Evaluation of MRN complex and ATM protein-protein interactions using cleavable DSSO crosslinking and mass spectrometry

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Harvey, Antony Martin

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Ataxia telangiectasia (A–T) is an inherited autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The canonical role of ATM is to induce cell checkpoint arrest following DNA damage. When cells experience DNA double-strand breaks (DSBs), the Mre11-Rad50-Nbs1 (MRN) complex senses the damage, activating ATM, which mobilizes a protective signaling cascade, activating the cell cycle checkpoint, arresting cell growth. If the extent of damage is excessive, the apoptotic pathway is activated; otherwise DNA repair is initiated using either homologous recombination (HR) or through non-homologous end joining (NHEJ). ATM also plays a key role in redox homeostasis. Loss of this cellular function results in the misfolding and aggregation of proteins. Misfolded proteins are degraded by three pathways: the ubiquitin proteasome system (UPS), macroautophagy and chaperone-mediated autophagy (CMA). Aggregated proteins that are resistant to these protein clearance mechanisms can form inclusion bodies, which can lead to neurodegeneration. The primary approach used for investigating protein interactions in this research was with the DSSO cleavable crosslinking technique: first to evaluate the MRN protein complex structure and protein-protein interactions; then to elucidate ATM binding partners in cells undergoing oxidative stress. The results of the MRN complex in vitro crosslinking experiment were that 53 unique crosslinks were detected. These were subsequently evaluated by calculating molecular distances between identified crosslinked residues using known crystal structures of homologous proteins. Conversely, the results of the ATM in vivo crosslinking experiment were that, while informative crosslinks were not detected between ATM and other proteins, certain pathways were over-, and under-represented in the Co-IP of cells expressing the ATM constructs. Specifically, the parkin-ubiquitin proteasomal system pathway was over-represented, while the proteasome degradation pathway was under-represented. Additionally, the small heat shock protein 27 (Hsp27) was found to be enriched, a chaperone known for its protective role in protein aggregation diseases.


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