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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

Blockage of CMA increases cellular susceptibility to genotoxicitya,b. Viability of mouse fibroblasts (NIH3T3) Ctr, L2A(−) or knock-down for Atg7 (Atg7 (−)) after 24h of etoposide treatment (a) or the indicated genotoxic insults (b), n=6 wells in 3 independent experiments. c. Representative immunoblot in the same cells as in a (top) and quantification of phosphorylated γH2AX relative to untreated cells (bottom), n=5 independent experiments. d. Immunofluorescence for γH2AX and nuclear staining by DAPI in the same cells. Scale bar: 5μm. e. Percentage of cells shown in d, with nuclear γH2AX foci (n = 3 independent experiments where the number of total cells counted per experimental condition were more than 100 (>5 fields, average 20 cells/field)). f. Neutral comet assay in the same cells, after 24h etoposide treatment. Scale bar: 10μm.g. Quantification of tail moment in the same cells (n = 3 experiments where more than 35 cells (>7 fields, ~.5cells/field) were counted per experiment. h. Immunofluorescence for γH2AX in L2A(−) cells transfected with GFP-hL2A and treated as in f. Nuclei are highlighted with DAPI. Individual and merged channels are shown. Dashed lines: Cell profiles. Scale bar: 10μm. Quantification of percent of cells with nuclear foci after GFP or GFP-hL2A transfection is shown, n=3 independent experiments. 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 1: Blockage of CMA increases cellular susceptibility to genotoxicitya,b. Viability of mouse fibroblasts (NIH3T3) Ctr, L2A(−) or knock-down for Atg7 (Atg7 (−)) after 24h of etoposide treatment (a) or the indicated genotoxic insults (b), n=6 wells in 3 independent experiments. c. Representative immunoblot in the same cells as in a (top) and quantification of phosphorylated γH2AX relative to untreated cells (bottom), n=5 independent experiments. d. Immunofluorescence for γH2AX and nuclear staining by DAPI in the same cells. Scale bar: 5μm. e. Percentage of cells shown in d, with nuclear γH2AX foci (n = 3 independent experiments where the number of total cells counted per experimental condition were more than 100 (>5 fields, average 20 cells/field)). f. Neutral comet assay in the same cells, after 24h etoposide treatment. Scale bar: 10μm.g. Quantification of tail moment in the same cells (n = 3 experiments where more than 35 cells (>7 fields, ~.5cells/field) were counted per experiment. h. Immunofluorescence for γH2AX in L2A(−) cells transfected with GFP-hL2A and treated as in f. Nuclei are highlighted with DAPI. Individual and merged channels are shown. Dashed lines: Cell profiles. Scale bar: 10μm. Quantification of percent of cells with nuclear foci after GFP or GFP-hL2A transfection is shown, n=3 independent experiments. 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 investigate if CMA confers cellular resistance against DNA damage, we used etoposide, an agent that induces DNA double strand breaks (DSBs)18, in mouse fibroblasts control (Ctr) or knocked down for LAMP-2A (L2A(−) cells) or for Atg7 (Atg7(−) cells) to reduce CMA or macroautophagy activity, respectively19. L2A(−) cells were significantly more vulnerable to etoposide than control or Atg7(−) cells (Fig. 1a and Supplementary Fig. 1a). Embryonic fibroblasts from mouse for L2A displayed similar higher sensitivity to etoposide, discarding possible off-target effects of the knock-down (Supplementary Fig. 1b). Exposure to other genotoxic agents including alkylating agents (methylmethanesulfonate (MMS) and cisplatin), pro-oxidant agents (paraquat), inhibitors of DNA synthesis (hydroxyurea) and a class I topoisomerase inhibitor, revealed that L2A(−) cells were consistently more sensitive to all these agents than control cells (Fig. 1b). Cells with compromised macroautophagy also showed higher sensitivity to the two alkylating agents but not to the other genotoxic insults (Fig. 1b).


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

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

Blockage of CMA increases cellular susceptibility to genotoxicitya,b. Viability of mouse fibroblasts (NIH3T3) Ctr, L2A(−) or knock-down for Atg7 (Atg7 (−)) after 24h of etoposide treatment (a) or the indicated genotoxic insults (b), n=6 wells in 3 independent experiments. c. Representative immunoblot in the same cells as in a (top) and quantification of phosphorylated γH2AX relative to untreated cells (bottom), n=5 independent experiments. d. Immunofluorescence for γH2AX and nuclear staining by DAPI in the same cells. Scale bar: 5μm. e. Percentage of cells shown in d, with nuclear γH2AX foci (n = 3 independent experiments where the number of total cells counted per experimental condition were more than 100 (>5 fields, average 20 cells/field)). f. Neutral comet assay in the same cells, after 24h etoposide treatment. Scale bar: 10μm.g. Quantification of tail moment in the same cells (n = 3 experiments where more than 35 cells (>7 fields, ~.5cells/field) were counted per experiment. h. Immunofluorescence for γH2AX in L2A(−) cells transfected with GFP-hL2A and treated as in f. Nuclei are highlighted with DAPI. Individual and merged channels are shown. Dashed lines: Cell profiles. Scale bar: 10μm. Quantification of percent of cells with nuclear foci after GFP or GFP-hL2A transfection is shown, n=3 independent experiments. 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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Related In: Results  -  Collection

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Figure 1: Blockage of CMA increases cellular susceptibility to genotoxicitya,b. Viability of mouse fibroblasts (NIH3T3) Ctr, L2A(−) or knock-down for Atg7 (Atg7 (−)) after 24h of etoposide treatment (a) or the indicated genotoxic insults (b), n=6 wells in 3 independent experiments. c. Representative immunoblot in the same cells as in a (top) and quantification of phosphorylated γH2AX relative to untreated cells (bottom), n=5 independent experiments. d. Immunofluorescence for γH2AX and nuclear staining by DAPI in the same cells. Scale bar: 5μm. e. Percentage of cells shown in d, with nuclear γH2AX foci (n = 3 independent experiments where the number of total cells counted per experimental condition were more than 100 (>5 fields, average 20 cells/field)). f. Neutral comet assay in the same cells, after 24h etoposide treatment. Scale bar: 10μm.g. Quantification of tail moment in the same cells (n = 3 experiments where more than 35 cells (>7 fields, ~.5cells/field) were counted per experiment. h. Immunofluorescence for γH2AX in L2A(−) cells transfected with GFP-hL2A and treated as in f. Nuclei are highlighted with DAPI. Individual and merged channels are shown. Dashed lines: Cell profiles. Scale bar: 10μm. Quantification of percent of cells with nuclear foci after GFP or GFP-hL2A transfection is shown, n=3 independent experiments. 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 investigate if CMA confers cellular resistance against DNA damage, we used etoposide, an agent that induces DNA double strand breaks (DSBs)18, in mouse fibroblasts control (Ctr) or knocked down for LAMP-2A (L2A(−) cells) or for Atg7 (Atg7(−) cells) to reduce CMA or macroautophagy activity, respectively19. L2A(−) cells were significantly more vulnerable to etoposide than control or Atg7(−) cells (Fig. 1a and Supplementary Fig. 1a). Embryonic fibroblasts from mouse for L2A displayed similar higher sensitivity to etoposide, discarding possible off-target effects of the knock-down (Supplementary Fig. 1b). Exposure to other genotoxic agents including alkylating agents (methylmethanesulfonate (MMS) and cisplatin), pro-oxidant agents (paraquat), inhibitors of DNA synthesis (hydroxyurea) and a class I topoisomerase inhibitor, revealed that L2A(−) cells were consistently more sensitive to all these agents than control cells (Fig. 1b). Cells with compromised macroautophagy also showed higher sensitivity to the two alkylating agents but not to the other genotoxic insults (Fig. 1b).

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