Fig. 4. ABHD11 loss impairs lipoylation of the OGDHc.

a OGDHc compromises 3 subunits: OGDH (E1) catalyses the oxidation of 2-OG to form the succinyl moiety, releasing carbon dioxide (CO₂) and reducing the lipoate to form a succinyl-dihydrolipoate intermediate. DLST (E2) catalyses transfer of the succinyl moiety to Coenzyme A (CoA), forming succinyl CoA and releasing dihydrolipoylated DLST. Dihydrolipoate is oxidised by DLD (E3) to reform lipoate, with the free electrons reducing NAD⁺ to NADH. Thus 2-OG oxidation is coupled to cyclical lipoate reduction/oxidation and NADH formation. b, c Immunoblot and quantification of OGDHc subunits and lipoylation in ABHD11 or LIAS deficient cells. HeLa cells were transduced with sgRNA targeting ABHD11 or LIAS to generate mixed KO populations, and probed for OGDHc components (b). Lipoate (Lp) antibody detects lipoylated proteins. The predominant lipoylated proteins, DLAT and DLST, are indicated. ImageJ quantification of lipoyl-DLST (left) and DLST (right), n = 5 biologically independent samples (c). Mean ± SEM, ***p = 0.0004, ns:p = 0.19, two-tailed one sample t test. Ct = control (d, e) Immunoblot OGDHc subunits and lipoylation in ABHD11 or LIAS deficient THP-1 or MCF-7 cells. THP-1 (d) or MCF-7 (e) cells were transduced with sgRNA targeting ABHD11 or LIAS to generate mixed KO populations, and probed for OGDHc components or lipoate. β-actin served as a loading control. f Reconstitution of mixed KO population of ABHD11 with exogenous ABHD11-GFP, or enzymatic inactive mutants. g ML226 treatment in vitro. Purified wildtype ABHD11-FLAG was incubated with p-nitrophenyl acetate and hydrolysis measured by rate of increase in absorbance at 405 nm (37 °C for 30 min), with addition of ML226 at the indicated concentration. Hydrolysis activity was subtracted from a background control without ABHD11-FLAG, and is normalised to the activity of ABHD11-FLAG with vehicle control. ϕ = vehicle control; n = 3. h, i ML266 treatment in HeLa cells (h) and C2C12 myoblasts (i). Cells were treated for 24 h with ML226 at the indicated concentrations and lipoylation measured by immunoblot. j HeLa cells were treated with 1μM ML226, or 0.075% DMSO as a vehicle (veh) control. ML226 was washed out after 24 h and lipoylation recovery measured by immunoblot.