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Title: Regulation of inflammatory TLR signalling by the BCAP adaptor
Author: Lauenstein, Johannes
ISNI:       0000 0004 8501 3205
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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BCAP is a PI3K adaptor protein in TLR, BCR and IL-1R and TCR signalling. In TLR signalling, BCAP acts as a negative regulator of inflammatory signalling. Regulation of TLR signalling is essential as overstimulation can cause excessive and pathological inflammation, which leads to serious illnesses such as sepsis and rheumatoid arthritis. Through an N-terminal TIR domain, BCAP interacts with the MAL and MyD88 adaptors downstream of TLR activation. BCAP then recruits PI3K and PLC-γ2 to the TLR signalosome. Although these proteins can facilitate negative regulation of TLRs by endocytosis, the mechanism by which BCAP dampens TLR signalling remains elusive. The first aim of this thesis is to determine the minimal domain requirements and stoichiometry of BCAP TIR domain interactions. The second aim is to characterise and further explore the BCAP interactome, in the context of TLR and BCR signalling. Identification of novel interaction partners and a more detailed understanding of existing interactions, including PI3K and PLC-γ2, is crucial in understanding the function of BCAP in various signalling pathways. An integrated structural and functional approach was used to study the mechanism of BCAP TIR domain interactions. Co-immunoprecipitation was used to show that the BCAP TIR domain associates with MAL, but does not dampen NF-κB signalling in reporter assays. For this negative regulation of TLR signalling, the BCAP DBB domain is essential. A crystal structure of the DBB domain in combination with SEC-MALS revealed that the DBB domain functions as a dimerisation region. First shown in NF-κB reporter assays, DBB domain dimerisation also prevents MAL TIR domain interactions in vitro. These results support a new model of steric inhibition of TIR domain interactions, by which BCAP negatively regulates TLR signalling. An investigation of BCAP post-translational modifications revealed that the tyrosine kinases BTK, LYN and SYK, and the serine kinase CSNK2A1 contribute to BCAP hyperphosphorylation. A virotrap interaction screen identified a number of proteins including Grb2 and CRKL as new adaptor proteins in the BCAP interactome. Subsequent validation in mammalian cells via co-immunoprecipitation showed that Grb2 is a direct interaction partner of BCAP. Further characterization of all SH2 and SH3 domain-containing proteins in the BCAP interactome revealed an extensive network of SH2 and SH3 domain interactions as well as the specific interaction sites. In conclusion, these results indicate that BCAP is a complex hub that integrates multiple immune signalling pathways.
Supervisor: Gay, Nicholas John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Toll-like receptor ; Innate immunity ; PI3K ; Crystallography ; Cell signalling