Extended Data Fig. 2: ABHD5 possesses intrinsic serine-protease activity.
From: The lipid-droplet-associated protein ABHD5 protects the heart through proteolysis of HDAC4
a, Prediction of 3D structure of ABHD5 using SWISS-MODEL. Highlighted are aa predicted to serve as putative catalytic triad (Asn153, Ser298 and His327) and the putative phosphorylation site Ser-237. Ser-237 and Ser-298 of ABHD5 lie in spatial proximity to each other. Lys 51 represents the N-terminal starting aa of the peptide sequence used for modelling. Pink, cap domain; green, α/β-hydrolase core. b, Multiple sequence alignment of ABHD5 protein of different species as indicated by UniProt online tool (https://uniprot.org/align/). Red boxes denote aa N153, S298 and H327 of the predicted serine-protease catalytic triad, green box denotes aa S237 as reported PKA phosphorylation site (aa numbering corresponds to human ABHD5 protein). c, Tryptophan fluorescence decay plot upon thermal denaturation of rec-ABHD5-WT, -N153D, -S237A, -S237E -S298A and -H327A (1 °C min–1). Left, The ratio of fluorescence at 350 nm/330 nm was plotted against the temperature. Right, Equivalent analysis to identify the melting temperature (Tm) of the rec-proteins. Measurements were performed once. d, Coomassie staining after protease assay using recombinant purified HDAC4 (rec-HDAC4) and increasing amounts of recombinant purified ABHD5 (rec-ABHD5) as indicated. e, Coomassie staining after protease assay using rec-HDAC4 and rec-ABHD5 in the presence or absence of serine-protease-inhibitor AEBSF as indicated. f, Coomassie staining after protease assay using rec-HDAC4 and rec-ABHD5 WT or putative catalytic-triad mutants as indicated. The data in d–f represent results of three independent experiments. g, Immunoblotting and Coomassie staining for HDAC4 and ABHD5 after in vitro proteolysis assays involving rec-HDAC4, rec-ABHD5 WT or Ser-237 phospho-death (S237A) and phospho-mimetic (S237E) mutants as indicated. Coomassie staining was used to quantify the proteolytic activity of ABHD5. Statistical analysis: Values are presented as mean ± s.e.m., n = 3 (n represents independent experiments); by one-way ANOVA, P < 0.05 considered as significant. h, Immunoblotting for HDAC4 and ABHD5 in yeast-cell extracts after proteolysis assay in a yeast expression system as indicated. Because HDAC4 contains the Protein-A (PRA; 15 kDa) tag, HDAC4-NT is expected to migrate at 43 kDa. The experiment was performed at least three times with similar results.
