Fig 2: A schematic diagram of proposed crosstalk among acetylation, phosphorylation, and ubiquitination events in dynamic regulation of MCL1 protein stabilityMCL1 is an unstable protein that is targeted for ubiquitination. p300-directed MCL1 acetylation at K40 leads to enhanced interaction with USP9X, facilitating MCL1 deubiquitination and stabilization. SIRT3 is the potential deacetylase counteracting the acetylation-dependent MCL1 stabilization. Phosphorylation also plays important roles in regulating the interaction with USP9X and certain E3s. Elevated MCL1 acetylation status may correlate with sensitivity to USP9X and p300 inhibitors.

Fig 3: p300 binds and acetylates MCL1 at K40(A) Endogenous MCL1 is targeted by acetylation in HeLa cells. Shown is immunoblot (IB) analysis of whole-cell lysates (WCLs) and anti-MCL1 immunoprecipitates derived from HeLa cells.(B) p300 promotes MCL1 acetylation in cells. Shown is IB analysis of WCLs and anti-Myc tag immunoprecipitates derived from 293T cells transfected with Myc-MCL1 along with the indicated hemagglutinin (HA)-tagged lysine acetyltransferase constructs.(C and D) Endogenous interaction between MCL1 and p300. Shown is IB analysis of WCLs (input) and anti-p300 immunoprecipitates (C) or anti-MCL1 immunoprecipitates (D) derived from HeLa cells.(E) Knockdown of p300 reduces MCL1 acetylation. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells stably expressing the lentiviral short hairpin RNA (shRNA) specific for GFP or p300. Cells were treated with MG132 (10 µM) for 6 h before harvesting to stabilize and accumulate MCL1 protein.(F) Treatment with the p300/CBP inhibitor A-485 reduces MCL1 acetylation. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells. Cells were co-treated overnight with A-485 at the indicated concentration and MG132 (5 µM) before harvesting.(G) Acetylation-deficient K40R markedly diminishes p300-mediated MCL1 acetylation. Shown is IB analysis of WCLs and anti-Myc tag immunoprecipitates derived from 293T cells transfected with the indicated Myc-MCL1 constructs.(H) K40 of endogenous MCL1 is targeted by acetylation in SKBR3 breast cancer cells. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from SKBR3 cells.(I) MCL1 and p300 are present together in cytoplasmic and nuclear compartments. Shown is IB analysis of WCLs and cytoplasmic, mitochondrial, and nuclear fractions derived from BT-20 cells. Data are representative of at least two independent experiments. See also Figure S1.

Fig 4: SIRT3 negatively regulates MCL1 stability through deacetylation(A) SIRT1, SIRT2, and SIRT3 efficiently deacetylate MCL1 in vitro. Shown is IB analysis of MCL1 acetylation after the in vitro deacetylation reaction and WCLs derived from HeLa cells transfected with the indicated Myc-MCL1 and FLAG-SIRT constructs for MCL1 and SIRT protein purification by FLAG and Myc immunoprecipitates (STAR Methods).(B) MCL1 interacts with SIRT3 at the endogenous level. Shown is IB analysis of WCLs (input) and anti-MCL1 immunoprecipitates derived from 293T cells.(C) SIRT3 depletion results in the accumulation of MCL1 protein abundance. Shown is IB analysis of WCLs derived from HeLa cells stably expressing the lentiviral shRNA specific for GFP, SIRT1, SIRT2, or SIRT3.(D) SIRT3 depletion extends MCL1 protein half-life. HeLa cells stably expressing the lentiviral shRNA specific for GFP or SIRT3 presented in (C) were treated with 100 µg/mL CHX for the indicated period before harvesting.(E) SIRT3 depletion results in impairment of MCL1 poly-ubiquitination. HeLa cells stably expressing the lentiviral shRNA specific for GFP, SIRT1, SIRT2, or SIRT3 were transfected with Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated with MG132 (10 µM) overnight before harvesting.(F) SIRT3 depletion accumulates endogenous Ac-K40-MCL1. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells stably expressing the indicated lentiviral shRNA specific for GFP or SIRT3.(G) Ectopic SIRT3 expression increases ubiquitination of WT MCL1 but not K40R. 293T cells were transfected with Myc-MCL1, FLAG-SIRT3, and His-Ub constructs as indicated. 36 h after transfection, the cells were treated with MG132 (20 µM) for 5 h before harvesting. Left: His-Ub-conjugated proteins were captured with Ni-NTA agarose beads. Right: a schematic model of SIRT3-mediated MCL1 ubiquitination through K40 deacetylation followed by USP9X dissociation.(H and I) SIRT3 depletion confers resistance to doxorubicin-induced apoptosis through MCL1 stabilization. CRISPR-Cas9-mediated MCL1 KO and its parental HeLa cells were infected with the lentiviral shRNA specific for GFP or SIRT3. These cells were treated with the indicated concentrations of doxorubicin for 24 h and then subjected to IB analysis (H) and a cell viability assay (I). Data are presented as mean ± SD; n = 3 biological replicates; **p < 0.01, ***p < 0.001.Data in (A)–(H) are representative of at least two independent experiments. See also Figure S5.

Fig 5: MCL1 acetylation promotes interaction with USP9X, resulting in MCL1 deubiquitination and stabilization(A) Acetylation-mimetic MCL1 K40Q enhances the interaction between MCL1 and USP9X. Shown is IB analysis of WCLs and anti-FLAG immunoprecipitates derived from 293T cells transfected with the indicated constructs.(B) USP9X depletion reverses the ubiquitination levels of acetylation-mimetic MCL1 K40Q. HeLa cells stably expressing the indicated lentiviral shRNA were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated overnight with MG132 (10 µM) before harvesting.(C) Treatment with the USP9X inhibitor reverses ubiquitination of acetylation-mimetic MCL1 K40Q. HeLa cells were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, the cells were treated with MG132 (10 µM) in the presence or absence of WP1130 (5 µM) for 4 h before harvesting. Left: His-Ub-conjugated proteins were captured with Ni-NTA agarose beads. Right: quantification of the Myc-poly-ubiquitination band intensities of IB replicates. Data are presented as mean ± SD, n = 3 independent experiments, *p < 0.05, **p < 0.01.(D) USP9X depletion abolishes stabilization of MCL1 K40Q. HeLa cells stably expressing the lentiviral shRNA specific for GFP or USP9X were transfected with the indicated Myc-MCL1 constructs. 36 h after transfection, cells were treated with 100 µg/mL CHX for the indicated periods before harvesting.(E) Quantification of the Myc band intensities of IB replicates in (D). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01.(F and G) Treatment with the USP9X inhibitor WP1130 efficiently downregulates acetylation-mimetic MCL1 K40Q. Shown is IB analysis of WCLs derived from the MCL1-reintroduced CRISPR-Cas9-mediated MCL1 knockout (KO) HeLa (F) and HCT116 (G) cells presented in Figure 3. These cells were treated with WP1130 (10 µM) for the indicated periods before harvesting.(H and I) Treatment with the USP9X inhibitor WP1130 abrogates the anti-apoptotic effect of acetylation-mimetic MCL1 K40Q. These cells were treated with the indicated concentrations of WP1130 for 24 h and then subjected to cell viability assays. Data are presented as mean ± SD; n = 3 biological replicates.Data in (A), (B), (F), and (G) are representative of at least two independent experiments. See also Figure S4.