The structural and cellular biology of endogenous TLR4 activators in inflammatory disease

Thesis

Toll-like receptors (TLRs) are critical to detecting the presence of dangerous infections, initiating a protective innate immune response, and triggering a long-lasting adaptive immune response. TLRs initiate defensive, pro inflammatory signalling following direct binding to pathogen-associated molecular patterns (PAMPs), which are exogenous molecular fingerprints derived from infectious pathogens like bacteria, viruses, and fungi. In addition to this role, more recent evidence demonstrates that TLRs can also initiate pro-inflammatory responses upon detection of damage-associated molecular patterns (DAMPs), which are endogenous self molecules released at sites of tissue damage or stress. Under normal conditions, TLR activation via DAMPs contributes to tissue repair; however, during autoimmune diseases like rheumatoid arthritis, persistent DAMP accumulation creates a chronically pro-inflammatory extracellular milieu that drives disease progression. Despite the therapeutic importance of the DAMP:TLR axis, the molecular mechanism behind TLR-mediated detection of DAMPs remains unclear: while there are dozens of structures of TLRs bound to PAMPs, there are no structures of any TLRs bound to any DAMPs from any organism. In order to clarify these molecular details, herein I employ primarily biophysical techniques to probe a model TLR:DAMP interaction between TLR4 and a class of proteins known as the fibrinogen-related proteins (FRePs). After presenting novel expression and purification methodologies for both TLR4 and the FRePs, I demonstrate with an assortment of biophysical techniques that TLR4 does not directly bind to the FRePs. Next, using an unbiased proteomics workflow to probe the cell-surface FReP interactome, I provide evidence to suggest that integrins and lipoprotein receptors, not TLR4, may be the direct FReP receptors. Based on my data and the existing literature, I synthesize these results into a broader general hypothesis regarding TLR-mediated detection of DAMPs which I call the "receptorome". Taken together, this work presents an updated model for DAMP activation of innate immunity: rather than a direct TLR:DAMP binding event, I contest that context-specific receptor mega-complexes are required to indirectly communicate the presence of DAMPs to TLRs. This conclusion clarifies some conflicting reports in the DAMP literature and provides a route to understanding the fundamental molecular mechanism behind the important but mysterious activation of TLRs in the presence of endogenous DAMPs.

Authors: Coker, J

• DOI: 10.5287/ora-9gqaeegkv
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English