Fig. 4: TANGO2 associates with CRYAB and regulates the protein-folding of desmin.

a Protein fragments of CRYAB that interact with TANGO2 identified from yeast two-hybrid screens of a human heart cDNA library. The common region found in different clones encoding CRYAB includes, at a minimum, amino acids 28-168. b Interaction of wild-type or mutant CRYAB and TANGO2 proteins in yeast two-hybrid assays. Interactions were assessed by survival on media lacking histidine and methionine (SC-L-T-M-H). The SMAD-SMURF interaction was used as a positive control. c AlphaFold model of the TANGO2 (green)—CRYAB (purple) interaction. d Confirmation of a direct interaction between recombinant proteins. SH3GL2 was used as a negative control. The results are representative of three independent experiments. e Protein binding affinity between TANGO2 and hemin (grey) or TANGO2 and CRYAB (pink) (n = 3 biological replicates). Values are means ± SD. f Immunoblots of CAL51 cells, heart and skeletal muscle lysates probed for CRYAB, desmin or vimentin. GAPDH was used as a loading control (n = 3 biological replicates). All values are means ± SD *p < 0.05, **p < 0.01 ***p < 0.001, Student’s two-tailed t test. g In vitro co-sedimentation assay of desmin, CRYAB and TANGO2. Filament assembly was initiated at 22 °C, 37 °C and 44 °C, and pellet (P) or aggregated fractions and supernatant (S) or soluble fractions analysed by SDS-PAGE and Coomassie Blue staining. Results are representative of three independent experiments. h Split-GFP protein-protein interaction assay for desmin and CRYAB variants (S59E, R120G and T170A) binding in control and TANGO2^–/– cells (n = 3 biological replicates). All values are means ± SD ***p < 0.001, ****p < 0.0001, Student’s two-tailed t test. (i) BioID identification of CRYAB client proteins in control and TANGO2^–/– cells (n = 3 biological replicates). Values are fold changes in abundance relative to adjusted p values, listed in Supplementary Data 2; significantly increased or decreased binding proteins are shown in red and blue, respectively; unique client proteins in each of the cell lines are shown to the left and right of the volcano plot; mitochondrial and cytoskeletal organisation proteins are highlighted in green and yellow, respectively. Source data are provided as a Source Data file.