SIRT3-dependent GOT2 acetylation status affects the malate-aspartate NADH shuttle activity and pancreatic tumor growth

Abstract

The malate-aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate-aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD(+) redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth in vivo. Finally, we show that GOT2 K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate-aspartate NADH shuttle activity and oxidative protection.

Keywords: GOT2; acetylation; malate–aspartate NADH shuttle; pancreatic cancer.

Figure 1

Acetylation of GOT2 at K159, K185, and K404 enhances its protein interaction with MDH2

1. A Cartoon representation of the malate–aspartate shuttle. As shown, this shuttle is operated by two pairs of enzymes, cytosolic GOT1 and MDH1 as well as mitochondrial GOT2 and MDH2, which act in concert to transfer reducing equivalents across the mitochondrial membrane.

2. B,C GOT2 can be acetylated. Flag-tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag-bead-purified GOT2 were determined by Western blot analysis using a pan-anti-acetyllysine antibody (α-Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag.

3. D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag-GOT2 and Myc-MDH2 were co-overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag-bead-purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis.

4. E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag-GOT2 was co-overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag-GOT2 and its association with endogenous MDH2 were determined by Western blot analysis.

5. F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag-MDH2 was co-overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag-MDH2 and endogenous GOT2 was determined by Western blot analysis.

6. G Cartoon representation of GOT2 structure (PDB ID: 3PDB) (Han et al 2011) made by Pymol (www.pymol.org). Three putative lysine residues (i.e., K159, K185, and K404) were labeled in red. See also Supplementary Fig S3.

7. H Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K-to-R mutants were examined for their protein association with ectopically expressed Myc-MDH2 by Western blot analysis.

8. I GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag-GOT2 was overexpressed in HEK293T cells without or with co-overexpression of Myc-MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag-GOT2 from the Myc-MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag-GOT2 by Western blot analysis.

9. J The 3KQ/R mutations do not change GOT2 enzyme activity. Wild-type and 3K mutant GOT2 proteins were overexpressed and purified from E. coli, and the enzyme activity of GOT2 was determined as described in “Materials and Methods”. Shown are average values with standard deviation (SD) of triplicated experiments. n.s. = not significant for the indicated comparison.

Figure 2

Glucose and glutamine promote GOT2 acetylation and GOT2–MDH2 association

1. A,B Glucose and glutamine increase GOT2 K159 acetylation and GOT2–MDH2 association in HEK293T cells. Flag-GOT2 and Myc-MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc-MDH2 were determined by Western blot analysis.

2. C,D Glucose and glutamine increase GOT2 K159 acetylation in Panc-1 cells. Flag-GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flag protein levels.

3. E,F Quantification of the percentage of K159-acetylated endogenous GOT2 in Panc-1 cells. Recombinant fully K159-acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc-1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels.

4. G,H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc-1 cells with GOT2 knockdown and re-expression of wild-type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag-tagged wild-type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.

Figure 3

SIRT3 is the major deacetylase of GOT2

1. A GOT2 interacts with SIRT3, but not SIRT4 and SIRT5. Flag-tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA-tagged SIRT as indicated. Proteins were purified by IP with Flag beads, following Western blot to detect SIRTs with an HA antibody.

2. B Endogenous proteins of GOT2 and SIRT3 interact with each other. SIRT3 protein in HEK293T cells was purified by IP with an anti-SIRT3 antibody, following Western blot to detect GOT2 with an anti-GOT2 antibody.

3. C GOT2 deacetylation is dependent on SIRT3 catalytic activity. Flag-tagged GOT2 was ectopically expressed in HEK293T cells together with HA-tagged wild-type SIRT3 and a catalytically inactive mutant, SIRT3 H248Y. GOT2 proteins were purified by Flag beads, following Western blot to detect GOT2 K159 acetylation.

4. D SIRT3 impairs the protein interaction between GOT2 and MDH2. HEK293T cells were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and their protein interaction with Myc-MDH2 was determined by Western blot.

5. E Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2–MDH2 association. HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis.

6. F,G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation. Flag-tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown. These cells were treated with different concentrations of glucose (F) and glutamine (G) as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis.

7. H,I Up-regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting. C57BL/6 mice (male, 20 weeks; n = 2 per group) were fed a normal chow diet or fasted overnight. Upon sacrifice, mouse liver and white adipose tissues were harvested, and the levels of the indicated proteins were determined by Western blot analysis.

Figure 4

GOT2 acetylation regulates mitochondrial NADH/NAD⁺ redox, cellular ATP production, and NADPH homeostasis

1. A The malate–aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc-1 cells. Cells were treated with AOA (1.6 mM, 20 min), oxamate (5 mM, 20 min), and EGCG (0.5 mM, 20 min). The cytosolic NADH level was measured as described in Materials and Methods.

2. B,C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria. In Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins, the NADH level in the cytosol (B) and mitochondrion (C) was determined in cell extracts as described in Materials and Methods.

3. D GOT2 3K acetylation regulates mitochondrial NADH/NAD⁺ redox in the cell. In Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins, the ratio of NADH/NAD⁺ in the mitochondria of cells was measured as described in Materials and Methods.

4. E GOT2 3K acetylation promotes ATP production. In Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins, ATP production was determined in cell extracts as described in Materials and Methods.

5. F GOT2 3K acetylation promotes NADPH production. In Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins, the ratio of NADPH/NADP⁺ was determined in cell extracts as described in Materials and Methods.

6. G GOT2 3K acetylation promotes GSH production. In Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins, the GSH/GSSG ratio was determined in cell extracts as described in Materials and Methods.

7. H GOT2 3K acetylation suppresses cellular ROS levels. In Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins, ROS was determined as described in Materials and Methods in cells under non-stressed condition or exposed to menadione (50 μM for 30 min).

8. I GOT2 3K acetylation protects cells from oxidative damage. Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated, and cell viability was determined by counting the remaining adherent cells.

9. J,K Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins were seeded in a 6-well plate, and the cells were maintained under the condition of low glucose (0.5 mM, J) or high glucose (12 mM, K). Culture medium was refreshed every day, and cell numbers were counted every 1–2 days over a period of 7 days.

10. L,M Panc-1 stable cells in (J, K) were grown under low glucose (0.5 mM) or high glucose (12 mM) for 7 days. The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis (L). The percentages of living cells and apoptotic cells were determined by flow cytometry as described in Materials and Methods (M).

Data information: Shown are average values with standard deviation (SD) of triplicated experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 for the indicated comparison; n.s. = not significant.

Figure 5

GOT2 acetylation promotes tumor growth, and GOT2 K159 acetylation is increased in pancreatic cancer

1. A–C GOT2 3K acetylation promotes xenograft tumor growth. Panc-1 stable cells with GOT2 knockdown and re-expressing the indicated proteins (2 × 10⁶ cells) were injected subcutaneously into the flanks of nude mice. The tumor volume was carefully monitored over the indicated time period (A). At 8 weeks after injection, tumors from six mice were extracted, photographed, and weighted (B, C).

2. D GOT2 3K acetylation promotes tumor cell proliferation. Tumor sections from xenografts were prepared for IHC staining, and the PCNA-stained tumor sections were analyzed by quantifying the PCNA-positive area.

3. E Clinical cases with increased GOT2 K159 acetylation in human pancreatic tumor tissues. 10 pairs of human pancreatic tumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The acetylation level of GOT2 at K159 was compared against GOT2 protein by Western blot.

4. F Clinical cases with decreased SIRT3 protein expression in human pancreatic tumor tissues. 10 pairs of human pancreatic tumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The SIRT3 protein levels were compared against tubulin by Western blot.

5. G,H Quantification of GOT2 K159 acetylation and SIRT3 protein expression in clinical cases as described in (E) and (F).

Data information: Shown are average values with standard deviation (SD) of triplicated experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 for the indicated comparison; n.s. = not significant.

Figure 6

A working model illustrating regulation of the malate–aspartate shuttle by GOT2 acetylation Shown is a working model depicting how acetylation of GOT2 at 3K regulates the malate–aspartate shuttle and promotes pancreatic tumor proliferation. In pancreatic tumor cells, reduced SIRT3 expression leads to an increase of GOT2 acetylation. GOT2 acetylation at 3K enhances the protein interaction between GOT2 and MDH2, thereby stimulating the net transfer of cytosolic NADH into mitochondria to support ATP production. Meanwhile, GOT2 3K acetylation promotes cellular NADPH production to suppress ROS that are produced during rapid cell proliferation. Glc, glucose; Gln, glutamine; Asp, aspartate; and Mal, malate.