Ligase-specific recognition of alpha-synuclein filaments.

<p>(A) Cartoon of the modular architecture of Nedd4-type E3 ligases and overview of the constructs used in this study. (B) Western blots of <i>in vitro</i> ubiquitination assays comparing different Itch versions (full-length, ΔC2, WW mutants [175nM, left panel]) and full-length or HECT-only versions of Nedd4, Nedd4L and Smurf2 [100nM, right panel] in their ability to ubiquitinate alpha-synuclein [600nM, upper panels]. The lower panels (anti-ubiquitin blot) show overall ubiquitination, demonstrating the activity of the E3 ligase constructs by their ability to auto-ubiquitinate. Note that full-length Smurf2 is auto-inhibited in the presence of monomeric alpha-synuclein. (C) Activation of autoinhibited Smurf2 by alpha-synuclein filaments. (D) Western blots of <i>in vitro</i> ubiquitination assays comparing full-length, ΔC2 or HECT-only versions of Nedd4, Itch and Smurf2 [100nM] in their ability to ubiquitinate alpha-synuclein [400nM], indicating ligase-specific recognition of filaments (antibody Syn-1). Three sections of the same exposure of the same gel are shown.</p

Efficient <i>in vitro</i> ubiquitination of alpha-synuclein filaments by Nedd4.

<p>Ubiquitination assays of Nedd4 with alpha-synuclein (antibody Syn-1). (A) Coomassie-stained gels of purified wild-type (WT) and A53T mutant alpha-synuclein. (B) Time course of polymerization of WT and mutant alpha-synuclein assayed by thioflavin dye fluorescence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200763#sec007" target="_blank">Methods</a>). (C) Coomassie-stained gel of purified Nedd4 ligase. (D) <i>In vitro</i> ubiquitination assays using Nedd4 [200nM] with alpha-synuclein [200nM] showing efficient ubiquitination of filamentous (fil.) wild-type alpha-synuclein with strongly reduced modification of the filamentous A53T mutant and hardly any detectable ubiquitination of the monomeric forms (mo.). (E) Ubiquitination assays of Nedd4 [150nM] with alpha-synuclein filaments [600nM] in the presence of ubiquitin chain-type specific deubiquitinases as indicated [UbiCREST, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200763#pone.0200763.ref014" target="_blank">14</a>]] demonstrating mainly K63, but also some K29 and K33, linkages.</p

Clustering of alpha-synuclein does not result in efficient ubiquitination.

<p>(A) Schematic of the polymerization domains (DIX and TPR) fused to a soluble substrate, either the cytoplasmic domain of Ndfip2 containing three PY motifs or full-length alpha-synuclein with mutants that were used in this study. The location of the A53T mutation is indicated, but mutant protein was not used in Fig 4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200763#pone.0200763.g005" target="_blank">Fig 5A</a>). (B) <i>In vitro</i> ubiquitination assays using Smurf2 with purified recombinant proteins as indicated above the panels, showing efficient Smurf2 autoubiquitination when incubated with polymerization-competent DIX-TPR-Ndfip2 with intact PY motifs (D-T-N2), but not when all three PY motifs were mutated (D-T-PPAG). No Smurf2 autoubiquitination was observed with DIX-TPR-alpha-synuclein (D-T-Syn) and only modest effects were observed with an introduced weak PY motif (D-T-Syn PY1). (C) Western blots of His pull downs from HEK293T cells co-transfected with His-Ub, HA-tagged Nedd4, WWP2 and the GFP-tagged highly efficient polymerizing DIX-TPR fusions as indicated. N2-Pym, Ndfip2 with all three PY motifs mutated; N2-1xPY, Ndfip2 with one remaining LPxY motif. (D) Live-cell images showing single confocal sections of representative HeLa cells transfected with DIX-TPR-alpha-synuclein-EGFP either with polymerization defective M4 mutant (top left), wild-type alpha-synuclein (middle left) or introduced weak PY motif (bottom left panel) and co-transfected with mCherry-Nedd4 (center panels). Recruitment of Nedd4 into larger punctate aggregates was observed only in the presence of the weak PY motif (see arrows). Insets show enlarged versions of the outlined areas.</p

<i>In vitro</i> ubiquitination of alpha-synuclein by different Nedd4-type ligases.

<p>Western blots of <i>in vitro</i> ubiquitination assays using different members of the Nedd4 family [250nM] with alpha-synuclein [600nM] (antibody Syn-1). (A) Comparing the ability of the Nedd4-type E3 ligases to ubiquitinate monomeric (mo.) or filamentous (fil.) alpha-synuclein indicates a general preference for filaments. (B) Ubiquitination assays of different Nedd4 family members with filamentous alpha-synuclein with or without the ubiquitin chain-type specific deubiquitinase Trabid as indicated above the panel. In each case the ubiquitin chains were sensitive to Trabid, implying similar linkages.</p

Immunoblotting analysis of abnormal tau in the sarkosyl-insoluble fraction.

<p>(A) Immunoblotting analysis was visualized using AT8 antibody for the sarkosyl-insoluble fraction. The numbers indicate individual mice: 1–15, MB 1 mg/kg/day group; 16–30, MB 0.3 mg/kg/day group; and 31–44, water only group. Molecular weight markers are shown on the right (kDa). P, positive control (P301L tau transgenic mouse, 20 month-old female). (B) A comparison of relative AT8 expression levels of the MB-treated groups and the water only group. The data were compared with the AT8 band intensity. (C) A comparison of relative phosphorylated tau (AT8)/total tau (HT7) levels of the MB-treated groups and the water only group. The data were compared with the AT8 band intensity, which was normalized with the total tau (HT7) band intensity. The central lines indicate medians and the vertical lines represent 25^th and 75^th percentiles. P<0.01 was considered to represent a statistically significant difference. a.u., arbitrary unit. N.S., no significant difference.</p

Immunoblotting analysis of total tau in the Tris-soluble fraction.

<p>(A) Immunoblotting analysis was visualized using HT7 antibody for the Tris-soluble fraction. The numbers indicate individual mice: 1–15, MB 1 mg/kg/day group; 16–30, MB 0.3 mg/kg/day group; and 31–44, water only group. Molecular weight markers are shown on the right (kDa). For quantitative measure of band intensity, α-tubulin was used as an internal control for protein concentration. (B) A comparison of the relative total tau (HT7) expression levels of the MB-treated groups and the water only group. The data were compared with the HT7 band intensity, which was normalized with α-tubulin. The central lines indicate medians and the vertical lines represent 25^th and 75^th percentiles. a.u., arbitrary unit. N.S., no significant difference.</p

Immunoblotting analysis of total tau in the sarkosyl-insoluble fraction.

<p>(A) Immunoblot analysis was visualized using HT7 antibody for the sarkosyl-insoluble fraction. The numbers indicate individual mice: 1–15, MB 1 mg/kg/day group; 16–30, MB 0.3 mg/kg/day group; 31–44, water only group. Molecular weight markers are shown on the right (kDa). (B) A comparison of relative total tau (HT7) expression levels in the sarkosyl-insoluble fraction of the MB-treated groups and the water only group. The data were compared with the HT7 band intensity. The central lines indicate medians and the vertical lines represent 25^th and 75^th percentiles. a.u., arbitrary unit.</p

Immunohistochemical staining of abnormal tau.

<p>(A) An AT8 immunoreaction was observed only in spinal cord, medulla oblongata and pons of a mouse with a low AT8/HT7 ratio. (B) AT8-positive cells were seen in spinal cord, medulla oblongata, pons, midbrain, hypothalamus and cerebral cortex of a mouse with a high AT8/HT7 ratio. Each insert shows the cerebral cortex of the mouse brain.</p

Immunoblotting analysis of phosphorylated tau in the Tris-soluble fraction.

<p>(A) Immunoblot analysis was visualized using AT8 antibody for the Tris-soluble fraction. The numbers indicate individual mice: 1–15, MB 1 mg/kg/day group; 16–30, MB 0.3 mg/kg/day group; and 31–44, water only group. Molecular weight markers are shown on the right (kDa). P, positive control (P301L tau transgenic mouse, 20 month-old female). (B) A comparison of relative phosphorylated tau (AT8) expression levels of the MB-treated groups and the water only group. The data were compared with the AT8 band intensity, which was normalized with α-tubulin. (C) A comparison of the relative phosphorylated tau (AT8)/total tau (HT7) levels of the MB-treated groups and the water only group. The data were compared with the AT8 band intensity, which was normalized with the total tau (HT7) band intensity. The central lines indicate medians and the vertical lines represent 25^th and 75^th percentiles. a.u., arbitrary unit. N.S., no significant difference.</p

Immunohistochemical staining with a conformational antibody that recognizes aggregated tau.

<p>MC-1-positive neurons and cellular processes were seen in the motor cortex (A and B), prepotic area (C and D), posterior hypothalamus (E and F) and pons (G and H). A, C, E, G, mouse with a low AT8/HT7 ratio; and B, D, F, H, mouse with a high AT8/HT7 ratio. The calibration bar in H applies to all photomicrographs (50 µm).</p