Molecular model of the RAGE dimer.

<p>(<b>A</b>) Dimer constructed by molecular modeling of the VC1 3CJJ structure rotated by 180°. (<b>B</b>) Dimer from (<b>A</b>) with the C2 domain appended. Twenty snapshots from the last 20 ns of molecular dynamics are overlaid and two RAGE monomers marked with different colors. In (<b>A</b>), the C1-C2 linker residues become positioned in the swapped arrangement due to interactions with E-F loop of the C1 domain of the second molecule. When the C2 domain is appended in (<b>B</b>), the swapped arrangement of the linker and C2 domain is retained and the C2 domain accommodated well, supported with a set of favorable contacts between the linker and C2 domain residues with C1 domain loops E-F and C’-D. Orange bars mark the network of hydrogen bonds linking two molecules. See also Figure S4.</p

Molecular model of the RAGE tetramer.

<p>(<b>A</b>) Tetramerization scheme in which the zinc-stabilized dimer from ref[25]. (T-dimer) was rotated and docked, reconstructing a dimer of two C2 symmetry dimers (R-dimers). (<b>B</b>) A molecular model was constructed according this scheme and subjected to molecular dynamics. The last 20 snapshots are shown. The model shows good compatibility between two dimeric structures, retaining the network of hydrogen bonds in sheet 4 β-strands linking four molecules, marked by orange bars. The enforced hydrogen bonding in this region explains the stabilization of hydrogen bonds observed for sheet 4 peptides in HDex. (<b>C</b>) The tetramer exposes positively charged internal binding surfaces in the form of four VC1 domain arms capable of binding ligands of different sizes and symmetries, either rotational (as S100B tetramer) or translational (as Aβ oligomers).</p

Kinetics of HD exchange in four selected exRAGE peptides.

<p>(<b>A</b>) PRVWEPVPLEE, (<b>B</b>) SASEL, (<b>C</b>) VKEQTRRHPETGL, and (<b>D</b>) HLDGKPLVPNEKGVS fraction of exchanged proteins was measured for exRAGE peptides were measured three times after incubation in D₂O for 10 seconds, 60 seconds, and 1200 seconds and shown on a logarithmic timescale. Filled circles and lines represent data for monomeric RAGE, whereas filled rectangles and dashed lines represent data for dimRAGE. Significant differences were observed in the fraction of exchanged peptide amides at shorter incubation times in (<b>A</b>) and (<b>B</b>) only. Error bars are standard deviations calculated based on at least three experiments. See also Figure S2.</p

YexRAGE dimer formation <i>via</i> a dityrosine cross-link.

<p>(<b>A</b>) PAGE and (<b>B</b>, <b>C</b>) mass spectra of exRAGE (<b>A</b>, lanes 1-4, B, C) and YexRAGE (<b>A</b>, lanes 5-7) incubated with horseradish peroxidase in the presence of hydrogen peroxide for 15 or 30 minutes (lanes 2 and 6 and lanes 3 and 7, respectively). The oligomeric forms visible in lanes 6 and 7 with a mass >45 kDa correspond to YexRAGE dimers and trimers covalently linked by dityrosine and trityrosine bonds, respectively. Such oligomeric forms are absent if the substrate of the reaction is exRAGE instead of YexRAGE (lanes 2 and 3). Lanes 1 and 5 represent the sample before reaction, lane M is a protein ladder, and lane 4 shows the monomeric fraction after SEC. Gels were overloaded on purpose to show the lack of oligomeric forms in the reaction with exRAGE. (<b>B</b>) MALDI-ToF mass spectra of exRAGE before and (<b>C</b>) after HRP/H ₂O₂ incubation. (<b>D</b>) Fluorescence emission spectra in the range 350-550 nm obtained during incubation of YexRAGE in the presence of hydrogen peroxide and horseradish peroxidase for a specified period of time after excitation at 315 nm. The band with a maximum at 402 nm is expected for dityrosine species, whereas the band at 419 nm represents trityrosine or tetratyrosine species. In the course of the reaction, the fraction of species yielding a signal at 419 nm compared to a band at 402 nm increases, indicating facile formation of multityrosine species in YexRAGE. See also Scheme S1.</p

Serine-38 of Ana2 is phosphorylated in vitro by Plk4 as identified by mass-spectrometry. from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S2. Serine-38 of Ana2 is phosphorylated in vitro by Plk4 as identified by mass-spectrometry. Phospho-Mascot-interpreted fragmentation spectra of the peptide 13-LAPRP…EV (phospho-S-38) ILFG…SPR-65 with phosphorylated serine at the position 38 (highlighted red). Labelled peaks correspond to fragment ions. Despite the relatively long peptide, the fragmentation pattern unambiguously identifies the phosphorylation site

GST-Ana2-S38A can bind Sas6 after Plk4 phosphorylation from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S3: GST-Ana2-S38A can bind Sas6 after Plk4 phosphorylation. Sas6 specifically interacts with both Ana2-WT and Ana2-S38A when the proteins are pre-phosphorylated by Plk4. GST, GST-tagged wild-type (GST-Ana2-WT) or the S38A substitution mutant (GST-Ana2-S38A) were treated with either MBP-Plk4 or MBP-Plk4KD and incubated in vitro with 35S-Met-labeled Sas6 produced in a coupled in vitro transcription/translation reaction. The resulting complex was analyzed by SDS-PAGE and autoradiography

List of site-directed mutagenesis primers from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

List of site-directed mutagenesis primer

Plk4 phosphorylation induces a band-shift in Ana2 from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S1. Plk4 phosphorylation induces a shift in the electrophoretic mobility of Ana2. A. Incubation of Ana2 synthesized by coupled in vitro transcription and translation (IVTT) with active MBP-Plk4 in the presence of increasing ATP induces a distinct band-shift. B. The shift in mobility of Ana2-FLAG protein following its co-overexpression with active non-degradable Plk4 (Plk4ND) is abolished by treating the extract with λ-Phosphatase. C. Alanine substitutions in mass-spectrometry-identified phospho-sites with highest spectral count following in vitro phosphorylation, including those of the STAN motif, still retain the band-shift. D: Phosphorylation sites identified in Ana2 by mass-spectrometry. Serine or threonine residues identified following phosphorylation by MBP-Plk4 in vitro are highlighted in blue. Sites identified as phosphorylated in vivo on tagged-Ana2 (Protein A, FLAG, or GFP tags) purified from D.Mel-2 cells or early Drosophila embryos are highlighted in yellow. The STAN-motif is highlighted in grey. E. The phosphorylation-site responsible for the band shift is located in the N-terminal 280 amino acids of Ana2. Ana21-280, but not Ana2281-420 displays the band-shift in both the co-overexpression assay (upper) and the in vitro phosphorylation assay (lower). F. Band-shift assay using IVTT product of WT and non-phosphorylatable mutants of Ana2 for the sites highlighted in yellow within the N-terminal 280 amino-acid part. The band-shift is seen for each of these mutants

Expression levels in Ana2-Myc cell lines from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S4: Expression levels in Ana2-Myc cell lines: An immuno-blot showing comparable levels of expression between an Ana2-WT-Myc and an Ana2-S38A-Myc cell line. The same three cell lines are presented as in Fig. 6B: Untransfected D.Mel-2 cells (lane 1), pAct5-Ana2-WT-Myc (lane 2) and pAct5-Ana2-S38A-Myc (lane 3). Top panel: anti-Asl immuno-blot as a loading control. Bottom panel: anti-Ana2 immuno-blot, revealing endogenous Ana2 and overexpressed Ana2-Myc

Effect of β-escin on NFκB p50 and p65 activation.

<p>Abbreviations: C—untreated cells; TNF—TNF-α-treated cells; E– 3 μM-escin-treated cells. The results are expressed as the mean ±SEM of four independent experiments with HUVEC obtained from four donors. Data were analyzed by one-way analyses of variance (ANOVA) followed by the post hoc Tukey multiple range test. *p < 0.05; ***p < 0.001</p