Understanding the molecular basis of a ubiquitin-dependent kinase and ubiquitin ligase at the interface of inflammation and cell death

Thesis

Inflammation and programmed cell death (PCD) are seemingly opposing yet intricately linked processes controlled through degradative and non-degradative ubiquitin chains. In this study, I report on two crucial components within apoptosis, a form of PCD, and inflammation. Firstly, the giant inhibitor of apoptosis (IAP), BRUCE, and how it mediates caspase inhibition and secondly how the TAK1-TAB complex undergoes activation by Lys63-linked chains.

Here, I present the cryoEM structures of BRUCE alone and in complex with its regulator SMAC at 3.2 and 3.1 Å resolution, respectively. Complimentary biochemical characterisation shows the capability of BRUCE to ubiquitinate and directly inhibit caspases. This BRUCE-mediated caspase inhibition is released by SMAC, and using structural and biochemical approaches I demonstrate how SMAC utilises a sub nanomolar binding affinity to BRUCE leading to caspase displacement.

Secondly, I present my work on elucidating the structural basis for the TAK1- TAB complex. Through employing short constructs of TAK1 and TAB2 I show how complex integrity is maintained using biophysical techniques and how Lys6-linked diubiquitin binds to the TAB2 NZF domain using X-ray crystallography. In addition, biochemical assays investigating the activation mechanism of the full-length TAK1 complex suggests longer Lys63-linked ubiquitin chains are required for enhanced activation.

Authors: Dietz, L

• DOI: 10.5287/ora-nojqerpdq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Biochemistry