Lisha Zhou

Acetylation Regulates Gluconeogenesis by Promoting PEPCK1 Degradation via Recruiting the UBR5 Ubiquitin Ligase

Protein acetylation has emerged as a major mechanism in regulating cellular metabolism. Whereas most glycolytic steps are reversible, the reaction catalyzed by pyruvate kinase is irreversible, and the reverse reaction requires phosphoenolpyruvate carboxykinase (PEPCK1) to commit for gluconeogenesis. Here, we show that acetylation regulates the stability of the gluconeogenic rate-limiting enzyme PEPCK1, thereby modulating cellular response to glucose. High glucose destabilizes PEPCK1 by stimulating its acetylation. PEPCK1 is acetylated by the P300 acetyltransferase, and this acetylation stimulates the interaction between PEPCK1 and UBR5, a HECT domain containing E3 ubiquitin ligase, therefore promoting PEPCK1 ubiquitinylation and degradation. Conversely, SIRT2 deacetylates and stabilizes PEPCK1. These observations represent an example that acetylation targets a metabolic enzyme to a specific E3 ligase in response to metabolic condition changes. Given that increased levels of PEPCK are linked with type II diabetes, this study also identifies potential therapeutic targets for diabetes.

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

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.

SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense

Excess in mitochondrial reactive oxygen species (ROS) is considered as a major cause of cellular oxidative stress. NADPH, the main intracellular reductant, has a key role in keeping glutathione in its reduced form GSH, which scavenges ROS and thus protects the cell from oxidative damage. Here, we report that SIRT5 desuccinylates and deglutarylates isocitrate dehydrogenase 2 (IDH2) and glucose‐6‐phosphate dehydrogenase (G6PD), respectively, and thus activates both NADPH‐producing enzymes. Moreover, we show that knockdown or knockout of SIRT5 leads to high levels of cellular ROS. SIRT5 inactivation leads to the inhibition of IDH2 and G6PD, thereby decreasing NADPH production, lowering GSH, impairing the ability to scavenge ROS, and increasing cellular susceptibility to oxidative stress. Our study uncovers a SIRT5‐dependent mechanism that regulates cellular NADPH homeostasis and redox potential by promoting IDH2 desuccinylation and G6PD deglutarylation.

SIRT5 Desuccinylates and Activates Pyruvate Kinase M2 to Block Macrophage IL-1β Production and to Prevent DSS-Induced Colitis in Mice

LPS-activated macrophages undergo a metabolic shift from dependence on mitochondria-produced ATP to reliance on aerobic glycolysis, where PKM2 is a critical determinant. Here, we show that PKM2 is a physiological substrate of SIRT5 and that SIRT5-regulated hypersuccinylation inhibits the pyruvate kinase activity of PKM2 by promoting its tetramer-to-dimer transition. Moreover, a succinylation-mimetic PKM2 K311E mutation promotes nuclear accumulation and increases protein kinase activity. Furthermore, we show that SIRT5-dependent succinylation promotes PKM2 entry into nucleus, where a complex of PKM2-HIF1α is formed at the promoter of IL-1β gene in LPS-stimulated macrophages. Activation of PKM2 using TEPP-46 attenuates Sirt5-deficiency-mediated IL-1β upregulation in LPS-stimulated macrophages. Finally, we find that Sirt5-deficient mice are more susceptible to DSS-induced colitis, which is associated with Sirt5 deficiency prompted PKM2 hypersuccinylation and boosted IL-1β production. In conclusion, our findings reveal a mechanism by which SIRT5 suppresses the pro-inflammatory response in macrophages at least in part by regulating PKM2 succinylation, activity, and function.

Correction: Insulin and mTOR Pathway Regulate HDAC3-Mediated Deacetylation and Activation of PGK1

[This corrects the article DOI: 10.1371/journal.pbio.1002243.].

Abstract 5177: SIRT2 deacetylates and activates glucose-6-phosphate dehydrogenase (G6PD) to protect cells against oxidative stress.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first committed step in the pentose phosphate pathway (PPP), which converts β-D-glucose-6-phosphate into D-glucono-1,5-lactone-6-phosphate, while concomitantly producing reducing equivalents in the form of NADPH. NADPH is a required cofactor in reductive biosynthesis,and is also required for the generation of reduced glutathione, the major antioxidant responsible for preventing reactive oxygen species (ROS) damage. Lysine acetylation is an evolutionarily conserved post-translational modification in the regulation of a wide range of cellular processes. Several recent acetylome proteomic studies have identified more than 2000 potential acetylation substrates. Among these identified acetylated proteins is G6PD, implicating a novel modulation of G6PD at the post-translational level. However, our knowledge of how acetylation regulates G6PD activity is limited. This study is directed toward to understand the molecular mechanism by which acetylation regulates G6PD function to regulate cellular redox state in response to oxidative stress. Here, we found that G6PD activity is negatively modulated by acetylation, and the major regulated site of acetylation in G6PD was mapped to K403, an evolutionarily conserved lysine residue. Site-specific genetic incorporation of N e -acetyl-lysine into the position 403 of G6PD revealed that K403 acetylated G6PD is incapable to form active dimers and displays a complete loss in activity. Moreover, we found that the reversible acetylation of K403 is critical to modulate G6PD activity in response to different physiologic conditions, such as high glucose and oxidizing agents. Cytosolic SIRT2 deacetylated G6PD at K403, and greatly restored G6PD activity by inducing the formation of active dimers. Knockdown of endogenous G6PD led to higher cellular susceptibility to oxidant-induced cell death. Re-expression of wild-type G6PD, but not the acetylated mimic G6PD K403Q , could rescue cells from oxidative injury. Together these results uncover a previously unknown mechanism by which SIRT2 deacetylates and activates G6PD and thus protects cells against ROS damage. Citation Format: Yiping Wang, Lisha Zhou, Yuzheng Zhao, Shiwen Wang, Yi Yang, Dan Ye, Yue Xiong, Kun-Liang Guan. SIRT2 deacetylates and activates glucose-6-phosphate dehydrogenase (G6PD) to protect cells against oxidative stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2013-5177 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

P5-13-13: The Role of Topoisomerase IIa in Predicting Sensitivity to Anthracyclines in Breast Cancer Patients: A Meta-Analysis of Published Literatures.

Topoisomerase IIα is not only a proliferation marker of tumor cells but also a target for anthracycline-based chemotherapy. Both in vitro and in vivo studies have shown that there was a relationship between topo IIα and chemosensitivity to anthracyclines, but the predictive role of topo IIα is still controversial in breast cancer patients. A meta-analysis based on published studies was performed with the aim of obtaining an accurate evaluation of the association between topo IIα and sensitivity to anthracycline-based chemotherapy. A total of 13 eligible studies including 2,633 cases and 2,118 controls were identified. Topo IIα was associated with sensitivity to anthracyclines in locally advanced breast cancer patients who received neoadjuvant chemotherapy (RR = 1.93, 95%CI: 1.27−2.94, P=0.002; RR =1.98, 95%CI: 1.37−2.86, P Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-13.

P3-16-10: The Efficacy of Zoledronic Acid in Breast Cancer Adjuvant Therapy: A Meta-Analysis of Randomized Controlled Trials.

Background: The effect of zoledronic acid in breast cancer adjuvant therapy concerning improvement of patient survival has yet to be confirmed. We performed a meta-analysis of published and unpublished randomized controlled trials with the aim of accurate evaluation between clinical outcome and the association of the addition of zoledronic acid to adjuvant therapy. Methods: We searched Pubmed (from 1966 to present) and online abstracts from the proceeding Annual Meetings of the American Society of Clinical Oncology (ASCO) (years 1992–2010) and online abstracts from San Antonio Breast Cancer Symposium (years 2004–2010). A total of five eligible studies including 3676 subjects and 3678 controls met our search criteria and were evaluated. Random and fixed-effects meta-analytical models were used where indicated, and between-study heterogeneity was assessed. The primary study endpoints were the DFS. Secondary endpoints were OS, distant or loco-regional recurrence free survival and bone metastasis free survival. Results: Compared with the control arm, adjuvant breast cancer treatment with zoledronic acid did not significantly improve overall survival (OS), disease free survival (DFS), bone metastasis free survival, distant and locoregional recurrence free survival. However, in the postmenopausal subgroup, the addition of zoledronic acid to standard therapy could significantly improve DFS (RR=0.763, 95%CI 0.658−0.884, p Discussion: Adjuvant zoledronic acid may potentially improve the prognosis of postmenopausal patients. Additional studies are needed to evaluate the value of adjuvant treatment of zoledronic acid in premenopausal couterparts, differing disease stages, and various pathological types of breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-10.

Seroma formation after breast cancer surgery and its risk factors.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #4085 Background: . Seroma formation is one of the most common complications after breast cancer surgery including modified radical mastectomy and breast conservative surgery. Our study aims to investigate the risk factors of postoperative seroma in Chinese breast cancer patients. Methods: Clinical data of 158 women who underwent surgerical therapy for breast cancer in our hospital were collected prospectively and all patients were followed up. The risk factors for seroma occurrence were compared between the seroma group and control group using X 2 testor t test, as well as the logistic regression was used as multivariate analysis. Results: Univariate analysis showed that the average age of the seroma group was significantly higher than those without seroma formation(58.71 vs 51.00, P =0.0019), but the total serum protein and albumin content were lower (68.47g/L vs 72.53g/L, P =0.009 and 40.75g/L vs 42.52g/L, P =0.020, respectively). In seroma group, the drainage volume of the first three days, the total and daily drainage volume were all higher (all p values less than 0.01), as well as drainage duration and hospital stay were longer(8.3d vs 14.4d, P =0.000 and 11.5d vs 23.7d, P =0.000, respectively). Logistic regression showed that older patients ( OR =1.080, 95% CI 1.016∼1.148, P =0.013), lower total serum protein content( OR =0.814, 95% CI 0.705∼0.940, P =0.005)and higher drainage volume in d1( OR =1.009, 95% CI 1.001∼1.016, P =0.022) and d3 ( OR =1.017, 95% CI 1.005∼1.029, P =0.005) were all independent risk factors for subcutaneous seroma. The daily average drainage curve showed a gradually decreasing trend with a highest collections in the first three days. The seroma group had significantly higher average daily drainage volume( P =0.034) and longer duration ( P =0.000). Conclusion :The risk factors of seroma formation after breast cancer surgery are complicated. However in order to prevent its occurrence effectively, the factors including age, nutrition status and daily drainage volume should be taken into consideration. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4085.

Epidemiology of Triple Negative Breast Cancer in China.

Background:The classification of tumors based on the gene expression may define intrinsic breast cancer subtypes in which the effect of risk factors could be more obvious. We sought to assess the risk factors for triple negative breast cancer on Chinese population.Methods: A retrospective study of 5761 patients was carried out from a large database of breast cancer patients undergoing surgery between January 1, 1991 and June 31, 2008 in Cancer Hospital, Fudan University, Shanghai, China. Univariate analyses were performed by chi-square test of Student9s t test and multivariate analyses by logistic regression.Results: A total of 1108 women were identified as having triple negative breast cancer and were compared with the 4653 women with non-triple negative. Regardless the pathological discrepancy, women with triple negative breast cancers were significantly more likely to be younger age(P=0.001; OR=1.615, 95%CI=1.207-2.160), premenopausal (P = 0.003; OR = 1.570, 95% CI = 1.171-2.106) and parous women (P = 0.001; OR = 1.741, 95% CI = 1.269-2.387).There seems no associations with increasing risk of triple negative breast cancer for breastfeeding (P=0.126), younger age at menarche (P=0.129), first degree family history (P=0.111) and oral contraceptive usage (P=0.251).Conclusion:Based on this large population study in Chinese breast cancer patients, the risk factors for triple negative phenotype may be somewhat different from those for Western women. Therefore, a better knowledge of this issue is warranted due to it may have impact on clinical outcomes and may offer some insight into the process of carcinogenesis and therapeutic efficacy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2065.