Fig 2: TRIM32 stimulates ULK1 activity in an AMBRA1-dependent manner through unanchored K63-linked polyubiquitin.(A) Protein extracts from FLAG-TRIM32–transfected 293 T cells were subjected to immunoprecipitation using an anti-ULK1 antibody. Immunopurified complexes were analyzed by immunoblotting to detect ULK1 and K63-linked ubiquitin. Total extracts were also probed with an anti-FLAG antibody to verify TRIM32 transfection. (B) In vitro ULK1 ubiquitination assay using immunopurified Flag TRIM32 and recombinant ULK1, HA ubiquitin, E1 Ub–activating enzyme (UBE1), and E2 Ub–conjugating enzyme (UBE2N). Reactions were also performed in the absence of UBE1 to verify ubiquitination reaction specificity. At the end of the reaction, ULK1 was immunopurified and ubiquitination evaluated by immunoblotting using an anti-HA antibody (lanes 1 and 2). To verify whether ubiquitin was covalently linked, immunopurified ULK1 was boiled in 1% SDS after the ubiquitination reaction, reimmunoprecipitated (Re-IP), and analyzed by immunoblotting using anti-HA and anti-ULK1 antibodies (lanes 3 and 4). (C) shCtr or shAmbra1 293 T cells were transfected with a vector encoding FLAG-TRIM32 or an empty vector (-). Protein extracts were immunoprecipitated using an anti-ULK1 antibody. Immunopurified complexes were analyzed by immunoblotting to detect ULK1 and K63-linked ubiquitin. Total extracts were also probed with FLAG and AMBRA1 antibodies to verify TRIM32 transfection and AMBRA1 silencing, respectively. (D) 293 T cells were transfected with vectors encoding HA-tagged ubiquitin, FLAG-ULK1, and TRIM32, as indicated. Protein extracts were subjected to immunoprecipitation using an anti-FLAG antibody. Immunopurified complexes were analyzed by immunoblotting to detect K63-linked ubiquitin and ULK1 using specific antibodies. TRIM32 expression was also analyzed in total extracts. To verify whether ubiquitin was covalently linked, immunopurified FLAG-ULK1 was boiled in 1% SDS, reimmunoprecipitated, and analyzed by immunoblotting using anti–K63-linked ubiquitin and anti-ULK1 antibodies. (E) 293 T cells were transfected with vectors encoding FLAG-VPS34, alone or in combination with HA-TRIM32. TRIM32, VPS34, and phospho-VSP34 [pVPS34; Ser249 (S249)] were analyzed by immunoblotting. GAPDH was included as a loading control. (F) 293 T cells were transfected with vectors encoding FLAG–BECLIN 1, alone or in combination with HA-TRIM32. The levels of TRIM32, BECLIN 1, and phospho–BECLIN 1 [pBECLIN 1; Ser15 (S15)] were analyzed by immunoblotting. GAPDH was included as a loading control.

Fig 3: TRIM32 pathological mutants are unable to interact with ULK1 and promote autophagy.(A) 293 T cells were transfected with a vector encoding for MYC-ULK1, alone or in combination with HA-TRIM32 WT, or the indicated pathogenic TRIM32 mutants. Protein extracts were subjected to immunoprecipitation using an anti-HA antibody. Immunopurified complexes were analyzed by immunoblotting to detect the presence of TRIM32 and ULK1. (B) TRIM32 KO C2.7 cells were infected with retroviruses encoding WT FLAG-TRIM32 or the indicated pathogenic TRIM32 mutants. EMPTY, TRIM32 KO C2.7 cells infected with noncoding retroviruses. Upon 3 days in differentiation medium, cells were treated with dexamethasone for 4 hours and incubated with the lysosome inhibitors E64d and pepstatin A for 1 hour before lysis, as indicated. LC3-II and TRIM32 levels were analyzed by immunoblotting using specific antibodies. GAPDH was included as a loading control. (C) Fibroblasts from a WT donor and a patient with LGMD2H were transdifferentiated to myoblasts by MYOD (myogenic differentiation 1) ectopic expression. Cells differentiated for 3 days were treated, or not, with dexamethasone for 2 and 4 hours. One hour before lysis, cells were incubated with the lysosome inhibitors E64d and pepstatin A, as indicated. LC3-II and myosin heavy chain (MYOSIN HC) levels were analyzed by immunoblotting (bottom panel). GAPDH was included as a loading control.

Fig 4: TRIM32 binds to AMBRA1.(A) Protein extracts from MYC-AMBRA1– and FLAG-TRIM32–transfected 293 T cells were subjected to immunoprecipitation (IP) using an anti-FLAG antibody. Immunopurified complexes were analyzed by immunoblotting using anti-MYC and anti-TRIM32 antibodies. (B) Undifferentiated and differentiated C2.7 cells were lysed, and protein extracts were immunoprecipitated using an anti-TRIM32 antibody. An unrelated antibody was used as a negative control (IP Ctr). Immunopurified complexes were analyzed by immunoblotting using anti-AMBRA1 and anti-TRIM32 antibodies. (C) 293 T cells were cotransfected with vectors encoding HA-TRIM32 and the following MYC-AMBRA1 constructs: full length (FL), N-terminal (amino acids 1 to 532), central (amino acids 533 to 751), and C-terminal region (amino acids 767 to 1269). Protein extracts were immunoprecipitated using an anti-MYC antibody. Immunopurified complexes were analyzed by immunoblotting using anti-HA and anti-MYC antibodies. A scheme of the AMBRA1 domain architecture is shown (P-rich, proline-rich domain; S-rich, serine-rich domain; BH3, Bcl2 homology 3 domain). The red bar indicates the TRIM32-interacting domain. Asterisks indicate bands of AMBRA1 at the expected molecular weights. (D) TRIM32 KO 293 T cells were cotransfected with vectors encoding MYC-AMBRA1 and the following FLAG-TRIM32 constructs: full length, catalytic domain (RING/B-box, amino acids 1 to 136), central region containing the coiled-coil domain (amino acids 136 to 326), and NHL repeats (amino acid 327 to 653). Protein extracts were immunoprecipitated using anti-FLAG antibody. Immunopurified complexes were analyzed by immunoblotting using anti-FLAG and anti-MYC antibodies. A scheme of the TRIM32 domain architecture is shown (CC, coiled-coil domain). The red bar indicates the AMBRA1-interacting domain.

Fig 5: TRIM32 associates with ULK1 in an AMBRA1-dependent manner.(A) Protein extracts from 293 T cells transfected with vectors encoding HA-TRIM32, FLAG–BECLIN 1, and MYC-ULK1, as indicated, were subjected to immunoprecipitation using an anti-HA antibody. Immunopurified complexes were analyzed by immunoblotting using anti-FLAG, anti-MYC, and anti-HA antibodies. (B) TRIM32 KO 293 T cells were cotransfected with vectors encoding MYC-ULK1 and the following FLAG-TRIM32 constructs: full length, catalytic domain (RING/B-box, amino acids 1 to 133), central region containing the coiled-coil domain (amino acids 134 to 198), and NHL repeats (amino acids 199 to 325). Protein extracts were immunoprecipitated using an anti-FLAG antibody. Immunopurified complexes were analyzed by immunoblotting using anti-FLAG and anti-MYC antibodies. A scheme of the TRIM32 domain architecture is shown; the red bar indicates the ULK1-interacting domain. (C) Control shRNA and shAmbra1 293 T cells were transfected with a vector encoding MYC-ULK1, alone or in combination with FLAG-TRIM32. Cells were lysed, and protein extracts were subjected to immunoprecipitation using an anti-FLAG antibody. Immunopurified complexes were analyzed by immunoblotting using anti-MYC and anti-FLAG antibodies. Total extracts were also probed with an anti-AMBRA1 antibody to verify AMBRA1 silencing.