Limits...
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 leads to instability of the MRN DNA repair complexa,b. Representative immunoblots of cells control (Ctr) or knock-down for L2A (L2A−) or Atg7 (Atg7−) untreated (none) or treated with the indicated concentrations of etoposide for 12h. LAMP-2A (L-2A) is shown as control for knock-down. Right: Changes in levels of the indicated proteins calculated by immunoblot densitometry, n=4 independent experiments. c. Representative immunoblot of Ctr and L2A− cells untreated (unt) or treated with 100μM for the indicated times. Arrows: levels of shift in the protein molecular weight. Bottom: Changes in levels of the indicated proteins calculated by immunoblot densitometry, n= 2 independent experiments. d. Immunoblot for MRN complex proteins after extended electrophoretic run in Ctr, L2A(−) or cells knockdown for Atg7(Atg7(−)) treated as in b with etoposide for 12h. e. Immunoblot for Mre11 in the same cells at the indicated times after exposure to γ-irradiation. All values are mean+s.e.m except panel c that are average and range. (unpaired two-tailed t-test). *P <0.005. Full gels are shown in Supplementary Fig. 8.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400843&req=5

Figure 8: Blockage of CMA leads to instability of the MRN DNA repair complexa,b. Representative immunoblots of cells control (Ctr) or knock-down for L2A (L2A−) or Atg7 (Atg7−) untreated (none) or treated with the indicated concentrations of etoposide for 12h. LAMP-2A (L-2A) is shown as control for knock-down. Right: Changes in levels of the indicated proteins calculated by immunoblot densitometry, n=4 independent experiments. c. Representative immunoblot of Ctr and L2A− cells untreated (unt) or treated with 100μM for the indicated times. Arrows: levels of shift in the protein molecular weight. Bottom: Changes in levels of the indicated proteins calculated by immunoblot densitometry, n= 2 independent experiments. d. Immunoblot for MRN complex proteins after extended electrophoretic run in Ctr, L2A(−) or cells knockdown for Atg7(Atg7(−)) treated as in b with etoposide for 12h. e. Immunoblot for Mre11 in the same cells at the indicated times after exposure to γ-irradiation. All values are mean+s.e.m except panel c that are average and range. (unpaired two-tailed t-test). *P <0.005. Full gels are shown in Supplementary Fig. 8.

Mentions: To understand the mechanism by which nuclear accumulation of Chk1 upon CMA blockage leads to the higher DNA damage (Fig. 1) and delayed DSB repair (Fig. 4) observed in L2A(−) cells, we analyzed possible changes in the protein components involved in the early steps of the DSB repair pathway, in particular, the MRN (Mre11-Rad50-Nbs1) complex, which binds to DSB prior to DNA repair by recombination and signals to the cell cycle checkpoints. Etoposide exposure led to a dose-dependent reduction in the levels of Mre11, Rad50, and Nbs1 in L2A(−) cells as compared to Ctr cells (Fig. 8a), that inversely correlated with the increase in the levels of Chk1 and DNA DSBs (Fig. 8b). Blockage of macroautophagy did not significantly change the levels of these proteins (Fig. 8a). Time course analysis after etoposide exposure also revealed differences in the apparent molecular weight of MRN proteins in L2A(−) cells. Thus, etoposide induced a shift in the molecular weight of Mre11 and Nbs1 in Ctr cells (level “1” in the images) and both proteins returned to their basal molecular weight gradually with time (Fig. 8c). In contrast, in L2A(−) cells already at 2 hour post-etoposide exposure, both Mre11 and Nbs1 were shifted to a higher level of phosphorylation (level “2”) (Fig. 8c) that was not visible upon blockage of macroautophagy (Fig. 8d). Exposure of L2A(−) to a different genotoxic agent, γ-irradiation, resulted in similar changes in levels and molecular weight of MRN proteins (Mre11 shown in Fig. 8e).


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 leads to instability of the MRN DNA repair complexa,b. Representative immunoblots of cells control (Ctr) or knock-down for L2A (L2A−) or Atg7 (Atg7−) untreated (none) or treated with the indicated concentrations of etoposide for 12h. LAMP-2A (L-2A) is shown as control for knock-down. Right: Changes in levels of the indicated proteins calculated by immunoblot densitometry, n=4 independent experiments. c. Representative immunoblot of Ctr and L2A− cells untreated (unt) or treated with 100μM for the indicated times. Arrows: levels of shift in the protein molecular weight. Bottom: Changes in levels of the indicated proteins calculated by immunoblot densitometry, n= 2 independent experiments. d. Immunoblot for MRN complex proteins after extended electrophoretic run in Ctr, L2A(−) or cells knockdown for Atg7(Atg7(−)) treated as in b with etoposide for 12h. e. Immunoblot for Mre11 in the same cells at the indicated times after exposure to γ-irradiation. All values are mean+s.e.m except panel c that are average and range. (unpaired two-tailed t-test). *P <0.005. Full gels are shown in Supplementary Fig. 8.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400843&req=5

Figure 8: Blockage of CMA leads to instability of the MRN DNA repair complexa,b. Representative immunoblots of cells control (Ctr) or knock-down for L2A (L2A−) or Atg7 (Atg7−) untreated (none) or treated with the indicated concentrations of etoposide for 12h. LAMP-2A (L-2A) is shown as control for knock-down. Right: Changes in levels of the indicated proteins calculated by immunoblot densitometry, n=4 independent experiments. c. Representative immunoblot of Ctr and L2A− cells untreated (unt) or treated with 100μM for the indicated times. Arrows: levels of shift in the protein molecular weight. Bottom: Changes in levels of the indicated proteins calculated by immunoblot densitometry, n= 2 independent experiments. d. Immunoblot for MRN complex proteins after extended electrophoretic run in Ctr, L2A(−) or cells knockdown for Atg7(Atg7(−)) treated as in b with etoposide for 12h. e. Immunoblot for Mre11 in the same cells at the indicated times after exposure to γ-irradiation. All values are mean+s.e.m except panel c that are average and range. (unpaired two-tailed t-test). *P <0.005. Full gels are shown in Supplementary Fig. 8.
Mentions: To understand the mechanism by which nuclear accumulation of Chk1 upon CMA blockage leads to the higher DNA damage (Fig. 1) and delayed DSB repair (Fig. 4) observed in L2A(−) cells, we analyzed possible changes in the protein components involved in the early steps of the DSB repair pathway, in particular, the MRN (Mre11-Rad50-Nbs1) complex, which binds to DSB prior to DNA repair by recombination and signals to the cell cycle checkpoints. Etoposide exposure led to a dose-dependent reduction in the levels of Mre11, Rad50, and Nbs1 in L2A(−) cells as compared to Ctr cells (Fig. 8a), that inversely correlated with the increase in the levels of Chk1 and DNA DSBs (Fig. 8b). Blockage of macroautophagy did not significantly change the levels of these proteins (Fig. 8a). Time course analysis after etoposide exposure also revealed differences in the apparent molecular weight of MRN proteins in L2A(−) cells. Thus, etoposide induced a shift in the molecular weight of Mre11 and Nbs1 in Ctr cells (level “1” in the images) and both proteins returned to their basal molecular weight gradually with time (Fig. 8c). In contrast, in L2A(−) cells already at 2 hour post-etoposide exposure, both Mre11 and Nbs1 were shifted to a higher level of phosphorylation (level “2”) (Fig. 8c) that was not visible upon blockage of macroautophagy (Fig. 8d). Exposure of L2A(−) to a different genotoxic agent, γ-irradiation, resulted in similar changes in levels and molecular weight of MRN proteins (Mre11 shown in Fig. 8e).

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