Proteomic Analysis of Stress Granule Dynamics Under Oxidative Stress Conditions
DOI:
https://doi.org/10.5281/zenodo.22688101Keywords:
stress granules, oxidative stress, proximity labelling proteomics, G3BP1, sodium arsenite, RNA-binding proteinsAbstract
Stress granules are membraneless cytoplasmic condensates that assemble when translation initiation is blocked,
and while their core composition under heat shock and osmotic stress has been mapped in detail, how oxidative
stress -- a physiologically distinct and disease-relevant trigger -- shapes stress granule proteome composition over
time remains comparatively underexplored. We used doxycycline-inducible APEX2 fused to the canonical
nucleator G3BP1 in U2OS cells to biotinylate proteins within labelling range of assembling stress granules at 15,
45 and 90 minutes after sodium arsenite exposure, followed by streptavidin enrichment and tandem mass tag
quantitative mass spectrometry. Of 412 proteins significantly enriched at any time point relative to unstressed
controls, 187 were present across all three time points and were classified as core components, while 225 showed
time-restricted enrichment, appearing transiently at 15 or 45 minutes before depletion, or appearing only at 90
minutes as granules matured. Gene ontology analysis showed early time points enriched for translation initiation
factors and 40S ribosomal proteins, consistent with stalled pre-initiation complexes nucleating granule assembly,
while the 90-minute proteome was enriched for RNA helicases and ubiquitin-proteasome components, suggesting
a shift toward quality-control and potential disassembly machinery as granules matured. Comparison with a
parallel heat-shock APEX2-G3BP1 dataset generated under matched conditions identified 64 proteins specifically
and reproducibly enriched under oxidative but not heat stress, including three proteins, PRDX1, TXNRD1 and
GPX4, with established roles in redox homeostasis that were validated by immunofluorescence co-localisation and
by reduced stress granule formation following individual siRNA knockdown. These results indicate that stress
granules assembled under oxidative stress recruit a stimulus-specific redox-regulatory sub-proteome not captured
by heat-shock-based reference datasets, with potential relevance to the granule pathology implicated in
oxidative-stress-associated neurodegenerative disease.
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