Figure 4. TRIM11 recognizes misfolded proteins and responds to heat shock, and other TRIMs can also prevent and reverse protein aggregation.
(A) Co-localization of GFP-Atxn1 82Q and HA-TRIM11 in HEK293T cells. Scale bar, 5 μm
(B and C) Interactions of Atxn1 82Q with endogenous TRIM11 or Hsp70 (B), or exogenous TRIM11, TRIM112EA, or Hsp70 (C), in HEK293T cells.
(D, E, and L) Preferential binding of TRIM11 (D and E) and TRIM21 (L) to Atxn1 82Q over Atxn1 30Q (D), or to denatured (d) over native (n) luciferase (E and L).
(F and G) Localization (F; scale bar, 10 pm) and interaction (G) of GFP-Atxn1 82Q and Flag-TRIM11 proteins in HEK293T cells.
(H and I) Increase in TRIM11 protein and mRNA levels in HCT116 cells (H) and mouse primary HC neurons (I) that were heat shocked at 42 °C for 1 (H) or 0.5 (I) hr and recovered at 37 °C for the indicated times (H) or 3 h (I).
(J) Activity of luciferase (10 nM) when heated at 45 °C for 1 min in the presence of GST or GST-TRIM21 (200 nM).
(K and M) Solubilization and reactivation of preformed luciferase (K, 10 nM) and GFP (M, 0.45 μM) aggregates by TRIM21 (K) and TRIM19 (isoform VI) for 90 and 60 min, respectively.
(N and O) A nucleus-localized luciferase was transfected alone in control or TRIM19-knockdown HCT116 cells (N), or together with control vector or TRIM19 in HCT116 cells (O). Luciferase activity during heat shock at 42 °C for 60 min (O) or 45 °C for 30 min (N) and recovery at 37 °C was assayed.
(P) Localization of GFP-Atxn1 82Q and mCherry-TRIM19 (isoform VI) in HeLa cells that were untreated or extracted with cytoskeleton stripping buffer.
Data are mean ± SD (n = 3 biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001.