Thesis

Decoding protease activated receptor 4 (PAR4) protein interaction networks with proteomics and artificial intelligence

Downloadable Content

Download PDF
Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2025
Thesis identifier
  • T17198
Person Identifier (Local)
  • 202152459
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • INTRODUCTION: Protease-activated receptor 4 (PAR4) is a G-protein coupled receptor (GPCR) activated by thrombin and cathepsin G, playing key roles in thrombosis and inflammation. As the least characterized member of the PAR family, elucidating PAR4's intracellular interactions and mutations, such as the Y157C mutation, is crucial for understanding its biological functions and interaction network. We hypothesised that PAR4 C-tail harbours a short linear motif (SLiM) resembling a class 1 PDZ binding sequence. To test this idea an array of technologies including SILAC proteomics, AI tools and super resolution microscopy was employed. METHODS: This study utilized stable isotope labelling of amino acids in cell culture (SILAC) to analyse the proteome of HEK293 cells expressing wild-type PAR4-YFP, a PAR4 variant with a modified short linear motif (PAR4ΔSLiM-YFP), and a mutant PAR4-Y157C. Following GFP-trap affinity purification and LC MS/MS analysis, network analysis and gene ontology enrichment were conducted. Interactions with PDZ domain-containing proteins were predicted and validated computationally using AlphaFold Multimer. Additionally, super-resolution microscopy (STED and SIM) was employed to investigate the mitochondrial localization of PAR4. RESULTS: Across four datasets, 1056 unique proteins exceeded a 95% confidence threshold, with significant clusters of mitochondrial and ribosomal proteins highlighted. AlphaFold Multimer confirmed interactions between PAR4 variants and GIPC proteins, indicating specific PDZ domain involvements. Predicting protein-protein interactions (PrePPI) suggested five more interactions, which were also validated using AlphaFold multimer. Super-resolution microscopy provided insights into the subcellular localization of PAR4, particularly its association with mitochondria, though these results suggest further in vitro investigation. CONCLUSION: This thesis significantly advances our understanding of PAR4 by detailing its interactions with mitochondrial components and PDZ domain-containing proteins, such as GIPCs. The integration of proteomic analyses, AI-driven predictions, and advanced microscopy techniques elucidates PAR4's complex interaction networks and supports its potential involvement in mitochondrial functions. These findings not only refine our understanding of PAR4's functional landscape but also highlight the utility of combining omics technologies with AI and super-resolution microscopy to enhance the precision and depth of biological studies. This study provides a proof of concept for the interaction of PAR4 with members of the GIPC family. Future developments of this project would include validation of these findings in more physiologically relevant models. Lay abstract: Protease-activated receptor 4 (PAR4) is a cell surface protein implicated in blood clotting and inflammation, activated by enzymes such as thrombin and cathepsin G, and stands as the least understood member of its protein family. The project explored how PAR4 interacts with downstream proteins and the effects of structural changes like the Y157C mutation by utilizing a technique known as SILAC. The analysis revealed over a thousand proteins interacting with PAR4, including significant groups found within mitochondria. Advanced AI tools were then employed to predict these interactions, with AI tools like AlphaFold confirming likely contacts with a family of proteins known as GIPCs. Another AI tool, PrePPI, suggested additional potential interacting proteins, enriching the understanding of PAR4’s interaction network. Super-resolution microscopy techniques were then used to observe PAR4's potential association with mitochondrial proteins. Although microscopy indicated an association of PAR4 with mitochondria, further in vitro studies are needed for confirmation. This comprehensive approach has not only advanced understanding of PAR4's functionality, but also highlighted how combining various scientific techniques can provide detailed insights into complex networks of protein interactions, laying a foundation for future research in more complex biological models to verify these interactions.
Advisor / supervisor
  • Cunningham, Margaret
Resource Type
DOI

Relations

Items

Thumbnail Title Date Uploaded Visibility Actions
file details PDF of thesis T17198 2025-02-27 Public Download