AbstractProtein quality control mechanisms are vital in maintaining correct levels of functional proteins in a crowded intracellular milieu. These mechanisms recognise proteins in their non-native state and triage such candidates for either rescue, or channel them towards pathways that lead to their degradation. In the case of secretory and membrane proteins that mislocalize to the cytosol (MLPs), and of newly synthesised tail-anchored (TA) membrane proteins, their exposure of otherwise buried hydrophobic residues necessitates a mechanism of shielding such residues from the aqueous cytoplasm. This task is carried out by the collective actions of SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the heterotrimeric BAG6 (BCL2-associated athanogene 6) complex, that contribute to a SGTA/BAG6 quality control cycle that can direct hydrophobic substrates towards either ubiquitination and proteasomal degradation or productive membrane insertion.
SGTA is a modular protein consisting of three domains, an N-terminal dimerisation domain, a central TPR domain, and a substrate binding C-terminal domain. The aim of this thesis is to characterise full-length SGTA using a range of biophysical techniques, with an emphasis to understand its C-terminal domain, molecular details of which remain elusive. In addition, this work aims to uncover how SGTA interacts with its hydrophobic substrates, underlying its role in enforcing cytosolic quality control. To this end, a combination of nuclear magnetic resonance (NMR) spectroscopy and native mass spectrometry experiments have identified a constrained conformation of SGTA in solution mediated by C-terminal dimerisation, and circular dichroism (CD) spectroscopy has revealed the presence of alpha helical regions within the C-terminal domain. Furthermore, fluorine-19 NMR has been used to investigate the interaction of TA proteins with the C-terminal domain of SGTA. Finally, results presented herein establish that SGTA interacts with the intrinsic proteasomal ubiquitin receptor Rpn13 via a two-carboxylate clamp mode of molecular recognition, and this interaction has been characterised by solution NMR spectroscopy, size exclusion chromatography, isothermal titration calorimetry (ITC) and mutagenesis experiments, to uncover details of a potential SGTA/BAG6 quality control cycle operating at the 19S regulatory particle of the proteasome.
|Date of Award||2017|
|Supervisor||Rivka Isaacson (Supervisor) & Ulrike Eggert (Supervisor)|