Sirt1-deficiency causes defective protein quality control

Abstract

Protein quality control is an important mechanism to maintain cellular homeostasis. Damaged proteins have to be restored or eliminated by degradation, which is mainly achieved by molecular chaperones and the ubiquitin-proteasome system. The NAD(+)-dependent deacetylase Sirt1 has been reported to play positive roles in the regulation of cellular homeostasis in response to various stresses. However, its contribution to protein quality control remains unexplored. Here we show that Sirt1 is involved in protein quality control in both an Hsp70-dependent and an Hsp70-independent manner. Loss of Sirt1 led to the accumulation of ubiquitinated proteins in cells and tissues, especially upon heat stress, without affecting proteasome activities. This was partly due to decreased basal expression of Hsp70. However, this accumulation was only partially alleviated by overexpression of Hsp70 or induction of Hsp70 upon heat shock in Sirt1-deficient cells and tissues. These results suggest that Sirt1 mediates both Hsp70-dependent and Hsp70-independent protein quality control. Our findings cast new light on understanding the role of Sirt1 in maintaining cellular homeostasis.

Figure 1. Ubiquitinated proteins accumulate in Sirt1^–/– MEFs.

(A) Immunoblotting of immortalized MEF lysates of the indicated genotype. Actin serves as a loading control. Values represent the relative band intensities of ubiquitin (Ub) (normalized to GAPDH). (B) Lysates from wild-type and Sirt1^–/– MEFs were subjected to immunoprecipitation with anti-Rpt6 antibodies, followed by immunoblotting. Values represent the relative band intensities of Ub (normalized to GAPDH). (C) Immunoblotting of lysates from Flag-ubiquitin-introduced wild-type MEFs and Sirt1^–/– MEFs transfected with wild-type Sirt1 or H355Y Sirt1. Values represent the relative band intensities of Ub (normalized to Actin). Uncropped gel images are shown in Supplementary Figure.

Figure 2. Proteasome activities are not impaired in Sirt1^–/– MEFs.

(A) Lysates from wild-type and Sirt1^–/– MEFs were fractionated by 8–32% glycerol gradient centrifugation. An aliquot of each fraction was used for an assay of chymotryptic activity of proteasomes using succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin (Suc-LLVY-AMC) as a substrate and immunoblotting for Rpt6 and α6. (B) Ubiquitin-independent and -dependent protease activities of proteasomes. Lysates from wild-type and Sirt1^–/– MEFs were subjected to an in vitro protein degradation assay. Antizyme-dependent degradation of ³⁵S-labeled ODC and ubiquitin-dependent degradation of ³⁵S-labeled cIAP1 were measured. The data represent means ± standard error of the mean (SEM) from three independent experiments. Statistical comparisons were made by Student’s t-test for two tailed unpaired samples. N.S. indicates not significant. (C) Lysates of wild-type and Sirt1^–/– MEFs treated with or without 10 μM MG132 for 30 min were subjected to immunoblotting with the indicated antibodies. Values represent the relative band intensities of Ub (normalized to GAPDH). (D) Lysates of wild-type and Sirt1^–/– MEFs treated with or without 100 nM bafilomycin A1 for 20 h were subjected to immunoblotting with the indicated antibodies. Values represent the relative band intensities of Ub (normalized to GAPDH). Uncropped gel images are shown in Supplementary Figure.

Figure 3. Hsp70-dependent protein quality control is impaired in Sirt1^–/– MEFs.

(A) Immunoblotting of lysates from wild-type and Sirt1^–/– MEFs. (B) Degradation of pVHL was monitored by immunoblotting. HA-tagged pVHL was transfected into wild-type and Sirt1^–/– MEFs, and 100 μg/ml cycloheximide (CHX) was added prior to harvest at the indicated times. Actin serves as a loading control. (C) Cycloheximide chase as described in (B) was performed in wild-type, Sirt1^–/–, Sirt1^–/– transfected with wild-type Sirt1 or Hsp70, and wild-type MEFs treated with 10 μM MG132. Protein levels of HA-pVHL were quantified. Actin was used for normalization. The data represent means ± SEM from three independent experiments. Uncropped gel images are shown in Supplementary Figure.

Figure 4. Downregulation of Hsp70 is not the sole cause of aberrant protein quality control in Sirt1^–/– MEFs.

(A) Immunoblotting of lysates from wild-type and Sirt1^–/– MEFs transfected with empty vector or HA-tagged Hsp70. Values represent the relative band intensities of Ub (normalized to Actin). (B) Immunoblotting of lysates from wild-type, Sirt1^–/–, and Sirt1^–/– transfected with wild-type Sirt1 MEFs. Values represent the relative band intensities of Ub (normalized to GAPDH). Uncropped gel images are shown in Supplementary Figure.

Figure 5. Sirt1-deficiency does not lead to impairment in the induction of Hsp70 after heat shock.

(A) Immunofluorescence with anti-ubiquitin antibodies of wild-type and Sirt1^–/– MEFs treated with or without heat shock (42 °C for 30 min). Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. Values represent the relative intensity of Ub staining per cell. (B) Wild-type and Sirt1^–/– MEFs were treated with or without heat shock (42 °C for 30 min). Heated cells were then immediately harvested or allowed to recover at 37 °C for 5 h, and cell lysates were subjected to immunoblotting. Values represent the relative band intensities of Ub (normalized to GAPDH). (C,D) Wild-type and Sirt1^–/– mice were treated with or without heat shock (42 °C for 45 min). Each group consisted of four or six littermates. Heated mice were then immediately sacrificed or allowed to recover at room temperature for 7 h. The lysates from their lung (C) and liver (D) were subjected to immunoblotting (left panels). Values represent the relative band intensities of Ub (normalized to GAPDH). Statistical comparisons were made by Student’s t-test for two tailed unpaired samples. The data represent means ± SEM. N.S. indicates not significant. Uncropped gel images are shown in Supplementary Figure.