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. 2014 Nov:76:163-172.
doi: 10.1016/j.freeradbiomed.2014.08.001. Epub 2014 Aug 22.

SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells

Ozkan Ozden #  1 , Seong-Hoon Park #  1 , Brett A Wagner  2 , Ha Yong Song  1 , Yueming Zhu  1 , Athanassios Vassilopoulos  1 , Barbara Jung  3 , Garry R Buettner  2 , David Gius  1
Affiliations

SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells

Ozkan Ozden et al. Free Radic Biol Med. 2014 Nov.

Abstract

Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321), and a PDHA1 mutant mimicking a deacetylated lysine (PDHA1(K321R)) increases PDH activity, compared to the K321 acetylation mimic (PDHA1(K321Q)) or wild-type PDHA1. Finally, PDHA1(K321Q) exhibited a more transformed in vitro cellular phenotype compared to PDHA1(K321R). These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration.

Keywords: Acetylation; Carcinogenesis; Free radicals; PDHA1; Pyruvate dehydrogenase; SIRT3; Warburg.

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Figures

Figure 1
Figure 1. SIRT3 deacetylates PDHA1 and increases the PDH activity in vitro
(a) Sirt3+/+ and Sirt3−/− liver extracts (600 μg of protein) were IPed with anti-PDHA1 antibody, separated, and subsequently immunoblotted with an anti-pan acetyl-lysine and PDHA1 antibody. (b) Sirt3+/+ and Sirt3−/− MEFs extracts were cultured in high or no glucose and extracts were used to determine PDH activity using a PDH activity Microplate assay kit (Abcam, Inc). (c) The brains from the Sirt3+/+ and Sirt3−/− mice, on an ad libitum diet or after fasting (36 h), were harvested and PDH activities were determined as above. (d) Sirt3+/+ and Sirt3−/− mouse heart, liver, and kidney were isolated and extracts were used to determine PDH activity as above. PDH activities for all data panels are presented as relative changes compared to control cells or tissues. Error bars represent one standard deviation from the mean (*p<0.05 and **p<0.001).
Figure 2
Figure 2. Overexpression of SIRT3 increases PDH activity in vitro
(a-c) HCT116 cells were infected with Myc-Sirt3WT or shRNA and after 36 h cells were harvested and measured for (a) PDH activity, (b) lactate production, and (c) glucose uptake. (d-e) HeLa and MCF7 cells were infected with either a control lentivirus or lenti-shSIRT3, or lenti-SIRT3WT and after 48 h, cells were harvested and measured for PDH activity. (f-g) HeLa and MCF7 cells were infected with either a control lentivirus or lenti-shSIRT3, or lenti-SIRT3WT and after 48 h; cells were harvested and measured for lactate production. (h) Sirt3−/− MMT cells were infected with either lenti-SIRT3DN or lenti-SIRT3WT and after 48 h cells were harvested and measured for PDH activity. For all these experiments the change in PDH activity, lactate production, and glucose uptake are presented as relative changes compared to control cells or tissues. Error bars represent one standard deviation from the mean (*p<0.05 versus the control group).
Figure 3
Figure 3. SIRT3 physically interacts and co-localizes with PDHA1
(a-b) 293T cells were transfected with (a) Flag-PDHA1 or (b) Flag-SIRT3 and 48 h after transfection cell extracts were IPed with an anti-Flag antibody and subsequently immunoblotted with anti-SIRT3 and anti-PDHA1 antibodies. Input immunoblotted is shown as a control. (c-d) 293T cells here harvested and endogenous (c) PDHA1 or (d) SIRT3 were IPed, separated, and immunoblotted with an anti-SIRT3 and anti-PDHA1 antibody. (e) H1299 lung cancer cells were stained with anti-PDHA1 (red staining), anti-SIRT3 (green staining), or DAPI (purple staining) and subsequently merged. Scale bar, 10Pm. (f) HCT116 cells were infected with Myc-Sirt3WT or shRNA and after 36 h extracts were IPed with and anti-pan acetyl-lysine antibody and immunoblotted with anti-PDHA1. (g) Purified acetylated Flag-PDHA1 was mixed with recombinant SIRT3, without or with NAD+, and samples were immunoblotted with anti-pan acetyl-lysine and anti-PDHA1 antibodies. (h) PDHA1 was co-transfected with acetyl-transferases (Tip60 and CBP) as well as HA SIRT3WT or a deacetylation null mutant (HA-SIRT3DN) gene and extracts were IPed with an anti-Flag antibody. Samples were immunoblotted with anti-pan acetyl-lysine and anti-Flag antibodies.
Figure 4
Figure 4. Acetylation of PDHA1 lysine 321 directs PDH enzymatic activity
(a) Lysine 321 of PDHA1 is phylogenetically conserved in various species from human to the C. elegans. The amino acid sequence of PDHA1 between around 301 and 350 was compared among Homo sapiens (human), Pan troglodytes (chimpanzee), Mus musculus (house mouse), Xenopus laevis (African clawed frog), and Caenorhabditis elegans (roundworm). K321 in human PDHA1 sequence was conserved in all species investigated. In Xenopus and C. elegans, the sequence frame is slightly shifted. In Xenopus, K321 corresponds to K331; in C. elegans it corresponds to K315. (b) 293T cells were transfected with Flag-PDHA1 as well as PDHA1 lysine mutants (Flag-PDHA1K83R, Flag-PDHA1K321R, and Flag-PDHA1K336R) with Tip60 and CBP and TSA / NAM, IPed with an anti-Flag antibody, and subsequently immunoblotted with an anti-pan acetyl-lysine and anti-PDHA1 antibody. (c-i) 293T cells expressing shPDHA1 to knockdown endogenous PDHA1 were subsequently transfected with Flag-PDHA1K321K, Flag-PDHA1K321Q, or Flag-PDHA1K321R. Cell extracts were used to determine (c) PDH activity, (d) lactate production, (e) O2 consumption rates (pmol/min) per 106 cells were measured using the Seahorse Bioscience instrumentation and corrected for antimycin and rotenone. For 321K cells the basal OCR | 52 amol cell−1 s−1, (f) glucose uptake, and (g-h) relative oxidant production as determined by the fluorescence of MitoSOX E+ (g) relative oxidant production which was determined as in (g) was normalized by subtracting oxidant production from parallel samples grown MnSOD and mutant PDHA1 overexpressing cells (h). (i-j) For quantitative analysis of oxidation products derived from MitoSOX™ Red, HPLC electrochemical analysis was performed. For all these experiments the change in PDH activity, lactate production, glucose uptake, and oxidant production are presented as relative changes compared to control cells or tissues. Error bars represent one standard deviation from the mean (*p<0.05 versus the control group).
Figure 5
Figure 5. Acetylation of PDHA1 K321 alters the in vitro transformative properties of PDH
MCF7 cells expressing shPDHA1 to knockdown endogenous PDHA1 were subsequently transfected with Flag-PDHA1K321K, Flag-PDHA1K321Q, or Flag-PDHA1K321R. (a) Relative PDH activity was measured. (b) Relative lactate secreted into the media was measured. (c) O2 consumption rates (pmol/min) per 106 cells were measured using the Seahorse instrument and corrected for antimycin and rotenone. For 321K cells the basal OCR ≈ 20amol cell−1 s−1 consistent with previous published OCR for MCF7 cells, 30 amol cell−1 s−1 [27]. (d) Cell extracts were labeled with MitoSOX™ Red. Oxidant production was determined using a flow cytometer as determined by the fluorescence of MitoSOX E+. (e-f) MCF7 cells expressing shPDHA1 were infected with Flag-PDHA1K321K, Flag-PDHA1K321Q, or Flag-PDHA1K321R and the total number of cells was counted at days 1, 3, and 6 (e). Doubling times of these cells were determined (f). (g) MCF7 cells expressing shPDHA1 were infected with Flag-PDHA1K321K, Flag-PDHA1K321Q, or Flag-PDHA1K321R and plated at low cell densities (250 cells per 60 mm plate). After 21 days, plates were stained with crystal violet, and the number of colonies for each group was quantified. (h) The MCF7 cells described above were treated with and without 4 Gy of ionizing radiation and cell survival was determined using a colony formation assay. (i) The MCF7 cells as mentioned above were treated with DCA (10 mM) and cell growth, as measured by total cell number, were determined at 1, 3, and 4 days and normalized to the untreated cells. (j) HCT116 cells expressing shPDHA1 were infected with Flag-PDHA1K321K, Flag-PDHA1K321Q, or Flag-PDHA1K321R. Then, they were treated with 10 mM DCA and cell growth, as measured by total cell number, were determined at 1, 3, and 4 days, and normalized to the non-treated cells. Error bars represent one standard deviation (*p<0.05 versus the control group).
Figure 6
Figure 6. Summary: SIRT3 deacetylates PDHA1 at lysine 321 and decreases the Warburg effect
PDH complexes in the mitochondria constitute a pool of active and inactive forms of the enzyme whose overall activity depends on the cell types and different physiological conditions. SIRT3 acts as a sensor for specific stress conditions such as starvation. When it gets activated, it deacetylates PDHA1 subunit of PDH. Deacetylation by SIRT3 increases the portion of active PDH. High PDH activity might increase the oxidative phosphorylation and ROS production, and decrease glycolysis. In many cancer cells, such as breast tumors, SIRT3 expression is significantly decreased. This results in hyper-acetylation of lysine residues (K321, K336, and possibly some others) and a decrease in PDH activity. Reduction in PDH activity causes higher glycolytic rates because pyruvate is not converted to acetyl-CoA, and this may ultimately increase the Warburg effect and decrease the production of oxidants in cancer cells.
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References

    1. Linn TC, Pettit FH, Reed LJ. Alpha-Keto Acid Dehydrogenase Complexes. X. Regulation of the Activity of the Pyruvate Dehydrogenase Complex from Beef Kidney Mitochondria by Phosphorylation and Dephosphorylation. Proc Natl Acad Sci U S A. 1969;62(1):234–241. - PMC - PubMed
    1. Peters SJ. Regulation of PDH activity and isoform expression: diet and exercise. Biochem Soc Trans. 2003;31:1274–1280. - PubMed
    1. Koukourakis MI, Giatromanolaki A, Sivridis E, Gatter KC, Harris AL. Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in non small cell lung cancer and tumor-associated stroma. Neoplasia. 2005;7:1–6. - PMC - PubMed
    1. Pilegaard H, Birk JB, Sacchetti M, Mourtzakis M, Hardie DG, et al. PDH-E1alpha dephosphorylation and activation in human skeletal muscle during exercise: effect of intralipid infusion. Diabetes. 2006;55:3020–3027. - PubMed
    1. Knoechel TR, Tucker AD, Robinson CM, Phillips C, Taylor W, et al. Regulatory roles of the N-terminal domain based on crystal structures of human pyruvate dehydrogenase kinase 2 containing physiological and synthetic ligands. Biochemistry. 2006;45:402–415. - PubMed

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