The Role of Chaperone-mediated Autophagy in Genome Maintenance

Abstract

Preservation of cellular homeostasis requires the activities of both proteome and genome maintenance systems. However, during aging, the activity of these cellular quality control systems declines, which could contribute to the alterations in proteostasis and the increased genome stability of old organisms. Chaperone-mediated autophagy (CMA) is a highly selective type of autophagy that involves degradation in lysosomes of single cytosolic proteins that carry in their amino acid sequence a characteristic pentapeptide motif for targeting to the lysosomal membrane. CMA activity declines with age and the contribution of this CMA failure to deficient proteostasis has been well established. However, although most of the processes that assure genome maintenance are mediated by proteins, the possible impact of CMA failure in genome quality control had not been previously explored.;The goal of this thesis was to investigate the role of CMA in genome maintenance through regulated turnover of genome maintenance proteins. We hypothesized that reduced CMA activity with age could contribute to gradual deterioration of the subproteome responsible for genome maintenance.;In the first part of the study, we examined a possible link between CMA and genome maintenance by determining whether 1) CMA is activated in response to DNA damage and 2) if CMA is required for the cellular response to DNA damage. Using etoposide, a well-known DNA damaging agent that induces double-strand breaks (DSBs), we have found CMA activation in cultured mouse fibroblasts as well as in vivo, using lysosomes isolated from livers of etoposide-treated mice. Moreover, we found that blockage of CMA resulted in increased sensitivity to etoposide manifested as higher CMA and Genome Maintenance levels of DNA damage and increased cell death. These findings suggest that CMA is a necessary component of the cellular response to DNA damage. In fact, genetic or chemical upregulation of CMA led to protection against etoposide-induced genotoxicity as indicated by decreased levels of DNA damage and improved cell viability.;In the second part of the study, we have further investigated the mechanism behind the protective effect of CMA activation against DNA damage. We have found that CMA-compromised cells exhibit altered cell cycle progression due to nuclear accumulation of the cell cycle checkpoint kinase Chkl. We confirmed that Chkl accumulation in CMA-compromised cells was due to a failure to degrade this protein in response to DNA damage. In fact, we have identified Chkl as a novel bona fide CMA substrate since it is present and degraded in CMA active lysosomes and interacts with the key CMA component, Hsc70, the chaperone responsible for substrate targeting. Furthermore, targeted mutation of the KFERQ-like motif in Chk1prevented its degradation and resulted in its retention in the nucleus. Finally, we have also examined the consequences of CMA blockage in the DNA repair process and found that cells with compromised CMA display defective DNA repair due in part to instability of the DNA repair complex MRN. Interestingly, we also demonstrate that this instability is in part caused by accumulation of Chkl and dysregulation of the ATR-Chkl signaling axis.;Overall, our study presents the first connection between CMA and genome maintenance and reveals a novel regulatory role of CMA both in cell cycle control and DNA repair. The fact that the regulated degradation of Chkl by CMA in response to DNA damage permits the temporo-spatial coordination between cell cycle progression and DNA repair opens the possibility to target this pathway in disease conditions resulting from deficient DNA repair. For example, modulation of CMA activity could be an attractive therapeutic intervention to prevent age-associated diseases related to increased genome instability, such as cancer.