Deacetylation of ACSS2 by SIRT2 under nutrient and amino acid stress.

(A) Overexpression of SIRT2 decreases ACSS2 acetylation. Flag-tagged ACSS2 was co-transfected with Flag-tagged SIRT2 into HEK 293T cells. Acetylation was determined using acetyl lysine IP and then western blot for Flag-ACSS2. Relative acetylation/protein ratios were quantified. Error bars represent ±SD for experiments performed in triplicate. (B) ACSS2 is deacetylated by SIRT2 under nutrient stress. Flag-tagged ACSS2 was ectopically expressed in control and SIRT2 knockdown HEK 293T cells. SIRT2 knockdown only increased ACSS2 acetylation under nutrient stress. Relative acetylation/protein ratios were quantified. Error bars represent ±SD for experiments performed in triplicate ** indicating p<0.01. (C) ACSS2 is deacetylated by SIRT2 under amino acid deprivation. Flag-tagged ACSS2 was ectopically expressed in control and SIRT2 knockdown HEK 293T cells. One set was grown in normal media and one set in EBSS media (no amino acids). Changes in ACSS2 acetylation was determined by acetyl lysine IP and western blot for Flag.

K271 acetylation shields ACSS2 from proteasomal degradation by impeding K271 ubiquitination.

(A and B) Endogenous ACSS2 levels increase when SIRT2 is knocked down under nutrient and amino acid stress. SIRT2 control and knock down HEK 293T cells were maintained in normal and nutrient deprived (A) or EBSS (B) media and endogenous ACSS2 protein levels were determined by western blot. (C) Knockdown of SIRT2 stabilizes ACSS2 in amino acid deprived media. SIRT2 control and knockdown HEK293T cells were maintained under EBSS media and treated with CHX at the indicated time points. Endogenous ACSS2 level was determined by western blot. Knockdown of SIRT2 is shown by qPCR. (D) ACSS2 is degraded by the proteasomal pathway. Proteasome inhibitor MG132, but not lysosome inhibitor bafilomycin (Baf), increases the endogenous level of ACSS2. (E) ACSS2 is ubiquitinated. Flag-tagged ACSS2 was transfected into HEK 293T cells. The cells were then treated with MG132. Ubiquitination of immunoprecipitated ACSS2 was determined using a pan-ubiquitin antibody. (F) Knockdown of SIRT2 decreases ACSS2 ubiquitination. Flag-ACSS2 was co-transfected with HA-tagged K48 ubiquitin into SIRT2 control and knockdown HEK 293T cells. Ubiquitination of purified Flag-ACSS2 was analyzed by western blot. (G) Inhibition of SIRT2 with TM decreases ACSS2 ubiquitination. Flag-tagged ACSS2 was transfected into HEK 293T cells with or without TM treatment. Ubiquitination of immunoprecipitated protein was detected using K48 ubiquitin antibody. (H) SIRT2 deacetylates ACSS2 at K271. Flag-tagged ACSS2 wildtype and K271R mutant were ectopically expressed in control and SIRT2 knockdown HEK 293T cells and the acetylation levels were detected by western blot. (I) Knockdown of SIRT2 does not change K271R ACSS2 ubiquitination. Flag-tagged WT and K271R ACSS2 were co-transfected with HA-tagged K48 ubiquitin into SIRT2 control and knockdown HEK 293T cells. Ubiquitination of purified proteins was analyzed by western blot. (J) ACSS2 K271R mutant is more stable than WT. Flag-tagged ACSS2 WT or K271R mutant were ectopically expressed in HEK 293T cells. Cells were treated with CHX to inhibit protein synthesis. The levels of the WT and K271R mutant ACSS2 at different time points were determined by western blot.

Acetylation of ACSS2 at K271 promotes lipid accumulation.

(A) Endogenous ACSS2 was stably knocked down with shRNA in 3T3L1 cells and then wildtype or K271R mutant of ACSS2 was re-expressed in the knockdown cells to a level that is compatible with endogenous ACSS2. ACSS2 knockdown efficiency and re-expression levels were determined by western blot. (B) The cell lysate at day 0 and 7 were collected and ACSS2 level was measured by western blot. (C, D) Inhibition of SIRT2 with TM results in increased neutral lipid accumulation in cells expressing ACSS2 WT but not in cells expressing ACSS2 K271R mutant. The differentiation media was supplemented with 1.5 mM acetate. Accumulation of neutral lipids in differentiated adipocytes was measured using Oil Red O. Neutral lipid accumulation is quantified (C) by measuring the absorbance of stained neutral lipids at 500 nm on a plate reader. Representative cell imaging is shown in (D). Statistical analysis was performed using an unpaired two-tailed Student’s t-test, with * indicating p<0.05 and ** indicating p<0.01.

Working model. Under amino acid stress, SIRT2 actively deacetylates ACSS2 at K271, which reveals the site for ubiquitination and leads to the degradation of ACSS2. This downregulation of ACSS2 protein level results in decreased de novo lipogenesis (DNL). On the contrary, without amino acid stress, acetylation at K271 protects ACSS2 from degradation resulting in higher DNL. Created with BioRender.com