Fig. 2: hsPEX19 variants suppress mHttex1 aggregation in vitro and in mammalian cells.
From: Engineering a membrane protein chaperone to ameliorate the proteotoxicity of mutant huntingtin
a Multiple sequence alignment of the α4 helix sequences of PEX19 across various species. The alignment was performed using the Clustal Omega and displayed with ESPript 3101,102. Conserved sequences of scPEX19-L288 and scPEX19-E292 are highlighted as green boxes. b NMR structure of hsPEX19-CTD (161–299 aa) (PDB 5LNF)43. Two conserved residues, M255 and Q259, are located in the α4 helix of hsPEX19 and are shown in magenta. The M255 residue of hsPEX19 is known to bind its C-terminally modified farnesyl group (cyan)43. c, d In vitro aggregation assay of Httex1-51Q in the absence and presence of hsPEX19 proteins. 3 μM of GST-TEV-Httex1-51Q-Stag and 1.5 μM (0.5×) or 3 μM (1×) of PEX19 proteins were incubated at 30 °C, and after the addition of TEV protease, samples were quenched at the indicated time points. SDS-insoluble Httex1-51Q aggregates in (c) and their replicates were quantified and shown in (d) (n = 3, mean ± SD). e ThioflavinT fluorescence assay to measure fibril formation of Httex1-51Q. The fluorescence intensity was measured every 15 min. Data are shown as mean ± SD with n = 3 (technical replicates). f Negatively stained transmission electron micrograph (TEM) images of Httex1-51Q in the absence and presence of hsPEX19 proteins. Scale bar: 500 nm g, h (g) Confocal microscopy images of HEK293T cells coexpressing Httex1-19Q-GFP or Httex1-134Q-GFP and hsPEX19. Empty vector control (denoted as vector control) was used as a negative control. Scale bar: 50 µm. h The percentage of cells containing 134Q aggregates was quantified. i (top) Representative image of the filter trap assays monitoring the SDS-insoluble Httex1-134Q-GFP aggregates in HEK293T cells upon coexpression of hsPEX19 proteins. (bottom) Quantification of the images and their replicates. Data in (h, i) are shown as mean ± SD, with three biological replicates (n = 3). Pairwise comparisons are shown as indicated, where **p < 0.01, ***p < 0.001, ****p < 0.0001 by the ordinary one-way ANOVA with Tukey post-hoc test. Source data are provided as a Source Data file.
