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Cholesterol and fatty acids regulate cysteine ubiquitylation of ACAT2 through competitive oxidation

Nature Cell Biology volume 19, pages 808–819 (2017)Cite this article

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A Corrigendum to this article was published on 01 December 2017

This article has been updated

Abstract

Ubiquitin linkage to cysteine is an unconventional modification targeting protein for degradation. However, the physiological regulation of cysteine ubiquitylation is still mysterious. Here we found that ACAT2, a cellular enzyme converting cholesterol and fatty acid to cholesteryl esters, was ubiquitylated on Cys277 for degradation when the lipid level was low. gp78–Insigs catalysed Lys48-linked polyubiquitylation on this Cys277. A high concentration of cholesterol and fatty acid, however, induced cellular reactive oxygen species (ROS) that oxidized Cys277, resulting in ACAT2 stabilization and subsequently elevated cholesteryl esters. Furthermore, ACAT2 knockout mice were more susceptible to high-fat diet-associated insulin resistance. By contrast, expression of a constitutively stable form of ACAT2 (C277A) resulted in higher insulin sensitivity. Together, these data indicate that lipid-induced stabilization of ACAT2 ameliorates lipotoxicity from excessive cholesterol and fatty acid. This unconventional cysteine ubiquitylation of ACAT2 constitutes an important mechanism for sensing lipid-overload-induced ROS and fine-tuning lipid homeostasis.

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Figure 1: ACAT2 is stabilized by sterols and saturated fatty acids.
Figure 2: Lipid-regulated ACAT2 ubiquitylation occurs on Cys277.
Figure 3: ACAT2 is ubiquitylated by Lys48-specific ubiquitin linkages.
Figure 4: The ubiquitylation on Cys277 of ACAT2 is regulated by oxidative stress.
Figure 5: The gp78–Insigs E3 complex mediates the ubiquitylation and degradation of ACTA2.
Figure 6: ACAT2−/− mice are more susceptible to HFD-induced insulin resistance.
Figure 7: The stabilization of hepatic ACAT2 ameliorates lipotoxicity and protects HFD-induced insulin resistance in mice.

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  • 13 November 2017

    In the original version of this Article, the procedure for assessing the ubiquitylation assays by western blotting was inadvertently omitted from the Methods. This method was used in Figures 2b,d,g,h, 3a,b,d, 4f and 5h and Supplementary Figures 3b, 5b and 6b, and was designed to omit reagents such as BME and DTT that might break the thioester bond. The full method is given below, and has been added to the Methods section of the Article. Ubiquitylation assay western blot. For Figs 2b,d,g,h, 3a,b, 4f and 5h, and Supplementary Figs 5b and 6b, after the last wash of the beads, the supernatant was discarded and beads were boiled for 10 min in 100 μl 2× SDS loading buffer (75 mM Tris-HCl, pH 6.8, 6% SDS, 15% (v/v) glycerol, 0.01% (w/v) Bromophenol blue) and were then vortexed and centrifuged at 1,000 g for 2 min. 90 μl supernatant was mixed with 90 μl solubilization buffer (62.5 mM Tris-HCl, pH 6.8, 15% SDS, 8 M Urea, 10% glycerol) and incubated at 37 °C for 30 min. For Fig. 3d and Supplementary Fig. 3b, the beads were incubated with 100 μl Myc peptide (1 mg/ml) for 3 hrs at 4 °C, vortexed and centrifuged at 1,000 g for 2 min. 90 μl supernatant was mixed with 90 μl solubilization buffer (62.5 mM Tris-HCl, pH 6.8, 15% SDS, 8 M Urea, 10% glycerol) and 60 μl modified 4× SDS loading buffer (150 mM Tris-HCl, pH 6.8, 12% SDS, 30% (v/v) glycerol, 0.02% (w/v) Bromophenol blue) and incubated at 37 °C for 30 min. Samples were resolved by SDS–PAGE and transferred onto PVDF membranes. Immunoblots were blocked with 5% BSA in TBS containing 0.075% Tween (TBST) and probed with primary antibodies overnight at 4 °C. After washing in TBST 3 times, blots were incubated with secondary antibodies for 1 h at room temperature. After washing in TBST 3 times, bands were visualized by enhanced chemiluminescence (ECL).

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Acknowledgements

We thank W. Qi for helpful discussion and critical reading of the manuscript, and X.-Y. Yang, H.-H. Miao, L. Qian, Y.-X. Qu, J. Xu and J. Qin for technical assistance. This work was supported by the grants from NNSF of China (31690102, 31430044, 31470802, 31230020 and 31271377), MOST of China (2016YFA0500100 and 2014DFG32410), the 111 Project of Ministry of Education of China (B16036) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). T.-Y.C. and C.C.Y.C. are funded by NIH grant AG 037609.

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Authors and Affiliations

  1. The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, China

    Yong-Jian Wang, Yan Bian, Ming Lu, Ying Xiong, Qin Li & Bo-Liang Li

  2. Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China

    Yong-Jian Wang

  3. Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan, 430072, China

    Jie Luo & Bao-Liang Song

  4. Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China

    Shu-Yuan Guo, Hui-Yong Yin & Xu Lin

  5. Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, 03755, USA

    Catherine C. Y. Chang & Ta-Yuan Chang

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Contributions

B.-L.L. and B.-L.S. conceived the project. Y.-J.W., C.C.Y.C., T.-Y.C., B.-L.L. and B.-L.S. designed the experiments. Y.-J.W., Y.B., M.L., Y.X. and Q.L. performed the experiments. S.-Y.G. and H.-Y.Y. measured the FA compositions. X.L. performed SNP analysis. Y.-J.W., J.L., C.C.Y.C., T.-Y.C., B.-L.L. and B.-L.S. organized and analysed the data. Y.-J.W., J.L., B.-L.L. and B.-L.S. wrote the manuscript with input from all other authors. All authors approved the final version of the manuscript.

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Correspondence to Bo-Liang Li or Bao-Liang Song.

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Integrated supplementary information

Supplementary Figure 1 ACAT2 is stabilized by sterols and fatty acids.

(a) Structures of different sterols. Red denotes sterols that elevated ACAT2 protein level. (b) CHO/ACAT2-Myc cells were depleted of lipids and treated with different sterols at indicated concentration. 16 hrs later, cells were harvested for western blotting. (c,d) Different cell lines (HepG2, Huh7, Hepa1-6 and Caco2) were depleted of lipids and treated with OA (200 μM) or PA (200 μM). 16 hrs later, cells were harvested for western blotting and RT-qPCR (mean ± s.d., n = 3 independent experiments). The immunoblots are representative of 3 independent experiments. Uncropped blots are shown in Supplementary Fig. 8. Statistics source data for d can be found in Supplementary Table 2.

Supplementary Figure 2 Mapping the ubiquitination site of ACAT2.

(a) Schematic representation of human ACAT2 (WT) and its various truncations. Red box denotes the region (aa 252 and aa 279) essential for sterol-regulated ACAT2 stabilization. (b–e) CHO cells transiently transfected with plasmids expressing ACAT2 variants were depleted of sterols and then treated with vehicle or 25-HC (3 μg ml−1) for 16 hrs and harvested for western blotting. (f) Schematic representation of human ACAT2 showing all Cs in a broader region (aa 227–328) (WT) and alanine substitution for single or all Cs. (g,h) CHO cells transiently transfected with plasmids expressing Myc-tagged ACAT2 (WT) or Cys mutants were depleted of sterols and then treated with vehicle or 25-HC for 16 hrs and harvested for western blotting. (i) The alignment of mammalian ACAT1 and ACAT2 partial sequences. The asterisk indicates the ubiquitination site. The highly conserved Cys of ACAT2 is marked in red. The immunoblots are representative of 3 independent experiments. Uncropped blots are shown in Supplementary Fig. 8.

Supplementary Figure 3 ACAT2 is ubiquitinated by K48-specific ubiquitin linkages.

(a) 200 ng of M1-linked (M1), K6-linked (K6), K11-linked (K11), K27-linked (K27), K29-linked (K29), K33-linked (K33), K48-linked (K48) or K63-linked (K63) ubiquitin dimers were separated by SDS-PAGE and probed with different anti-ubiquitin antibodies as indicated. The coommassie brilliant blue-stained gel is shown as a loading control. This is an independent repeat of the experiments in Fig. 3c. (b) CHO cells were transfected with pCMV-HA-Ub together with the plasmid expressing Myc-tagged ACAT2 (WT), (K-null) or (C277A) and depleted of lipids for 16 hrs. Cells were treated with or without 25-HC for 11 hrs. Then MG132 was added into medium for another 5 hrs. Cells were harvested and lysed. The ACAT2 proteins were immunoprecipitated by anti-Myc antibodies coupled agarose and eluted with Myc peptides. Western blotting was carried out using different ubiquitin linkage antibodies or anti-Myc antibody as indicated. The di-ubiquitin was loaded about 30 min later than ACAT2 samples and served as controls. This is an independent repeat of the experiments in Fig. 3d. Uncropped blots are shown in Supplementary Fig. 8.

Supplementary Figure 4 The critical concentration and time course of different lipids to induce ROS production and elevate ACAT2 protein level.

(a–i) CHO/ACAT2-Myc cells were depleted of lipids and treated with cholesterol (a–c), 25-HC (d–f) or PA (g–i) at indicated concentration. 16 hrs later, cells were harvested for western blotting (a,d,g) and ROS measurement (b,e,h). The correlation between these two parameters was analyzed by GraphPad Prism and demonstrated by scatterdiagrams (c,f,i). (j–r) CHO/ACAT2-Myc cells were depleted of lipids and treated with cholesterol (10 μg ml−1, j–l), 25-HC (5 μg ml−1, m–o) or PA (200 μM, p–r) for indicated time duration. 16 hrs later, cells were harvested for western blotting analysis (j,m,p) and ROS measurement (k,n,q). The correlation between these two parameters was demonstrated by scatterdiagrams (l,o,r). The immunoblots are representative of 3 independent experiments. Blots were quantified and the mean intensity of ACAT2 normalized to beta-actin relative to control treated cells is indicated. Uncropped blots are shown in Supplementary Fig. 8. Data are presented as mean ± s.d., n = 3 independent experiments. Statistics source data for a–r can be found in Supplementary Table 2.

Supplementary Figure 5 Analysis of the ubiquitination, degradation and protein binding of ACAT2.

(a) CHO/ACAT2-Myc cells were depleted of lipids, treated with H2O2 for indicated time durations and harvested for western blotting. (b) CHO/ACAT2-Myc cells were depleted of lipids for 27 hrs. Cells were then treated with H2O2 for indicated times in the presence of 10 μM MG132 and harvested for ubiquitination assay. (c) CHO cells were transfected with plasmids expressing Myc-tagged ACAT2 and HA-tagged gp78 WT or its variants including C356S-RING finger domain mutation (RINGmut), or M467F, F468S, P469S-CUE domain mutation (CUEmut) as indicated. 48 hrs later, cells were harvested for western blotting. (d) CHO cells were transfected with plasmids expressing Myc-tagged ACAT2 (WT) or (C277A) and T7-tagged Insig1 and depleted of lipids for 27 hrs. Cells were then treated with MG132 for another 5 hrs. Cells were harvested and lysed in the non-denaturing IP buffer, and subsequently subjected to co-IP analysis. (e) CHO cells transfected with plasmids expressing Myc-tagged ACAT2, HA tagged-gp78 and T7-tagged-Insig1 were depleted of lipids and treated with 25-HC/PA or menadione as indicated. 11 hrs later, cells were treated with MG132. After incubation for another 5 hrs at 37 °C, cells were harvested, lysed in the absence (-) or presence (+) of 20 mM DTT, and subsequently subjected to co-IP analysis. (f) CHO cells transfected with plasmid expressing Myc-tagged ACAT2 (WT) or (C-null) were depleted of lipids and treated with vehicle or 25-HC/PA. Cells were harvested 16 hrs later for western blotting. (g) CHO cells transfected with plasmids expressing Myc-tagged ACAT2 (WT) or (C-null) in combination with HA tagged-gp78 and T7-tagged-Insig1 were depleted of lipids and treated with vehicle or menadione as indicated. 11 hrs later, cells were treated with MG132. After incubation for another 5 hrs, cells were harvested for co-IP analysis. The immunoblots are representative of 3 independent experiments. Uncropped blots are shown in Supplementary Fig. 8.

Supplementary Figure 6 Schematic representation for the pathway of lipid-induced ACAT2 stabilization via ROS-mediated C277 oxidation.

(a) CHO cells transfected with plasmid expressing ACAT2 or gp78 were depleted of lipids and treated with 25-HC/PA. Cells were harvested 8 hrs later for sulfenic acids detection as described in Methods. (b) CHO-7 (WT) and gp78−/− deficient CHO-7 cell line (gp78 KO) cells were transfected with indicated plasmids. Cells were depleted of lipids and treated with 25-HC/PA or menadione. 11 hrs later, cells were treated with MG132. After incubation for another 5 hrs, cells were harvested, lysed in the non-denaturing buffer, and subsequently subjected to co-IP analysis. (c–e) Male ACAT2−/− mice (8–12 weeks old) were randomly grouped (n = 5 mice) and intravenously injected with AAV2/8-ACAT2 (WT) or (C277A) at a dose of 1 × 1012 vg per mouse. Mice were then allowed ad libitum access to water and chow diet or HFD for 12 weeks. (c) The ACAT2 expression in various tissues of mice receiving AAV2/8-ACAT2 injection. (d) The relative mRNA level of ACAT2 in the livers of mice receiving AAV2/8-ACAT2 (WT)- or (C277A)-injections fed on chow diet and HFD normalized by endogenous ACAT2 mRNA level that was defined as 1. (e) Fast performance liquid chromatography (FPLC) analysis of CEs concentrations from the pooled serum of AAV2/8-ACAT2 (WT)- or (C277A)-injected mice fed on HFD. (f) In the presence of low levels of sterols and/or fatty acids, ACAT2 is ubiquitinated at C277 by the gp78/Insig E3 complex and targeted for proteasomal degradation. Lipid overloading, however, triggers ROS generation that further oxidizes C277 and prevents ACAT2 from ubiquitin-mediated enzyme degradation. The stabilized ACAT2 then converts unesterified cholesterol and fatty acids into cholesteryl esters and ameliorates insulin-resistance induced by lipotoxicity. The immunoblots are representative of 3 independent experiments. Uncropped blots are shown in Supplementary Fig. 8. Data are presented as mean ± s.d. Statistics source data for b,c can be found in Supplementary Table 2.

Supplementary Figure 7 Hepatic fatty acid composition in the CEs of WT mice fed on chow diet or HFD.

Male ACAT2−/− WT littermates (8–12 weeks) were randomly grouped and allowed ad libitum access to water and chow diet or HFD for 12 weeks. (a–c) Fatty acid composition of the chow diet and HFD revealed by LC/MS (mean, n = 2 independent experiments). (d–f) Hepatic fatty acid composition of the CEs in WT mice fed on chow diet or HFD revealed by LC/MS (mean ± s.d., n = 6 mice) n.s., no significance; ∗p < 0.05, ∗∗p < 0.01; unpaired two-tailed Student’s t test. Statistics source data for a–f can be found in Supplementary Table 2.

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Wang, YJ., Bian, Y., Luo, J. et al. Cholesterol and fatty acids regulate cysteine ubiquitylation of ACAT2 through competitive oxidation. Nat Cell Biol 19, 808–819 (2017). https://doi.org/10.1038/ncb3551

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