The proteasome is an essential multi-protein complex found within all eukaryotes. It degrades unwanted proteins and is involved in regulating many cellular functions, including cell homeostasis and apoptosis. Previous research in the field mainly focused on the mechanisms by which substrate proteins are specifically tagged for proteasome-mediated degradation and the subsequent mechanisms involved in substrate processing. However, the proteasome is known to interact with a wide range of ancillary proteins, and not all of these interactions have been characterised. The main focus of this thesis was therefore to identify and investigate the human proteasome interactions at a molecular level, with a focus on three specific proteins.
Firstly, I investigated the proteasome interaction with the UBR4 E3 ligase. I optimised the preparation of 26S proteasomes and found UBR4 co-purifying. I also showed that endogenously-tagged UBR4 conversely co-purifies with proteasome complexes. My biochemical and biophysical experiments indicate a strong interaction, as it was not possible to separate UBR4 from the proteasome in vitro. Increasing the yield of obtainable UBR4 to perform more detailed structural and functional analysis will be one of the future challenges.
Secondly, I studied the proteasome interaction with the P97 AAA+ ATPase. Using the baculovirus mediated insect cell overexpression system, I recombinantly co-expressed the human P97 and the 20S proteolytic core of the proteasome. Although P97 was shown to co-purify with affinity tagged 20S proteasomes, the data obtained suggest that any direct interaction appears to be of transient nature or may depend on other still unidentified proteins or cofactors. Finally, I focused on PI31, a poorly characterised protein that has been described as a proteasome inhibitor. Indeed, by adding purified PI31 to 20S proteasomes I showed that in the presence of PI31 the proteasome peptidase activities are decreased. Interestingly, cryo-EM analysis of affinity-tagged 20S proteasomes recombinantly co-expressed with PI31 showed the 20S proteasome bound to PA200 and PA28-like caps, with no evidence for the 26S proteasome 19S regulatory particle. There was also a low abundance complex with a smaller, less-resolved density, that could be assigned to PI31. Surprisingly, these experiments suggest a regulatory role of PI31 in the selective assembly of higher order proteasome complexes, rather than acting as an alternative cap directly modulating the proteasome proteolytic active sites. My results emphasise the importance of fully characterising the proteasome and its interactors, as regulation of this multi-protein complex is likely to occur on many different levels, all of which represent potential drug target sites.