Interactions between components of the protein homeostasis network

Thesis

An intricate set of processes responsible for maintaining the stability of the proteome forms a protein homeostasis network, which is crucial for cell viability and survival. This thesis describes insights into the interactions of the components that belong to that network. We focus on small heat-shock proteins (sHsps), significant members of the chaperone machinery, and glucosidase II (GluII), which is a key enzyme of the ER quality control system. We utilise native mass spectrometry, supported by other biophysical techniques, to broaden the understanding of studied protein-protein and protein-ligand complexes.

First, we characterise the properties of organelle sHsps, a family unique to plants. We show their structural diversification from cytosolic homologs. We find that two of the examined sHsps exhibit polydispersity, never reported in plant sHsps before. Further, we show evidence of dual monomer architecture within higher oligomers among organelle-targeted sHsps.

Then, we demonstrate that organelle-localised sHsps from different evolution- ary classes, as well as those from different cellular compartments, co-assemble with diverse modes of interactions. We quantify the energy biases of those interactions using a developed statistical-mechanical model. Studied co-assemblies exhibit a range from a slight to strong preference toward homo-interactions.

We next investigate the subunit exchange between the organelle and cytosolic sHsps. The results indicate that the diversification of the α-crystallin domain led to the incompatibility in the dimerisation interfaces between these two groups. However, more flexible C-terminal and N-terminal regions maintain the assembly of homo-dimer subunits into higher hetero-oligomers.

Lastly, we determine the mode of interactions between GluII and a thiopyridone ligand, a potential host-targeted antiviral drug. Based on the obtained results, we propose a ‘dock, lock and latch’ molecular mechanism, where the ligand accesses the binding pocket near the dimerisation interface and then binds to the available cysteine through a disulfide bond. This binding induces structural rearrangement followed by dimer dissociation.

Authors: Sadowska, WL

• DOI: 10.5287/ora-pv0b26r8q
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English