Abstract
A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.
Highlights
The PI3K-like serine/threonine checkpoint kinase ataxia telangiectasia mutated (ATM)functions as a central regulator of the DNA damage response (DDR), and is recruited early to DNA double-strand breaks (DSBs) by the MRE11/RAD50/NBS1 (MRN) complex (Shiloh andZiv 2013)
2018), we find that knocking out ATM in both U2OS and HEK293 results in an increase in protein aggregation (Figure 1A-C, Figure 1 – figure supplement 1A, B)
This reveals that the increased aggregation occurs only late, and argues that it does not stem from any immediate, unknown damaging effect of either CPT or Etop on mRNA or protein molecules. Together, these data indicate that the increased protein aggregation triggered by targeting ATM, ATR and topoisomerases is a late consequence of genotoxic stress. 110 Camptothecin and ATM loss drive aggregation in a cell-type dependent 111 manner To investigate the nature of the proteins that become aggregated after genotoxic stress, we subjected the SDS-insoluble protein aggregate fractions and whole cell lysates (WCL) of CPT114 treated HEK293T cells to label-free proteomics (Figure 2 – figure supplement 1A)
Summary
The PI3K-like serine/threonine checkpoint kinase ataxia telangiectasia mutated (ATM)functions as a central regulator of the DNA damage response (DDR), and is recruited early to DNA double-strand breaks (DSBs) by the MRE11/RAD50/NBS1 (MRN) complex (Shiloh andZiv 2013). Impaired function of ATM has been linked to a disruption of protein homeostasis and increased protein aggregation (Corcoles-Saez et al 2018; Lee et al 2018; Liu et al 2005). Protein homeostasis is normally maintained by protein quality control systems, including chaperones and proteolytic pathways When the capacity of protein quality control systems becomes overwhelmed during (chronic) proteotoxic stress, the stability of the proteome can no longer be sufficiently guarded, causing proteins to succumb to aggregation more readily. A loss of protein homeostasis and the accompanying widespread aggregation can have profound consequences, and is associated with a range of (degenerative) diseases, including neurodegeneration (Kampinga and Bergink 2016; Klaips, Jayaraj, and Hartl 2018; Ross and Poirier 2004)
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