Engineering of a functional γ-tocopherol transfer protein.

Area of Science:

• Biochemistry
• Structural Biology
• Cell Biology

Background:

• Alpha-tocopherol transfer protein (TTP) naturally forms 24-meric spheres (α-TTP_S) and exhibits transcytotic activity across endothelial cells.
• Understanding TTP's substrate specificity is crucial for elucidating tocopherol homeostasis.

Purpose of the Study:

• To characterize a functional TTP variant with altered selectivity towards γ-tocopherol.
• To investigate the structural and functional implications of the A156L mutation in TTP.

Main Methods:

• Site-directed mutagenesis to create the A156L TTP variant.
• X-ray crystallography to determine the structure of the γ-tocopherol specific particle (γ-TTP_S).
• In vitro assays including micro-differential scanning calorimetry and ligand-transfer activity measurements.
• Trans-well and cell culture experiments using human umbilical vein endothelial cells (HUVECs).

Main Results:

• The A156L mutation successfully shifted TTP's selectivity towards γ-tocopherol, yielding γ-TTP_S.
• γ-TTP_S maintained self-aggregation and transcytotic activity, similar to α-TTP_S.
• In contrast to α-TTP, γ-TTP protomers did not counteract cytokine-mediated inflammation transcriptionally.

Conclusions:

• The A156L substitution results in a fully functional γ-tocopherol-specific TTP variant.
• This engineered TTP provides a novel tool for exploring tocopherol homeostasis and potential therapeutic applications.