Journal: bioRxiv
Article Title: Tripeptidyl peptidase II is essential for maintaining cerebrovascular homeostasis of female mice and represents a novel therapeutic target for vascular dementia
doi: 10.64898/2026.05.05.722100
Figure Lengend Snippet: (A) Screening of various fluorescent choline analogs via a choline uptake assay revealed that the version with a Cy2 fluorophore conjugated to the hydroxyl group (Choline-O-Cy2) exhibits the highest uptake efficiency. Conversely, NBD fluorophore linked to the methyl group (Choline-CH3-NBD) completely abolishes choline incorporation, and Cy2 fluorophore attached to the carbon atom adjacent to the hydroxyl group (Choline-CO-Cy2) results in partial inhibition. (A1) Illustration of the molecular structures of fluorescently labeled cholines (left) and representative images of incorporated cholines (right). (A2) Statistical graphs of CTCF of the images. n = 6, ***p ≤ 0.001. Scale bar, 2.5 µm. T2KO cells were used as a control to ensure that measured choline uptake was TPP2-dependent and to rule out nonspecific background incorporation. (B) Ether phospholipids regulate choline incorporation strongly dependent on their ether-linked alkyl chain at sn-1 position and ester-linked acyl chain at sn-2 position. Either diacyl phospholipid (e.g. 16:0/18:0 PC) or dialkyl phospholipid (e.g. 16:0o/16:0o PC) prevents choline incorporation. Note that the polyunsaturated fatty acids (PUFAs) at sn-2 position may enhance choline incorporation (16:0/20:4 PC). (B1) Representative images of incorporated choline-O-Cy2 in WT and T2KO bEnd.3 cells with or without treatment with different phospholipids (PC-O, PC, or LPC). (B2) Statistical graphs of CTCF of the images. n = 12, ***p ≤ 0.001. Scale bar, 5 µm. (C) Dynamic analysis of choline uptake using live cell station revealed that TPP2 depletion drastically impairs this process ( bottom left ), which can be almost completely rescued by PC-O ( top right ) or E2 ( bottom left ) treatment. (D) TPP2 depletion significantly increases plasma membrane fluidity and metabolic dynamics and that PC-O (C18:0p/18:1 PC) treatment can strongly restore plasma membrane rigidity without altering incorporated ester-linked PC in plasma membrane. (D1) Transmission electron microscopic imaging revealed that the trilaminar or railroad track structure of the plasma membrane is interrupted in the absence of TPP2 and that PC-O treatment can significantly restore the trilaminar structure. The images below are enlarged versions of those above, presenting the regions of interest (ROI) in greater detail. Scale bar, 30 nm (upper), 10 nm (lower) . (D2) Steady-state fluorescence anisotropy using the gold-standard hydrophobic probe TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene) revealed that the surface fluidity of plasma membrane in the absence of TPP2 significantly increases and that PC-O treatment can almost completely restore the surface rigidity (upper). In addition, steady-state fluorescence anisotropy using the fluorescent PC analog BODIPY FL C5-HPC (1-Hexadecanoyl-2-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoyl)- sn -Glycero-3-Phosphocholine) revealed that the plasma membrane metabolic dynamics is more active in the absence of TPP2 compared to WT control and that PC-O treatment doesn’t significantly change incorporated PC analog in plasma membrane (lower). (E) Choline uptake is affected in cerebrovascular ECs specifically upon FLVCR2 depletion. (E1) RT-PCR analysis of choline transporter expression in primary ECs and bEnd.3 cells demonstrated that FLVCR2, CTL1, and CTL2 are all expressed. (E2) Choline uptake assay revealed that depletion of FLVCR2 specifically impairs choline uptake in bEnd.3 cells, whereas depletion of CTL1 had no effect. Choline uptake was not assessed for CTL2 transporter, as it is normally localized to the mitochondrial membrane rather than the plasma membrane. Representative results from three independent experiments are shown. Scale bar: 5 µm. (F) Molecular docking simulation revealed that the choline transporter FLVCR2 and PC-O (C18:0p/18:1 PC) exhibit high binding affinity through hydrogen-bond and Pi-alkyl interaction. The docking score reaches -11.125525 kcal/mol. (F1 ) The three-dimensional schematic depicts the deep view of the interaction between FLVCR2 and PC-O. Key residues are shown in a ball-and-stick model, the protein FLVCR2 in a colored cartoon representation, and the ligand PC-O in gray. Nitrogen and oxygen atoms are colored blue and red, respectively. (F2 ) The molecular interaction is shown in a two-dimensional diagram. In this conformation, the PC-O ligand mainly contacts FLVCR2 through hydrogen bonds and Pi-alkyl interaction. Specifically, FLVCR2 ASP147 forms two hydrogen bonds with the glycerol backbone of PC-O, and FLVCR2 PHE347 forms Pi-alkyl bond with the ether linked alkyl chain of PC-O. (G) Both FLVCR2 ASP147ALA (D147A) and FLVCR2 PHE347ALA (F347A) mutations resulted in complete loss of choline transport function. (G1) DNA sequencing results of FLVCR2 D147A and FLVCR2 F347A mutations . (G2) Choline uptake i n FLVCR2-depleted bEnd.3 cells was rescued by ectopic expression of FLVCR2 but not by ectopic expression of its D147A and F347A mutants. Representive images from more than 100 images taken are shown. Scale bar, 10 µm.
Article Snippet: Cell membrane fluidity TMA-DPH fluorescence detection kit (Bestbio, Shanghai, China) and BODIPY FL C5-HPC (MCE, Shanghai, China) were used according to manufacturer’s instruction.
Techniques: Inhibition, Labeling, Control, Clinical Proteomics, Membrane, Transmission Assay, Imaging, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Expressing, Binding Assay, DNA Sequencing
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