Abstract
dc:description.abstractToll-like receptors (TLRs) are crucial sensors in innate immunity against microbial infections. Their pro-inflammatory signal transduction typically involves the oligomerisation of downstream TIR-domain-containing adaptors MAL and MYD88. BCAP (B-cell adapter for PI3K) was identified as a negative regulator in the TLR4 MYD88-dependent signalling pathway. This study utilises biochemical, biophysical and structural biological techniques and focuses on two hypotheses to elucidate the role of BCAP in the TLR signalling pathways. The first hypothesis focuses on the steric inhibition of TLR signal transduction by BCAP and builds upon previous structural studies of BCAP, suggesting that it may adopt a unique TIR-domain conformation to disrupt the oligomerisation of MAL and MYD88 through direct TIR domain interactions with MAL and potentially MYD88. Recombinant BCAP was confirmed as a dimer in solution and was optimised for cryo-EM, although challenges of aggregation during vitrification persisted. The results of biophysical and functional analyses of MAL TIR filament formation in vitro with BCAP indicated no association between BCAP and MAL TIR or MYD88 TIR, suggesting an alternative mechanism of BCAP as the negative regulator. BCAP was identified with an extensive interactome, in which PI3K p85α/p110δ and PLCγ2 were associated with the negative regulation of the TLR4 pro-inflammatory response. It is hypothesised that BCAP may facilitate the recruitment and activation of PI3K and PLCγ2 to the TLR signalosome. In this study, a robust protocol was developed to phosphorylate BCAP in vitro by BTK, further examining critical post-translational modifications of BCAP. A novel tyrosine phosphorylation site was identified by mass spectrometry, in addition to eight previously identified sites. The tyrosine phosphorylation of BCAP significantly increased its binding affinity to PI3K and PLCγ2, allowing the formation of stable complexes of BCAP-p85α/p110δ and BCAP-PLCγ2 that are suitable for structural studies. BCAP-PI3K complexes provided a new approach to understanding the activation and regulation of PI3K, enabling comprehensive experimental applications beyond the scope of TLR signalling pathways.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2025
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Wu, Yuan
- Advisor dc:contributor.advisor
-
- Gay, Nicholas
Subjects
dc:subject × 7Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.119477
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/386115