Novel proteomics approaches to study S-acylation in biomolecular condensate regulation and disease
File(s)
Author(s)
Nigmatulina, Amina
Type
Thesis
Abstract
Biomolecular condensates are membraneless organelles formed through liquid-liquid phase separation (LLPS), driven by multivalent interactions. These dynamic compartments play key roles in a wide range of cellular processes, and their dysregulation is linked to numerous diseases. Whilst many protein post-translational modifications (PTMs) are known to regulate condensate assembly and disassembly, the role of lipid PTMs, particularly S-acylation, remains largely unexplored. S-acylation involves the covalent attachment of long-chain fatty acids (C16-C22) onto cysteines, mediated by zDHHC enzymes and reversed by acyl protein thioesterases (APTs). S-acylation regulates myriad cellular functions, and its dysregulation is implicated in many diseases. Given its hydrophobic and reversible nature, it was hypothesised that S-acylation could influence condensate dynamics via hydrophobic interactions. However, studying S-acylation is challenging due to its inherent biochemical properties, enzyme redundancy, and limited analytical tools.
Here, I initially developed a novel double enrichment proteomic method to systematically study S-acylation dynamics within stress granules (SGs), a model condensate system. This method combined APEX2 proximity labelling and S-acyl exchange (APEX-AX) techniques to investigate S-acylation changes during SG assembly upon stress. However, initial experiments revealed the incompatibility of the APEX2 approach with S-acylation profiling due to lability of the thioester bond. Separately, addressing the broader scarcity of analytical tools, I developed the S-acylation Profiling by Phosphonate Tagging Proteomics (SAPPHIRE) method, which enables high-throughput and site-specific analysis of S-acylation on a proteome-wide scale. SAPPHIRE demonstrated improved sensitivity and specificity compared to existing S-acylproteomic techniques, enabling the identification of >4,000 putative S-acylation sites across multiple human cell lines. Furthermore, SAPPHIRE facilitated pioneering analyses of S-acylation patterns in human patient tissues and provided the first site-specific S-acylation profile in T. gondii parasites. Collectively, this thesis addresses key challenges in studying S-acylation and establishes SAPPHIRE as a powerful platform for comprehensive analysis of S-acylation across diverse biological systems and disease contexts.
Here, I initially developed a novel double enrichment proteomic method to systematically study S-acylation dynamics within stress granules (SGs), a model condensate system. This method combined APEX2 proximity labelling and S-acyl exchange (APEX-AX) techniques to investigate S-acylation changes during SG assembly upon stress. However, initial experiments revealed the incompatibility of the APEX2 approach with S-acylation profiling due to lability of the thioester bond. Separately, addressing the broader scarcity of analytical tools, I developed the S-acylation Profiling by Phosphonate Tagging Proteomics (SAPPHIRE) method, which enables high-throughput and site-specific analysis of S-acylation on a proteome-wide scale. SAPPHIRE demonstrated improved sensitivity and specificity compared to existing S-acylproteomic techniques, enabling the identification of >4,000 putative S-acylation sites across multiple human cell lines. Furthermore, SAPPHIRE facilitated pioneering analyses of S-acylation patterns in human patient tissues and provided the first site-specific S-acylation profile in T. gondii parasites. Collectively, this thesis addresses key challenges in studying S-acylation and establishes SAPPHIRE as a powerful platform for comprehensive analysis of S-acylation across diverse biological systems and disease contexts.
Version
Open Access
Date Issued
2025-06-24
Date Awarded
2025-09-01
Copyright Statement
Attribution-Non Commercial-No Derivatives 4.0 International Licence (CC BY-NC-ND)
Advisor
Tate, Ed
Publisher Department
Department of Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)