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Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage.

Park C, Suh Y, Cuervo AM - Nat Commun (2015)

Bottom Line: Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing.Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability.We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA [2] Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461, USA [3] Institute for Aging Research, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

ABSTRACT
Chaperone-mediated autophagy (CMA) is activated in response to cellular stressors to prevent cellular proteotoxicity through selective degradation of altered proteins in lysosomes. Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing. Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability. This role of CMA in genome quality control is exerted through regulated degradation of activated checkpoint kinase 1 (Chk1) by this pathway after the genotoxic insult. Nuclear accumulation of Chk1 in CMA-deficient cells compromises cell cycle progression and prolongs the time that DNA damage persists in these cells. Furthermore, blockage of CMA leads to hyperphosphorylation and destabilization of the MRN (Mre11-Rad50-Nbs1) complex, which participates in early steps of particular DNA repair pathways. We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

No MeSH data available.


Related in: MedlinePlus

CMA blockage leads to inefficient DNA repair and alterations in cell cycle check pointa. Representative immunoblot for γH2AX in mouse fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)) untreated (−) or at the indicated times after treatment with etoposide. Right: Decay of γH2AX in both groups of cells. Half-lives are indicated, n=3 wells in 2 independent experiments. Values are average and range. b. Representative FACS analysis profile of the same cells after propidium iodine labeling. c. Cell cycle distribution of the cells subjected to FACS. Values are average and range of two different experiments. d,e. Levels of Chk1 proteins in the same cells and in cells knock-down for Atg7 (Atg7(−)). Representative immunoblot (d) and quantification of levels of pChk1 (S345) and total Chk1 (e), n=4 independent experiments. f,g. Immunoblot of the same cells untreated (−) or at the indicated times of etoposide treatment (f) and quantification of levels of pChk1 (S345) and total Chk1 (g), n=2 independent experiments. Values are average and range. h, Representative immunoblots for Chk1 at different times after exposure to 50Gy γ-irradiation in the indicated cell types. The experiments were repeated 2 times. i. Cell cycle distribution of Ctr and L2A(−) cells untreated (None) or treated with etoposide for 24h in presence or absence of ATR inhibitors. The experiments were repeated 2 times. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05, **P <0.005 or ***P <0.0005. Full gels are shown in Supplementary Fig. 8.
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Figure 4: CMA blockage leads to inefficient DNA repair and alterations in cell cycle check pointa. Representative immunoblot for γH2AX in mouse fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)) untreated (−) or at the indicated times after treatment with etoposide. Right: Decay of γH2AX in both groups of cells. Half-lives are indicated, n=3 wells in 2 independent experiments. Values are average and range. b. Representative FACS analysis profile of the same cells after propidium iodine labeling. c. Cell cycle distribution of the cells subjected to FACS. Values are average and range of two different experiments. d,e. Levels of Chk1 proteins in the same cells and in cells knock-down for Atg7 (Atg7(−)). Representative immunoblot (d) and quantification of levels of pChk1 (S345) and total Chk1 (e), n=4 independent experiments. f,g. Immunoblot of the same cells untreated (−) or at the indicated times of etoposide treatment (f) and quantification of levels of pChk1 (S345) and total Chk1 (g), n=2 independent experiments. Values are average and range. h, Representative immunoblots for Chk1 at different times after exposure to 50Gy γ-irradiation in the indicated cell types. The experiments were repeated 2 times. i. Cell cycle distribution of Ctr and L2A(−) cells untreated (None) or treated with etoposide for 24h in presence or absence of ATR inhibitors. The experiments were repeated 2 times. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05, **P <0.005 or ***P <0.0005. Full gels are shown in Supplementary Fig. 8.

Mentions: To determine whether higher levels of DNA DSBs in cells defective in CMA were due to increased DNA damage or delayed repair, we performed a time-course analysis post 24h etoposide treatment. While γH2AX levels gradually decreased in Ctrl cells as a result of DNA repair, the decrease in L2A(−) cells was markedly slowed down (Fig. 4a). A similar longer persistence of γH2AX was observed after γ-irradiation Supplementary Fig. 1d). These results suggest that the higher content of DNA DSBs in cells defective in CMA was due for the most part, to inefficient DNA repair.


Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage.

Park C, Suh Y, Cuervo AM - Nat Commun (2015)

CMA blockage leads to inefficient DNA repair and alterations in cell cycle check pointa. Representative immunoblot for γH2AX in mouse fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)) untreated (−) or at the indicated times after treatment with etoposide. Right: Decay of γH2AX in both groups of cells. Half-lives are indicated, n=3 wells in 2 independent experiments. Values are average and range. b. Representative FACS analysis profile of the same cells after propidium iodine labeling. c. Cell cycle distribution of the cells subjected to FACS. Values are average and range of two different experiments. d,e. Levels of Chk1 proteins in the same cells and in cells knock-down for Atg7 (Atg7(−)). Representative immunoblot (d) and quantification of levels of pChk1 (S345) and total Chk1 (e), n=4 independent experiments. f,g. Immunoblot of the same cells untreated (−) or at the indicated times of etoposide treatment (f) and quantification of levels of pChk1 (S345) and total Chk1 (g), n=2 independent experiments. Values are average and range. h, Representative immunoblots for Chk1 at different times after exposure to 50Gy γ-irradiation in the indicated cell types. The experiments were repeated 2 times. i. Cell cycle distribution of Ctr and L2A(−) cells untreated (None) or treated with etoposide for 24h in presence or absence of ATR inhibitors. The experiments were repeated 2 times. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05, **P <0.005 or ***P <0.0005. Full gels are shown in Supplementary Fig. 8.
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Figure 4: CMA blockage leads to inefficient DNA repair and alterations in cell cycle check pointa. Representative immunoblot for γH2AX in mouse fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)) untreated (−) or at the indicated times after treatment with etoposide. Right: Decay of γH2AX in both groups of cells. Half-lives are indicated, n=3 wells in 2 independent experiments. Values are average and range. b. Representative FACS analysis profile of the same cells after propidium iodine labeling. c. Cell cycle distribution of the cells subjected to FACS. Values are average and range of two different experiments. d,e. Levels of Chk1 proteins in the same cells and in cells knock-down for Atg7 (Atg7(−)). Representative immunoblot (d) and quantification of levels of pChk1 (S345) and total Chk1 (e), n=4 independent experiments. f,g. Immunoblot of the same cells untreated (−) or at the indicated times of etoposide treatment (f) and quantification of levels of pChk1 (S345) and total Chk1 (g), n=2 independent experiments. Values are average and range. h, Representative immunoblots for Chk1 at different times after exposure to 50Gy γ-irradiation in the indicated cell types. The experiments were repeated 2 times. i. Cell cycle distribution of Ctr and L2A(−) cells untreated (None) or treated with etoposide for 24h in presence or absence of ATR inhibitors. The experiments were repeated 2 times. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05, **P <0.005 or ***P <0.0005. Full gels are shown in Supplementary Fig. 8.
Mentions: To determine whether higher levels of DNA DSBs in cells defective in CMA were due to increased DNA damage or delayed repair, we performed a time-course analysis post 24h etoposide treatment. While γH2AX levels gradually decreased in Ctrl cells as a result of DNA repair, the decrease in L2A(−) cells was markedly slowed down (Fig. 4a). A similar longer persistence of γH2AX was observed after γ-irradiation Supplementary Fig. 1d). These results suggest that the higher content of DNA DSBs in cells defective in CMA was due for the most part, to inefficient DNA repair.

Bottom Line: Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing.Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability.We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA [2] Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461, USA [3] Institute for Aging Research, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

ABSTRACT
Chaperone-mediated autophagy (CMA) is activated in response to cellular stressors to prevent cellular proteotoxicity through selective degradation of altered proteins in lysosomes. Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing. Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability. This role of CMA in genome quality control is exerted through regulated degradation of activated checkpoint kinase 1 (Chk1) by this pathway after the genotoxic insult. Nuclear accumulation of Chk1 in CMA-deficient cells compromises cell cycle progression and prolongs the time that DNA damage persists in these cells. Furthermore, blockage of CMA leads to hyperphosphorylation and destabilization of the MRN (Mre11-Rad50-Nbs1) complex, which participates in early steps of particular DNA repair pathways. We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

No MeSH data available.


Related in: MedlinePlus