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Poly-ADP-ribosylation drives loss of protein homeostasis in ATM and Mre11 deficiency

Ji-Hoon Lee, Seung W. Ryu, Nicolette A. Ender, Tanya T. Paull

Preprint posted on 27 October 2020 https://www.biorxiv.org/content/10.1101/2020.10.27.357210v1

Article now published in Molecular Cell at http://dx.doi.org/10.1016/j.molcel.2021.01.019

Poly-ADP-ribosylation lies at the crossroads of oxidative stress, DNA damage, and protein homeostasis in ataxia-telangiectasia.

Selected by Sree Rama Chaitanya

Context1-4

Maintaining genome stability is central to cellular homeostasis, failure of which results in several human maladies. In eukaryotes, Ataxia-Telangiectasia Mutated (ATM), a master regulator of DNA damage response, swiftly supports genome stability by orchestrating the phosphorylation of several proteins either to arrest cell cycle or repair damaged DNA. ATM facilitates DNA damage response under the key upstream regulation by the Mre11-Rad50-Nbs1 complex (MRN). ATM also thwarts oxidative stress (reactive oxygen species, ROS), that can work independently of the DNA damage response pathway. ATM loss-of-function mutations or deletion causes a plethora of neurodegenerative disorders, predisposition to cancer, and immunodeficiency in humans.

Previous studies demonstrated a causative link between protein aggregation and neurotoxicity in many neurodegenerative disorders. In fact, the host lab found that cells derived from ataxia-telangiectasia (A-T) patients manifest rampant protein aggregation exaggerated by oxidative stress. Now in the current study, they extended their earlier findings and investigate the mechanistic details of protein aggregation in ATM deficiency and how this is linked to A-T.

Key findings

  1. First, the authors identified aggregated proteins in ATM depleted osteosarcoma (U2OS) and glioblastoma (U87-MG) cell lines using biochemical fractionation followed by label-free mass spectrometry. They found an overlap of 84% of aggregated polypeptides between the two cell types. Furthermore, protein aggregation diminished with anti-oxidant treatment (N-acetyl cysteine, NAC).
  2. ATM-deficient cells accumulate poly-ADP-ribosylated (PAR) proteins5. As PARylation by poly-ADP-ribopolymerases (PARPs) coalesce intrinsically disordered proteins at the sites of DNA damage6, the authors envisaged a possibility of PARPs in mediating protein aggregation in ATM deficiency. They tested this by inhibiting PARPs using genetic manipulation (via shRNA) and pharmacological (veliparib). They found that PARP inhibition is sufficient to prevent protein aggregation of two tested proteins PSMB2 and CK2β, under ATM deficiency.

    (1) PARylation sensor, (2) PBZ-CK2β PARylation system. (3) Proposed model. Taken and modified from Fig 2 and 4 of Lee JH et. al., 2020 under a CC-BY 4.0 international license.
  3. The authors then utilized a fluorescent PAR sensor that constitutes an engineered PAR binding domain (PBZ) with a split-venus protein fluorescence detection system7 (fig.1). Using this PARylation imaging sensor, they demonstrated prominent pan-nuclear foci of PARylated proteins in cells inhibited of ATM or treated with arsenite (to induce protein aggregation) but not in NAC treated cells. They also custom engineered PBZ and CK2β domains (fig.2) and figured that PBZ-CK2β PARylated fluorescent loci pop up in the nucleus when ATM is inhibited but not in NAC treated cells. Intriguingly, they observed PBZ-CK2β PARylated foci in ATM mutant deficient in ROS-mediated activation (C2991L, CL) and kinase-dead (D2889A, DA) but not in the mutant deficient in MRN-driven pathway (2RA).
  4. As PARP is implicated in DNA damage response, the authors evaluated the nature of DNA breaks in ATM mutants. They found that ATM mutants deficient in ROS-pathway – CL, and R3047X (RX) – manifested higher levels of single-stranded DNA breaks that are reduced by NAC (assessed by alkaline comet assay). Note that ATM mutant RX is also found in some individuals suffering from A-T. Furthermore, DNA breaks in these mutants were reduced under transient inhibition of transcription (using 5,6-dichloro-1-β-D-ribofuranosyl benzimidazole, DRB).
  5. Transcription-associated DNA damage is driven by co-transcriptionally formed RNA-DNA hybrids (also called R-loops)8. So, the authors ectopically expressed RNA-DNA helicase Senataxin (SETX) that is known to resolve R-loops.  They found that SETX expression reduced the DNA breaks in the ATM mutants. Moreover, NAC treatment and SETX expression reduced aberrant R-loops formed at R-loop prone gene loci (BTBD19, ACTB, EGR1) in cells expressing ATM mutants (assayed by DNA-RNA immunoprecipitation, DRIP-qPCR). Moreover, resolving R-loops (SETX) and inhibiting transcription (DRB) also reduced PSMB2 and CK2β protein aggregation and PARylation.
  6. Surprisingly, they also found that mutations in Mre11 (MRN complex) that cause an A-T like disorder also induced widespread protein aggregation,  PARylation, and DNA breaks on par with ATM loss, albeit independent of ROS. Thus, the authors suggest that ROS-dependent ATM mutants induce transcription and R-loop associated DNA damage that triggers PARylation and protein aggregation.
  7. The authors then extended the above findings to individuals suffering from A-T disorder. For this purpose, they collected fresh-frozen cerebellum tissue from 21 individuals battling with A-T and 21 healthy individuals that acted as control matched to age, sex, and ethnicity. They report a core set of 189 aggregation-prone proteins when comparing the clinical and cellular data (by mass-spectrometry). The aggregation-prone protein list includes heat-shock proteins, metabolic enzymes, oxidoreductases, ion-channel transporters, ataxin-10, and spinocerebellar ataxia type 10 that support neuronal health. Notably, the A-T derived cerebellum tissues revealed prominent anti-PAR signal (assayed by immunohistochemistry).

Conclusion and perspective

In the current preprint, the authors demonstrate that ATM leads to aberrant R-loop-mediated DNA damage in a ROS-dependent pathway (fig.3). They suggest that R-loops and DNA damage can instigate widespread PARylation and protein aggregation. Loss of protein homeostasis was observed in many neurodegenerative diseases; however, the authors report dysregulated protein homeostasis for the first time in A-T (and A-T like disorder).

Aberrant R-loops and R-loop mediated DNA damage is implicated in the etiology of many neurological diseases, cause of mutations in proteins that maintain R-loop homeostasis9,10. Here the authors propose a model where ROS-mediated R-loops and DNA damage could coalesce PARylated protein aggregates that can cause neurotoxicity (at least in A-T).

Acknowledgments

Thanks to all the authors for their support, especially Tanya Paull, for taking the time to comment on the highlight and replying promptly.

References

  1. https://doi.org/10.1186/s13023-016-0543-7
  2. https://doi.org/10.1146/annurev-biochem-060614-034335
  3. https://doi.org/10.1126/science.1192912
  4. https://doi.org/10.1126/scisignal.aan5598
  5. https://doi.org/10.1016/j.cmet.2016.09.004
  6. https://doi.org/10.1038/ncomms9088
  7. https://doi.org/10.1038/s41467-018-04466-4
  8. https://doi.org/10.1016/j.cell.2019.08.055
  9. https://doi.org/10.1007/s12035-018-1246-y
  10. https://dx.doi.org/10.1016%2Fj.jmb.2016.08.031
  11. https://doi.org/10.1073/pnas.1611673113
  12. https://doi.org/10.1242/jcs.244129

 

Posted on: 13 November 2020 , updated on: 17 November 2020

doi: https://doi.org/10.1242/prelights.25680

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Author's response

Tanya Paull (TP) shared

1) The authors’ data suggest that R-loops are upstream of protein aggregation and PARylation, at least in the A-T disease spectrum. However, in Amyotrophic lateral sclerosis (ALS), aggregation of FUS and TDP-43 seem to prevent their binding to RNA that leads to R-loop mediated DNA damage11,12. How do the authors reconcile their data compared to other neurological disorders (like ALS)?

TP: ALS and A-T are quite different; the cerebellum specificity of A-T and the nature of the genetic defects are distinct. So, there may be different cellular responses to RNA-associated damage in these contexts.

2) The authors demonstrated the dependency of R-loops further pathological effects on ROS in ATM deficiency. However, how do the authors speculate the R-loops come from? Do the authors think that ATM deficiency or ROS production triggers transcription anomalies?

TP: We do not know. There is evidence for a role for ATM in regulating splicing, so this may be part of the mechanism or other pathways not yet identified.

3) The authors propose that PARylation at the sites of DNA damage could incite protein aggregation. Although the authors’ clearly demonstrated DNA breaks using the alkaline comet assay, would the authors consider using proximity-ligation-based assays to identify R-loops or DNA damage markers at the sites of the PAR sensor? (The authors’ data related to Fig.4 and S3 could support this.)

TP: Perhaps.

4) R-loops have been implicated in neurological disorders9,10. But it is not clear how protein aggregation or neurotoxicity is linked to R-loops. The authors’ data elegantly raises many interesting hypotheses. What do the authors think is necessary to bridge this gap of knowledge? It would be interesting to hear the authors’ perspective.

TP: One thing we need to know is whether the observed proteostasis deficiencies are present in tissues other than the cerebellum in A-T patients. We would also like to know if there are transcriptional changes in the A-T patient brain tissues.

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