Fig. 2. ABHD5 possesses intrinsic serine protease activity.

(a) Recombinant purified HDAC4 (rec-HDAC4) was incubated with increasing amounts of recombinant purified ABHD5 (rec-ABHD5) for 4 hrs at 37 °C. Proteolysis was analyzed by immunoblotting using antibodies directed against HDAC4 and by Coomassie staining, detecting absolute changes of full length HDAC4 (HDAC4-FL). rec-ABHD5 was detected by immunoblotting and Coomassie staining. The amounts of HDAC4-FL were determined in the Coomassie gel by densitometry. The ratio of HDAC4-FL in the presence of ABHD5 to HDAC4-FL in the absence of ABHD5 was subtracted from 1 and expressed as percent to calculate the proteolytic activity of ABHD5. (b) Similar assay as in ‘a’ was performed using rec-HDAC4 and rec-ABHD5 in the presence or absence of the serine protease inhibitor AEBSF. Proteolysis was analyzed by immunoblotting and Coomassie staining was used to quantify the proteolytic activity of ABHD5. (c) Similar assay as in ‘a’ and ‘b’ was performed using ABHD5 wild type (WT) and the ABHD5 mutants N153D (N/D), S298A (S/A) and H327A (H/A). Proteolysis was analyzed by immunoblotting, and Coomassie staining was used to quantify the proteolytic activity of ABHD5. (a-c) Statistical analysis: Values are presented as Mean±SEM, n=3 (n represents independent experiments); by one-way ANOVA, P<0.05 considered as significant. (d) ABHD5-WT, ABHD5-S298A and luciferase (Luc; control) were expressed with AAV9 vectors in cardiac-specific ABHD5 knockout mice (cKO) to replace endogenous ABHD5. HDAC4 and ABHD5 levels were analyzed by immunoblotting. Corresponding band intensities were quantified, and the ratio between HDAC4-NT and HDAC4-FL was determined as compared to Luc in cKO. Statistical analysis: Values are presented as Mean±SEM, n=3 (n represents different mice); by one-way ANOVA, P<0.05 considered as significant.