Figure 1. TRIM11 prevents the formation of amorphous and fibrillar aggregates.
(A and B) Inactivation of luciferase (10 nM) at 45 °C in the presence of GST, GST-TRIM11, or Hsp70 at 200 nM for the indicated time (A), or at the indicated concentration for 2 min (B). Samples with Hsp70 also contained 5 mM ATP and an ATP regeneration system (same below).
(C and D) Aggregation of luciferase (200 nM, C) and citrate synthase (400 nM, D) at 42 °C and 40 °C, respectively, in presence of GST or GST-TRIM11 at indicated molar ratios.
(E to G) Luciferase was transfected into HCT116 cells that were treated with control or TRIM11 siRNA (E), or stably expressed mCherry or mCherry-TRIM11 (F and G). Cells were heat shock (HS) and recovered at 37 °C. Shown are luciferase activity and protein expression with recombinant GST-TRIM11 and Hsp70 as protein standards.
(H and I) ThT-binding (H, left), sedimentation (H, right), and electron microscopy (I; Scale bar, 100 nm) assays of fibrilization of α-Syn monomers (70 μM) in the absence or presence of GST or GST-TRIM11 at indicated concentrations (H) or at 2 μM (I).
(J) ThT-binding assay of α-Syn (70 μM) fibrillization in the absence or presence of GST (1 μM), GST-TRIM11 (0.5 μM), Hsp70 (0.5 μM), and/or Hsp40 (0.25 μM).
(K) RT-QuIC assay of PFFs (133 nM)-seeded α-Syn (13.3 μM) aggregation in the presence of GST-TRIM11 (0.94, 1.88, 3.75, 7.5, and 15 μM, respectively). Results are representatives of three independent experiments done in 4 technical repeats. Blank, buffer only. Negative control (Neg Con), monomeric α-Syn.
(L) Atxn1 82Q (0.1 μM) fibrillization in the presence of indicates amounts of 6xHis-TRIM11 for 24 h.
(M) Suppresses Atxn1 82Q aggregation by GST-TRIM11. The three parts of SR were from the same exposure of the same blot. Relative amounts of Atxn1 82Q in each fraction are shown.
(N) Binding of 6xHis-TRIM11 and Hsp70 with agarose beads conjugated with ATP via different moiety. Molecular Weight markers (in KDa) are shown.
Data are mean ± SD (n = 3 biological replicates unless otherwise indicated). *P < 0.05, **P < 0.01, ***P < 0.001.