Fig 1: HSD17B10 acetylation affects its enzymatic activity and the formation of mt-tRNA splicing complex.a Flag-tagged HSD17B10 was transfected into HEK293T cells, and cells were treated with or without 10 mM NAM for 6 h for deacetylated and hyperacetylated HSD17B10 purification. The L-3-hydroxyacyl-CoA dehydrogenase activity assay of HSD17B10 was conducted with acetoacetyl-CoA as the substrate and NADH as the cofactor, and changes of the absorbance at 340 nm were measured to show the decrease of NADH. kcat/KM was calculated to represent catalytic efficiency. Data were shown as mean ± S.D. (n = 3). b Flag-tagged HSD17B10 WT and KR mutants were individually transfected, and cells were treated with 10 mM NAM for 6 h. The L-3-hydroxyacyl-CoA dehydrogenase activity of HSD17B10 was measured as previously described. Data were shown as mean ± S.D. (n = 3). c Flag-tagged HSD17B10 WT and KR mutants were co-transfected with HA-tagged TRMT10C into HEK293T cells followed by immunoprecipitation with M2 beads. d Flag-tagged HSD17B10 WT and 3KR were transfected into HEK293T cells, and cells were treated with 10 mM NAM for 6 h. Relative HSD17B10 acetylation ratios were calculated after normalizing against Flag. e Flag-tagged HSD17B10 WT, 3KR and 3KQ were individually transfected. The L-3-hydroxyacyl-CoA dehydrogenase activity of HSD17B10 was measured as previously described. Data were shown as mean ± S.D. (n = 3). f Flag-tagged HSD17B10 WT and 3KR were co-transfected with HA-tagged TRMT10C into HEK293T cells followed by immunoprecipitation with M2 beads. g Flag-tagged HSD17B10 WT and 3KQ were co-transfected with HA-tagged TRMT10C into HEK293T cells followed by immunoprecipitation with M2 beads.
Fig 2: HSD17B10 acetylation affects cell growth and cell resistance under stresses.a 5 × 104 HSD17B10 knockdown HCT116 re-expressed with vector, Flag-tagged HSD17B10 WT, 3KR and 3KQ cells were seeded into six-well plates. Cell number was counted every 24 h. Data were shown as mean ± S.D. (n = 3). b 5 × 103 HSD17B10 knockdown HCT116 re-expressed with vector, Flag-tagged HSD17B10 WT, 3KR and 3KQ cells were seeded into 96-well plates. Cell growth was measured by CCK-8 assay after 48 h. Data were shown as mean ± S.D. (n = 3). c 5 × 103 HSD17B10 knockdown HCT116 re-expressed with vector, Flag-tagged HSD17B10 WT, 3KR and 3KQ cells were seeded into 6 cm plates. Colony formation was shown after crystal violet staining. d Flag-tagged HSD17B10 was transfected into HCT116 cells, and cells were treated with different stresses for the indicated time. HSD17B10 was IPed with M2 beads. e 2 × 105 HSD17B10 knockdown HCT116 re-expressed with vector, Flag-tagged HSD17B10 WT, 3KR and 3KQ cells were seeded into six-well plates. After 24 h, cells were treated with different stresses for the indicated time, and the number of living cells was counted. Data were shown as mean ± S.D. (n = 3).
Fig 3: Identify the main acetylation sites of HSD17B10.a Flag-tagged HSD17B10 was co-transfected with HA-tagged CBP, and cells were treated with 10 mM NAM for 6 h. Whole-cell lysate was collected, IPed with M2 beads and eluted by Flag peptide. After SDS-PAGE gel separating and Coomassie blue staining, the specific band for HSD17B10 was analyzed by mass spectrometry. b HSD17B10 GST N-terminal (NT), middle (M), C-terminus (CT) and full-length (FL) plasmids were constructed, and in vitro acetylation assay was conducted. GST protein was used as a negative control. c HSD17B10 amino acid sequence conservation was analyzed via sequence analyzing software MEGA. d HSD17B10 KR mutant (mimicking the deacetylation status) plasmids were constructed. Flag-tagged HSD17B10 WT and KR mutants were individually transfected and cells were treated with 10 mM NAM for 6 h. Relative HSD17B10 acetylation ratios were calculated after normalizing against Flag.
Fig 4: HSD17B10 acetylation regulates the intracellular functions.a Vector, Flag-tagged HSD17B10 WT, 3KR, 3KQ and K172A were individually transfected into HSD17B10 knockdown HCT116 cells. Endogenous and re-expressed HSD17B10 were detected with HSD17B10 antibody. b The absorbance at 450 nm was measure to quantify NADH and NADtotal in HSD17B10 knockdown HCT116 re-expressed with vector, Flag-tagged HSD17B10 WT, 3KR and 3KQ cells. NAD+/NADH = (NADtotal − NADH)/NADH. Data were shown as mean ± S.D. (n = 3). K172A was used as an enzymatic negative control. c The amounts of representative mt pre- and total tRNAs in HSD17B10 knockdown HCT116 re-expressed with vector, Flag-tagged HSD17B10 WT, 3KR and 3KQ cells were confirmed by qPCR. Data were shown as mean ± S.D. (n = 3). d HSD17B10 knockdown HCT116 re-expressed with vector, Flag-tagged HSD17B10 WT, 3KR, and 3KQ cells were stained with TMRM (10 nM) and Mitotracker (200 nM) for 0.5 h.
Fig 5: HSD17B10 interacts with SIRT3.a Flag-tagged HSD17B10 and HA-tagged SIRT3 were transfected individually or together into HEK293T cells followed by immunoprecipitation with M2 beads. b Flag-tagged SIRT3 and HA-tagged HSD17B10 were transfected individually or together into HEK293T cells followed by immunoprecipitation with M2 beads. c Endogenous SIRT3 was IPed from HCT116 cells. Rabbit IgG was used as a control. d Endogenous HSD17B10 was IPed from HCT116 cells. Mouse IgG was used as a control. e Flag-tagged HSD17B10 was transfected into HEK293T cells followed by immunoprecipitation with M2 beads. Purified Flag-tagged HSD17B10 was incubated with GST/GST-SIRT3 and was IPed with GST-agarose beads. f Flag-tagged SIRT3 was transfected into HEK293T cells followed by immunoprecipitation with M2 beads. Purified Flag-tagged SIRT3 was incubated with GST/GST-HSD17B10 and was IPed with GST-agarose beads.
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