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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

Activation of CMA protects against double strand DNA damagea. Representative immunoblot for LAMP-2A in mouse fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)) after 24h of treatment with etoposide. b. L2A mRNA levels cells treated with etoposide for 24h and 24h after treatment. Paraquat (PQ) is shown as positive control, n=3 wells from 3 independent experiment. c,b. Immunofluorescence for γH2AX in Ctr cells transfected with GFP or GFP-hL2A and exposed to etoposide for 24h. n = 3 independent experiments where the number of total cells counted per experimental condition were more than 100 (>5 fields, average 20 cells/field) (c) and representative images in single and merged channels images (d). Dashed lines: Cell profiles. e–g. Viability (e) and γH2AX levels (f,g) of cells pretreated or not with the CMA activator AR7 after 24h of etoposide treatment (damage) or 24h after removing the drug (recovery), n=3 independent experiments. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05 or **P<0.005. Scale bar: 10μm. Full gels are shown in Supplementary Fig. 8.
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Figure 3: Activation of CMA protects against double strand DNA damagea. Representative immunoblot for LAMP-2A in mouse fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)) after 24h of treatment with etoposide. b. L2A mRNA levels cells treated with etoposide for 24h and 24h after treatment. Paraquat (PQ) is shown as positive control, n=3 wells from 3 independent experiment. c,b. Immunofluorescence for γH2AX in Ctr cells transfected with GFP or GFP-hL2A and exposed to etoposide for 24h. n = 3 independent experiments where the number of total cells counted per experimental condition were more than 100 (>5 fields, average 20 cells/field) (c) and representative images in single and merged channels images (d). Dashed lines: Cell profiles. e–g. Viability (e) and γH2AX levels (f,g) of cells pretreated or not with the CMA activator AR7 after 24h of etoposide treatment (damage) or 24h after removing the drug (recovery), n=3 independent experiments. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05 or **P<0.005. Scale bar: 10μm. Full gels are shown in Supplementary Fig. 8.

Mentions: Upregulation of CMA activity in response to etoposide coincided with an increase in LAMP-2A levels, both at the protein (Fig. 3a) and mRNA levels (Fig. 3b, the pro-oxidant paraquat, a well-characterized activator of CMA21 is shown as positive control). Under our experimental conditions, this increase was not observed for LAMP1 or LAMP2B, upon exposure to etoposide or γ-radiation (Supplementary Fig. 4c). Furthermore, genetic and chemical enhancements of CMA activity were protective against etoposide. Overexpression of LAMP-2A, shown to enhance CMA activity in cultured cells13, reduced the percentage of cells with γH2AX foci after exposure to etoposide (Fig. 3c,d), whereas overexpression of LAMP-2B at similar levels did not resulted in noticeable reduction in γH2AX foci (Supplementary Fig. 2d). Likewise, treatment of cultured cells with AR7, a novel retinoic acid derivative that selectively activates CMA22, significantly improved cellular viability upon etoposide treatment (Fig. 3f) and reduced DNA damage (Fig. 3g). Overall, our findings demonstrate that CMA is upregulated as part of the cellular response to DNA damage and that increased CMA activity is effective in reducing DNA damage.


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

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

Activation of CMA protects against double strand DNA damagea. Representative immunoblot for LAMP-2A in mouse fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)) after 24h of treatment with etoposide. b. L2A mRNA levels cells treated with etoposide for 24h and 24h after treatment. Paraquat (PQ) is shown as positive control, n=3 wells from 3 independent experiment. c,b. Immunofluorescence for γH2AX in Ctr cells transfected with GFP or GFP-hL2A and exposed to etoposide for 24h. n = 3 independent experiments where the number of total cells counted per experimental condition were more than 100 (>5 fields, average 20 cells/field) (c) and representative images in single and merged channels images (d). Dashed lines: Cell profiles. e–g. Viability (e) and γH2AX levels (f,g) of cells pretreated or not with the CMA activator AR7 after 24h of etoposide treatment (damage) or 24h after removing the drug (recovery), n=3 independent experiments. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05 or **P<0.005. Scale bar: 10μm. Full gels are shown in Supplementary Fig. 8.
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Figure 3: Activation of CMA protects against double strand DNA damagea. Representative immunoblot for LAMP-2A in mouse fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)) after 24h of treatment with etoposide. b. L2A mRNA levels cells treated with etoposide for 24h and 24h after treatment. Paraquat (PQ) is shown as positive control, n=3 wells from 3 independent experiment. c,b. Immunofluorescence for γH2AX in Ctr cells transfected with GFP or GFP-hL2A and exposed to etoposide for 24h. n = 3 independent experiments where the number of total cells counted per experimental condition were more than 100 (>5 fields, average 20 cells/field) (c) and representative images in single and merged channels images (d). Dashed lines: Cell profiles. e–g. Viability (e) and γH2AX levels (f,g) of cells pretreated or not with the CMA activator AR7 after 24h of etoposide treatment (damage) or 24h after removing the drug (recovery), n=3 independent experiments. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05 or **P<0.005. Scale bar: 10μm. Full gels are shown in Supplementary Fig. 8.
Mentions: Upregulation of CMA activity in response to etoposide coincided with an increase in LAMP-2A levels, both at the protein (Fig. 3a) and mRNA levels (Fig. 3b, the pro-oxidant paraquat, a well-characterized activator of CMA21 is shown as positive control). Under our experimental conditions, this increase was not observed for LAMP1 or LAMP2B, upon exposure to etoposide or γ-radiation (Supplementary Fig. 4c). Furthermore, genetic and chemical enhancements of CMA activity were protective against etoposide. Overexpression of LAMP-2A, shown to enhance CMA activity in cultured cells13, reduced the percentage of cells with γH2AX foci after exposure to etoposide (Fig. 3c,d), whereas overexpression of LAMP-2B at similar levels did not resulted in noticeable reduction in γH2AX foci (Supplementary Fig. 2d). Likewise, treatment of cultured cells with AR7, a novel retinoic acid derivative that selectively activates CMA22, significantly improved cellular viability upon etoposide treatment (Fig. 3f) and reduced DNA damage (Fig. 3g). Overall, our findings demonstrate that CMA is upregulated as part of the cellular response to DNA damage and that increased CMA activity is effective in reducing DNA damage.

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