The AAA (ATPase involved in various cellular activities) protein p97 is a versatile chaperone in the UPS (ubiquitin-proteasome system). As such, it is involved in many cellular pathways, for example membrane fusion, NF-κB activation, and DNA-damage repair. In these pathways, p97 engages an array of adaptor proteins to allow it to bind ubiquitylated substrates, process them and pass them to the proteasome for degradation. p97 forms a series of dedicated complexes that allow it to perform such a large number of tasks. The largest family of p97 adaptor proteins is the UBX (ubiquitin regulatory X) protein family, which includes p47 and FAF1 (Fas-associated factor 1). This thesis aims to probe the structures and biochemical properties of p97-UBX protein complexes to better understand how they help regulate p97 function. Recent advances in single-particle cryo-EM data processing of heterogeneous p97 datasets were utilised to improve the resolution of the cryo-EM model of p97-p47 in the presence of ADP. The method successfully removed unbound p97 and yielded a model of improved resolution. The main focus of the project was on FAF1. It was found to have a propensity to oligomerise via its coiled-coil domain, and bind p97 with a stoichiometry of 3:6. FAF1 decreased p97 ATPase activity, which may also be linked to its oligomerisation. A cryo-EM reconstruction of p97-FAF1 was obtained. It showed that p97 adopted a similar conformation to that seen in p97-UN. These data led to a proposed mechanism for how p97-FAF1 may be regulated. Furthermore, this data showed that p97- FAF1, p97-p47, and potentially other UBX proteins, share a trimer-hexamer binding mode. These common features impact the way they accomplish their tasks, indicating that p97 may utilise them by a common mechanism.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:616776 |
| Date | January 2013 |
| Creators | Ewens, Caroline Anne |
| Contributors | Freemont, Paul ; Zhang, Xiaodong |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/14416 |
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