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. 2016 Aug 11:7:12497.
doi: 10.1038/ncomms12497.

Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome

Lianhui Sun  1   2 , Guangjian Fan  1   2 , Peipei Shan  1   2 , Xiaoying Qiu  2 , Shuxian Dong  2 , Lujian Liao  2 , Chunlei Yu  2 , Tingting Wang  1 , Xiaoyang Gu  1 , Qian Li  3 , Xiaoyu Song  4 , Liu Cao  4 , Xiaotao Li  2   5 , Yongping Cui  6 , Shengping Zhang  1 , Chuangui Wang  1   2
Affiliations

Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome

Lianhui Sun et al. Nat Commun. .

Abstract

Maintenance of energy homeostasis is essential for cell survival. Here, we report that the ATP- and ubiquitin-independent REGγ-proteasome system plays a role in maintaining energy homeostasis and cell survival during energy starvation via repressing rDNA transcription, a major intracellular energy-consuming process. Mechanistically, REGγ-proteasome limits cellular rDNA transcription and energy consumption by targeting the rDNA transcription activator SirT7 for ubiquitin-independent degradation under normal conditions. Moreover, energy starvation induces an AMPK-directed SirT7 phosphorylation and subsequent REGγ-dependent SirT7 subcellular redistribution and degradation, thereby further reducing rDNA transcription to save energy to overcome cell death. Energy starvation is a promising strategy for cancer therapy. Our report also shows that REGγ knockdown markedly improves the anti-tumour activity of energy metabolism inhibitors in mice. Our results underscore a control mechanism for an ubiquitin-independent process in maintaining energy homeostasis and cell viability under starvation conditions, suggesting that REGγ-proteasome inhibition has a potential to provide tumour-starving benefits.

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Figures

Figure 1
Figure 1. REGγ deficiency promotes energy consumption and starvation-induced cell death.
(a–c) REGγ regulates cellular energy homeostasis under normal growth conditions. (A,B) MEF cells from wild-type (+/+, WT) and REGγ knockout (−/−, KO) mice were cultured in DMEM-high glucose medium. The relative intracellular ATP levels (a) and the cellular ADP/ATP ratios (b) were detected. To determine the specific effect of REGγ, REGγ-KO MEF cells were infected with lentiviral vectors expressing REGγ. Western blots show REGγ expression. (c) The relative intracellular ATP levels in HeLa, HCT116 and HCT116−/− (p53 null) cancer cells with stable knockdown of REGγ (ShR1 or ShR2) or a vector control (ShN) cultured in DMEM-high glucose medium. Western blots show the knockdown efficiency. (d,e) REGγ deficiency promotes energy consumption. Indicated cell lines were cultured in glucose-free DMEM (glucose deprivation, GD) for the indicated time periods, or re-supplemented with glucose (+gluc.) for 4 or 6 h after GD. The relative intracellular ATP levels were analysed. (f-l) REGγ deficiency promotes energy-dependent cell death. (f) REGγ-WT and -KO MEF cells were treated with GD for 16 h and apoptosis was analysed by FACS. Quantitative data show percentage of apoptosis. (g) REGγ-WT and -KO MEF cells were treated with GD for 16 h and analysed for activated caspase-3 and poly (ADP-ribose) polymerase cleavage by western blotting. (h) REGγ-WT and -KO MEF cells were treated with GD for the indicated times and the large-scale DNA fragmentation was determined by agarose gel electrophoresis. (i,j) Indicated cell lines were treated with GD, or re-supplemented with glucose (+ gluc.) for the indicated time periods after GD. Cell viability was determined using MTT assay. (k,l) Indicated cells were treated with GD in the presence or absence of methyl pyruvate (MP, 10 μM) for the indicated time periods, and cell viability was analysed using MTT assay. All experiments were repeated three times, data represent mean±s.d., *P<0.05, **P<0.01; Student's t-test is used throughout. See also Supplementary Fig. 1.
Figure 2
Figure 2. REGγ limits rDNA transcription.
(a–c) REGγ deficiency enhances rDNA transcription. (a) Indicated stable cell lines were transfected with the rDNA-promoter luciferase reporter plasmid for 24 h and the relative luciferase activity levels were determined. (b,c) Relative pre-rRNA (47S/45S) expression levels in indicated cell lines were analysed by qRT-PCR (b) and northern blot (c). (d–f) REGγ overexpression decreases rDNA transcription. (d) HeLa cells were co-transfected with rDNA-promoter luciferase reporter and GFP-tagged REGγ plasmids for 24 h. The relative luciferase activity was analysed. (e,f) Levels of pre-rRNA in HeLa cells with or without GFP-REGγ overexpression were analysed by qRT-PCR (e) and northern blot (f). (g,h) REGγ deficiency delays starvation-induced pre-rRNA reduction. Indicated cell lines were treated with GD for the indicated time periods. The relative pre-rRNA transcript levels were analysed by qRT-PCR. All experiments were repeated three times; data represent mean±s.d. *P<0.05, **P<0.01, Student's t-test.
Figure 3
Figure 3. REGγ regulates SirT7 subcellular distribution and degradation.
(a) REGγ overexpression causes SirT7 redistribution. Flag-SirT7 and GFP-REGγ (wild type, aa1-103, or aa66-161) plasmids were cotransfected to HeLa cells, and Flag-SirT7 was immunostained with anti-Flag antibody (red) and visualized by fluorescence microscopy (scale bar, 10 μm). GFP-REGγ was detected by the intrinsic GFP fluorescence. Nuclei were stained with DAPI. Graph shows the percentage of cells displaying prominent nucleolar SirT7 staining. Data represent mean±s.d., n=3, >100 cells were counted per replicate. NS=not significant, **P<0.01, Student's t-test. (b–d) REGγ interacts with SirT7. (b) Indicated Flag-Sirtuins and GFP-REGγ were cotransfected into 293 T cells followed by immunoprecipitation using FLAG-M2 beads and western blot using anti-GFP antibody. (c) REGγ-SirT7 interaction domain in REGγ was determined by cotransfection of indicated GFP-REGγ deletion mutants with Flag-SirT7 into 293 T cells followed by immunoprecipitation using FLAG-M2 beads and western blot using anti-GFP antibody. The schematic diagram indicates the region of REGγ required for SirT7 interaction. (d) Endogenous REGγ in HeLa cells was precipitated using anti-REGγ antibody or with IgG (Mock IP), and coprecipitated SirT7 was detected by western blot. (e) REGγ interacts with SirT7 in vitro. Recombinant His-tagged REGγ was incubated with GST-SirT7 or GST proteins at 4 °C for 4 h followed by GST pull-down and western blot. (f–k) REGγ destabilizes SirT7. (f) Western blot analysis of SirT7 expression in the indicated cell lines. (g) Semiquantitative RT-PCR analysis of relative SirT7 mRNA in REGγ-WT and -KO MEF cells. (h) REGγ-KO MEF cells were infected with lentivirus expressing GFP-REGγ-WT, -1-103 and -66-161 for 48 h, and endogenous SirT7 protein was detected by western blot. (i,j) Western blot analysis of lysates of indicated cell lines treated with translation inhibitor cycloheximide (CHX, 50 μg ml−1). Relative SirT7 band intensities were quantified through densitometry and presented. (k) 293 T cells transfected with His-ubiquitin, Flag-SirT7 and GFP-REGγ plasmids were treated with or without MG132 (25 μM, 4 h). Ubiquitinated proteins were precipitated using Ni-NTA beads. SirT7 ubiquitination was detected by western blot using anti-Flag antibody. To ensure equal expression of SirT7, a higher amount of SirT7 plasmid DNA (++) was cotransfected with REGγ. See also Supplementary Fig. 2.
Figure 4
Figure 4. REGγ regulates rDNA transcription, energy consumption and cell death via SirT7.
(a–c) REGγ inhibits SirT7 activity in rDNA transcription. (a) rDNA-promoter luciferase reporter plasmid was cotransfected with SirT7 and REGγ plasmids into HeLa cells for 24 h and relative luciferase activity was analysed. Representative western blots show the expression levels of SirT7 and REGγ. (b,c) HeLa cells were transfected with SirT7 and REGγ plasmids as indicated, and levels of pre-rRNA were analysed by qRT-PCR (b) and northern blot (c). Representative western blots show the expression levels of SirT7 and REGγ (upper panels). (d) REGγ deficiency attenuates starvation-induced reduction of pre-rRNA via SirT7 induction. REGγ-ShN and-ShR HCT116−/− cells infected with or without SirT7 small hairpin RNA (SirT7-Si) expression lentivirus were treated with glucose deprivation (GD) for 6–12 h. The relative pre-rRNA levels were analysed by qRT-PCR. SirT7 knockdown efficiency was detected by western blot (right panel). (e,f) REGγ deficiency promotes starvation-induced energy-consumption and cell death via SirT7. Cells in d were starved for glucose for indicated time periods. The ATP consumption (e) and cell viability (f) were analysed. (g,h) SirT7 overexpression enhances starvation-induced energy consumption and cell death in REGγ-knockdown cells. Stable REGγ-ShN and -ShR HCT116−/− cell lines were infected with lentivirus expressing SirT7 and cultured for 20 h, and then treated with or without GD for the indicated time periods. The relative intracellular ATP (g) and cell viability (h) were analysed. All experiments were repeated three times; data represent mean±s.d. *P<0.05, **P<0.01, Student's t-test.
Figure 5
Figure 5. REGγ causes SirT7 nuleoplasmic redistribution and degradation under starvation.
(a) REGγ is required for starvation-induced SirT7 nuleoplasmic redistribution. Flag-SirT7 transfected REGγ-ShN and -ShR HeLa cells were treated with glucose deprivation (GD, 12 h) or AICAR (0.5 mM, 12 h), and immunostained with anti-Flag (red) and anti-UBF (green) antibodies and visualized by fluorescence microscopy (scale bar, 10 μm). Nuclei were stained with DAPI. Graph shows the percentage of cells displaying prominent nucleolar SirT7 staining. Data represent mean±s.d., n=3, and at least 100 cells were counted per replicate. NS=not significant, **P<0.01, Student's t-test. (b,c) Energy stress increases REGγ-SirT7 association. 293 T cells were cultured in DMEM medium with different glucose concentrations (0–30 mM) for 4 h (b), or treated with glycolytic inhibitor 2DG in high-glucose DMEM medium for indicated times (c), followed by immunoprecipitation using anti-SirT7 antibody and western blot with anti-REGγ antibody. (d,e) AMPK is required for starvation-induced REGγ-SirT7 association. 293 T cells were treated with AMPK activator AICAR (0.5 mM) for indicated time periods (d), or with AMPK inhibitor Compound C (10 μM, 1 h) followed by glucose deprivation (GD, 1-2 h) (e). Cell lysates were immunoprecipitated with anti-SirT7 antibody followed by western blotting using anti-REGγ antibody. (f,g) Glucose starvation induces REGγ-dependent SirT7 degradation. Indicated cell lines were treated with GD for the indicated time periods. SirT7 degradation was analysed by western blot. See also Supplementary Fig. 3.
Figure 6
Figure 6. REGγ regulates SirT7 under starvation in a phosphorylation-dependent manner.
(a) Cell lysates from GD (1–4 h)-treated 293 T cells were immunoprecipitated with SirT7 followed by western blotting using anti-phospho-Ser/Thr antibody (P-Thr/Ser). (b) 293 T cells transfected with indicated Flag-SirT7 T153 mutants were immunoprecipitated with FLAG-M2 beads followed by REGγ western blotting. (c) 293 T cells transfected with indicated Flag-SirT7 plasmids were treated with cycloheximide (CHX, 50 μg ml−1) and analysed for SirT7 stability by anti-FLAG western blot. Relative SirT7 band intensities were quantified through densitometry and presented. (d) REGγ-ShN and -ShR HeLa cells transfected with indicated Flag-SirT7 mutants were immunostained with anti-Flag antibody (red) and visualized by microscopy (scale bar, 10 μm). Graph shows the percentage of cells displaying prominent nucleolar SirT7 staining. Data represent mean±s.d., n=3, >100 cells were counted per replicate. NS=not significant, **P<0.01, Students t-test. (e) Characterization of SirT7 T153 phosphorylation antibody (SirT7 T153p). Cell lysates from 293 T cells transfected with wild-type or mutant forms of Flag-SirT7 were immunoprecipated with FLAG-M2 beads followed by western blot using antibody against SirT7 T153p or Flag. (f,g) Cell lysates from GD (f) or AICAR (0.5 mM) (g) treated 293 T cells were immunoprecipitated with anti-SirT7 antibody followed by immunoblot with anti-SirT7 T153p or anti-SirT7 antibodies. HC, heavy chain. (h) Flag-SirT7-WT or -T153A mutant transfected HeLa cells were treated with or without GD (12 h) followed by immunostaining with anti-Flag antibody (red). Scale bar, 10 μm. Graph shows the percentage of cells displaying prominent nucleolar SirT7 staining. Data represent mean±s.d., n=3, >100 cells were counted per replicate. NS=not significant, **P<0.01, Students t-test. (i) Stable REGγ-ShN and REGγ-ShR HeLa cells transfected with Flag-SirT7-WT or -153A mutant were treated with or without GD (12 h), then immunostained with anti-FLAG (red) and anti-SirT7 T153p (green) antibodies and visualized by microscopy (scale bar, 10 μm). Nuclei were stained with DAPI. The SirT7-T153A mutant was used to evaluate the specificity of the SirT7 T153p antibody. (j) 293T cells transfected with indicated Flag-SirT7 plasmids were treated with or without GD (1 h). Cell lysates were immunoprecipitated with FLAG-M2 beads and probed with anti-REGγ or anti-Flag antibodies. See also Supplementary Fig. 4.
Figure 7
Figure 7. AMPK directly regulates SirT7 phosphorylation and subcellular distribution under starvation.
(a) 293T cells transfected with Flag-SirT7 and HA-AMPKα plasmids were treated with or without GD (4 h), followed by immunoprecipitation with FLAG-M2 beads. The precipitated proteins were analysed by western blot using anti-Flag or anti-HA antibody. (b) In vitro phosphorylation of SirT7 by activated AMPKα. GST-SirT7-WT or -153A mutants were expressed in E.coli and purified with GST beads. Activated Flag-AMPKα was precipitated from GD (6 h) treated Flag-AMPKα-overexpressing 293T cells using FLAG-M2 beads and eluted with Flag peptide. GST-SirT7-WT or -153A proteins were incubated with or without Flag-AMPKα in the presence or absence of ATP as indicated. The reaction product was separated by SDS-PAGE and analysed by western blot. (c) Similar in vitro kinase assay was performed as detailed for (b), except that the kinase-dead AMPKα D159A (AMPK-DN) mutant was used. (d) HeLa cells with AMPKα knockdown (AMPKα-Si) or control Si-RNA (Ctrl-Si) were transfected with Flag-SirT7 following GD (12 h) treatment, then immunostained with anti-Flag (red) or anti-UBF (green) antibodies (scale bar, 10 μm). Nuclei were stained with DAPI. (e) HeLa cells with AMPKα knockdown or control Si-RNA were treated with GD (4 h) followed by immunoprecipitation with anti-SirT7 antibody and western blot analysis. AMPK activation was confirmed by its phosphorylation at Thr-172.
Figure 8
Figure 8. REGγ deficiency benefits 2DG treatment in tumour starvation.
(a,b) REGγ knockdown enhances 2DG-induced cell death and ATP consumption. Stable REGγ-ShN and-ShR HCT116−/− cells were treated with 2DG (12.5 mM) for the indicated time periods, and relative cell viability (a) and intracellular ATP levels (b) were determined. Data represent mean±s.d., n=3, *P<0.05, **P<0.01, Student's t test. (c-e) REGγ knockdown sensitizes the tumour to 2DG treatment in mice. (c,d) Mice with xenograft tumours originated from stable HCT116−/− cells with REGγ-ShN or -ShR were treated with 2DG or PBS by intraperitoneal injection. Images show tumours after 32 days of PBS or 2DG treatment (c). The tumour size was measured every 5 days and tumour volume was calculated (d). Data represent mean±s.d., n=7, **P<0.01, Student's t test. (e) Tissue sections of xenograft tumours of mice on day 32 were analysed by TUNEL staining (Scale bar, 200 μm). Nuclei were stained with DAPI. (f) stable REGγ-ShN and-ShR HCT116−/− cells infected with or without SirT7 knockdown (Si) lentivirus were treated with 2DG (12.5 mM) for the indicated times, and cell viability was determined using MTT assay. Data represent mean±s.d., n=3, *P<0.05, **P<0.01, Student's t test.
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