Probing the role of protein S-acylation in biomolecular condensate regulation

Biomolecular condensates have emerged as novel membraneless compartments that
regulate cellular biochemistry. Whilst the role of protein post-translational modifications in
condensate regulation is an area of intensive interest, the potential role of protein fatty
acylation remains largely unexplored. Given the inherent hydrophobicity of this class of
PTMs, we hypothesise that protein S-acylation - a reversible PTM involving the attachment
of a fatty acyl chain (usually C16) onto >3000 cysteine residues in the human
proteome - regulates biomolecular condensate formation and disassembly through
multivalent hydrophobic interactions. We are investigating this hypothesis in a model of
stress granule (SG) formation to undertake the first comprehensive analysis of regulated
S-acylation in a biomolecular condensate and investigate how changes in S-acylation affect
SG dynamics. We first developed a novel site-specific S-Acylation Profiling by PHosphonate
taggIng pRotEomics platform (SAPPHIRE), enabling sensitive and quantitative mass
spectrometry-based analysis of S-acylation. Using SAPPHIRE for global S-acylation
profiling in HEK293T cells, we identified 2419 S-acylation sites, 2226 of which are not
previously reported in SwissPalm S-acylation database. We then couple SAPPHIRE to
APEX2 proximity-dependent biotinylation (APEX-AX) to map changes in the S-acylation
state of the SG proteome within a single proteomics workflow. These findings will enhance
our understanding of the molecular grammar governing condensates, offering novel insights
into basic biology and mechanisms of disease, and in the longer term enable the
development of tools to regulate condensate formation through protein lipidation, for
research and therapeutic purposes.