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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 is upregulated in response to double strand DNA damagea. Lysosomal protein degradation in mouse fibroblasts treated with etoposide for 24h, n=6 wells in 3 independent experiments. b,c. CMA activity in cells expressing a photoactivatable CMA reporter and treated with increasing concentrations of etoposide for the indicated times (b) or for different times at the indicated concentrations of etoposide (c). Arrow indicates etoposide removal after 12h of treatment (grey lane) Right: Representative images of the indicated conditions. Inserts in b show higher magnification images. n=3 independent experiments, >200 cells/experiment were counted. d,e. CMA activity measured as in b at the indicated times after UV exposure (d) or at 12 or 24h after the indicated treatments (e). n=3 independent experiments, >200 cells/experiment. f. CMA activity against a pool of radiolabeled cytosolic proteins of lysosomes isolated from livers of mice untreated (none) or at different times after a single etoposide injection. n=3 independent experiments and the means of two animals per group are shown with average value (red line). All values are mean+s.e.m except panel F that are mean and range. (unpaired two-tailed t-test). *P<0.005 or ***P<0.0005. Scale bar: 10μm.
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Figure 2: CMA is upregulated in response to double strand DNA damagea. Lysosomal protein degradation in mouse fibroblasts treated with etoposide for 24h, n=6 wells in 3 independent experiments. b,c. CMA activity in cells expressing a photoactivatable CMA reporter and treated with increasing concentrations of etoposide for the indicated times (b) or for different times at the indicated concentrations of etoposide (c). Arrow indicates etoposide removal after 12h of treatment (grey lane) Right: Representative images of the indicated conditions. Inserts in b show higher magnification images. n=3 independent experiments, >200 cells/experiment were counted. d,e. CMA activity measured as in b at the indicated times after UV exposure (d) or at 12 or 24h after the indicated treatments (e). n=3 independent experiments, >200 cells/experiment. f. CMA activity against a pool of radiolabeled cytosolic proteins of lysosomes isolated from livers of mice untreated (none) or at different times after a single etoposide injection. n=3 independent experiments and the means of two animals per group are shown with average value (red line). All values are mean+s.e.m except panel F that are mean and range. (unpaired two-tailed t-test). *P<0.005 or ***P<0.0005. Scale bar: 10μm.

Mentions: Since the connection between CMA and DNA damage had not been studied before, we next determined if genotoxic stress induced CMA. Etoposide induced a dose-dependent increase in lysosomal degradation (Fig. 2a) but not by macroautophagy, because flux of LC3 and p62 did not change significantly under these experimental conditions (Supplementary Fig. 3). In contrast, a CMA-specific fluorescent substrate (photoactivable KFERQ-dendra reporter (modified from20) that highlights lysosomes as fluorescent puncta when CMA is activated, revealed that CMA activity increased in a dose-dependent manner during the etoposide treatment (Fig. 2b) and remained upregulated even 12h after the removal of etoposide (Fig. 2b). A similar dose- and time-dependent increase in CMA activity was observed upon exposure to other genotoxic agents (Fig. 2d,e and Supplementary Fig. 4a,b). We confirmed that upregulation of CMA in response to DNA damage also occurs in vivo since intact lysosomes isolated from the livers of mice treated with a single intraperitoneal injection of etoposide showed a marked increase in their ability to take up and degrade radiolabeled cytosolic CMA substrates, the most direct assay for quantification of CMA (Fig. 2f). Maximal activation of CMA was reached at 12h post injection and was still evident 24h after the treatment.


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

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

CMA is upregulated in response to double strand DNA damagea. Lysosomal protein degradation in mouse fibroblasts treated with etoposide for 24h, n=6 wells in 3 independent experiments. b,c. CMA activity in cells expressing a photoactivatable CMA reporter and treated with increasing concentrations of etoposide for the indicated times (b) or for different times at the indicated concentrations of etoposide (c). Arrow indicates etoposide removal after 12h of treatment (grey lane) Right: Representative images of the indicated conditions. Inserts in b show higher magnification images. n=3 independent experiments, >200 cells/experiment were counted. d,e. CMA activity measured as in b at the indicated times after UV exposure (d) or at 12 or 24h after the indicated treatments (e). n=3 independent experiments, >200 cells/experiment. f. CMA activity against a pool of radiolabeled cytosolic proteins of lysosomes isolated from livers of mice untreated (none) or at different times after a single etoposide injection. n=3 independent experiments and the means of two animals per group are shown with average value (red line). All values are mean+s.e.m except panel F that are mean and range. (unpaired two-tailed t-test). *P<0.005 or ***P<0.0005. Scale bar: 10μm.
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Figure 2: CMA is upregulated in response to double strand DNA damagea. Lysosomal protein degradation in mouse fibroblasts treated with etoposide for 24h, n=6 wells in 3 independent experiments. b,c. CMA activity in cells expressing a photoactivatable CMA reporter and treated with increasing concentrations of etoposide for the indicated times (b) or for different times at the indicated concentrations of etoposide (c). Arrow indicates etoposide removal after 12h of treatment (grey lane) Right: Representative images of the indicated conditions. Inserts in b show higher magnification images. n=3 independent experiments, >200 cells/experiment were counted. d,e. CMA activity measured as in b at the indicated times after UV exposure (d) or at 12 or 24h after the indicated treatments (e). n=3 independent experiments, >200 cells/experiment. f. CMA activity against a pool of radiolabeled cytosolic proteins of lysosomes isolated from livers of mice untreated (none) or at different times after a single etoposide injection. n=3 independent experiments and the means of two animals per group are shown with average value (red line). All values are mean+s.e.m except panel F that are mean and range. (unpaired two-tailed t-test). *P<0.005 or ***P<0.0005. Scale bar: 10μm.
Mentions: Since the connection between CMA and DNA damage had not been studied before, we next determined if genotoxic stress induced CMA. Etoposide induced a dose-dependent increase in lysosomal degradation (Fig. 2a) but not by macroautophagy, because flux of LC3 and p62 did not change significantly under these experimental conditions (Supplementary Fig. 3). In contrast, a CMA-specific fluorescent substrate (photoactivable KFERQ-dendra reporter (modified from20) that highlights lysosomes as fluorescent puncta when CMA is activated, revealed that CMA activity increased in a dose-dependent manner during the etoposide treatment (Fig. 2b) and remained upregulated even 12h after the removal of etoposide (Fig. 2b). A similar dose- and time-dependent increase in CMA activity was observed upon exposure to other genotoxic agents (Fig. 2d,e and Supplementary Fig. 4a,b). We confirmed that upregulation of CMA in response to DNA damage also occurs in vivo since intact lysosomes isolated from the livers of mice treated with a single intraperitoneal injection of etoposide showed a marked increase in their ability to take up and degrade radiolabeled cytosolic CMA substrates, the most direct assay for quantification of CMA (Fig. 2f). Maximal activation of CMA was reached at 12h post injection and was still evident 24h after the treatment.

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