The phosphoproteome of stress granules

Under stress conditions, eukaryotic cells rapidly partition mRNAs and proteins into condensates called stress granules (SGs) to protect these molecules from degradation, and to initiate changes in gene expression and signalling pathways. SGs are integral cellular structures involved in proteostasis and aging, and the persistence and dysfunction of SGs contributes to many cancers and neurodegenerative diseases.

The protein composition of SGs differs depending on the type of stress, however, the specific protein selection mechanisms are largely unknown. Protein phosphorylation can disrupt or promote interactions required for SGs and has been implicated in SG biogenesis. We hypothesize that phosphorylation plays a more substantial role in regulating SG interactions than previously thought, particularly mediating the inclusion or exclusion of proteins under different stress conditions. To understand this type of phosphoregulation, we measured the phosphoproteome of SGs under diverse stress conditions in human osteosarcoma cells (U-2 OS) with the goal of identifying general and stress-specific phosphorylation responses.

To confirm SG formation, we used an engineered cell line with a GFP-fused SG marker, Ras GTPase-activating protein-binding protein 1 (G3BP1). Using mass spectrometry, we quantified stress-specific changes to the total proteome and phosphoproteome in response to oxidative stress (sodium arsenite), osmotic stress (sorbitol), heat stress, and proteasome inhibition (bortezomib) in six biological replicates per condition. We quantified over ten thousand phosphosites, 150 of which were significantly regulated in stress. Comparing the identified phosphoproteins to published SG protein databases, we selected candidate phosphosites that localize to SGs and are likely involved in SG regulation. Localization of candidate phosphosites was performed by BioID proximity labelling using multiple SG baits. Overall, we characterized the phosphorylation-specific changes to proteins in SGs in multiple stress conditions to more fully understand the mechanisms regulating stress responses, SG persistence and SG dysfunction that may contribute to health and disease.