMC3496730 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Engineering tocopherol selectivity in α-TTP: a combined in vitro/in silico study.

Helbling Rachel E RE Aeschimann Walter W Simona Fabio F Stocker Achim A Cascella Michele M

PloS one 20121113 11

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of [Formula: see text]-tocopherol transfer protein ([Formula: see text]-TTP) towards [Formula: see text]-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for [Formula: see text]-tocopherol to [Formula: see text]-TTP 8.26[Formula: see text]2.13 kcal mol[Formula: see text] lower than that of [Formula: see text]-tocopherol. Our ...[more]

PMID: 23152872

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Project description:Vitamin E acts as an antioxidant and reduces the level of reactive oxygen species (ROS) in Alzheimer's disease (AD). Alpha-tocopherol (ATF) is the most widely studied form of vitamin E besides gamma-tocopherol (GTF) which also shows beneficial effects in AD. The levels of amyloid-beta (A?) and amyloid precursor protein (APP) increased in the brains of AD patients, and mutations in the APP gene are known to enhance the production of A?. Mitochondrial function was shown to be affected by the increased level of A? and may induce cell death. Here, we aimed to compare the effects of ATF and GTF on their ability to reduce A? level, modulate mitochondrial function and reduce the apoptosis marker in SH-SY5Y cells stably transfected with the wild-type or mutant form of the APP gene. The A? level was measured by ELISA, the mitochondrial ROS and ATP level were quantified by fluorescence and luciferase assay respectively whereas the complex V enzyme activity was measured by spectrophotometry. The expressions of genes involved in the regulation of mitochondrial membrane permeability such as voltage dependent anion channel (VDAC1), adenine nucleotide translocase (ANT), and cyclophilin D (CYPD) were determined by quantitative real-time polymerase chain reaction (qRT-PCR), while the expressions of cyclophilin D (CypD), cytochrome c, Bcl2 associated X (BAX), B cell lymphoma-2 (Bcl-2), and pro-caspase-3 were determined by western blot. Our results showed that mitochondrial ROS level was elevated accompanied by decreased ATP level and complex V enzyme activity in SH-SY5Y cells expressing the mutant APP gene (p?<?0.05). Treatment with both ATF and GTF reduced the mitochondrial ROS level with maximum reduction was observed in the cells treated with high concentrations of ATF and GTF (p?<?0.05). However, only GTF at 80?µM significantly increase the ATP level and complex V enzyme activity (p?<?0.05). VDAC1 and CYPD were downregulated and CypD protein was significantly overexpressed in cells transfected with the wild-type (WT) and mutant APP gene (p?<?0.05). Cytochrome c release, the ratio of BAX/Bcl-2, and pro-caspase-3 expression increased in cells expressing mutated APP gene (p?<?0.05). The expression of CypD and pro-caspase 3 protein, and the ratio of BAX/Bcl-2 were increased in the following order; SH-SY5Y-APP-WT?<?SH-SY5Y-APP Swe <SH-SY5Y-APP Swe/Ind. Treatment with both ATF and GTF reduced the release of cytochrome c and the ratio of BAX/Bcl-2. However, only GTF significantly reduced the expression of CypD and pro-caspase-3, suggestive of its unique role in AD. In conclusion, GTF has an effect that was not shown by ATF and thus suggest its potential role in the development of therapeutic agents for AD.

Dataset Information

Engineering tocopherol selectivity in ?-TTP: a combined in vitro/in silico study.

Publications

Engineering tocopherol selectivity in α-TTP: a combined in vitro/in silico study.

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