The Clusterin Enigma: Structure and Function Studies of the First Known Mammalian Secreted Chaperone

Proteostasis refers to a crucial act of fine-tuning the synthesis, folding, localisation and degradation of proteins, and this balance must be maintained both inside and outside cells. Clusterin (CLU) is the best characterised secreted mammalian chaperone that is a key component of extracellular proteostasis - it binds to misfolding proteins to stabilise them in a soluble state, inhibit their aggregation, and facilitate their clearance by cellular uptake and degradation. CLU has been shown to potently inhibit the aggregation of misfolding proteins that form both amorphous and amyloid aggregates. CLU is a disulphide-linked heterodimer that in solution exists in a range of different oligomeric states (monomer, dimer and higher multimers) which may be important in its chaperone action. However, the lack of a known three-dimensional structure for CLU and limited information about its secondary structures have been major roadblocks in identifying regions of the molecule that play critical roles in its biological functions. Furthermore, preliminary evidence indicates that during ER stress the intracellular trafficking of CLU deviates from the secretory pathway and CLU is released into the cytosol. However, there is very limited information about the mechanism of CLU release to the cytosol, or its biological roles in cells undergoing ER stress. The work summarised in this thesis suggests that (i) residues 360-427 in the CLU β-chain C terminus contain elements that are likely to be critical for self-oligomerisation of CLU heterodimers, and (ii) residues 347-354 and 420-427 are critical for binding to misfolded proteins and inhibiting their aggregation to form either amorphous aggregates or amyloid. Furthermore, the results also show that in ER stressed cells, CLU that is released to the cytosol contains an intact N-terminus similar to secreted CLU, and is comprised of both uncleaved and cleaved variants (probably released from the ER and Golgi respectively). Further findings are that CLU release to the cytosol is via the N-End Rule pathway, being dependent upon the expression of arginyl transferase 1 (encoded by ATE1) and CLU residues D1 and D6 (which may act as N-degrons). The results also suggest that cytosolic CLU forms complexes with misfolded proteins in the cytosol and traffics these to proteasomes and autophagosomes for degradation. Collectively, the results provide very significant new information about the structure-function relations of CLU and substantively extend understanding of an important role of cytosolic CLU as a key player in intracellular proteostasis during ER stress. In the longer term, these results may provide parts of the necessary foundation upon which novel therapies using CLU can be developed in the future to treat serious human diseases, including Alzheimer’s disease, systemic amyloidosis, preeclampsia, cancers and dry eye diseases