The unfolded protein response (UPR) is a cellular mechanism that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER). In mammalian cells, the UPR is mediated by three ER-transmembrane proteins: PERK, IRE1 and ATF6. Early studies in the field provided evidence for the role of BiP, the major ER Hsp70 chaperone, in UPR activation by binding to the luminal domains of PERK, IRE1 and ATF6 and maintaining them in an inactive state. However the underlying mechanism of ER stress sensing and UPR activation is not yet understood. This thesis presents (i) the novel X-ray crystal structure of PERK luminal domain and (ii) a biochemical study of the unconventional interaction between BiP chaperone and the luminal domains of PERK and IRE1 in vitro. Firstly, the structure of PERK luminal domain was solved in two oligomeric states: dimers and tetramers. Compelling evidence is provided for a role of tetramer formation in directing downstream UPR signalling. Secondly, the unprecedented and unconventional direct binding of PERK and IRE1 luminal domains to BiP is demonstrated. The binding surface was mapped to the nucleotide-binding domain (NBD) of BiP. As such, this points away from a substrate-chaperone interaction and rather implies BiP as an explicit UPR signalling component. Upon binding to CH1 unfolded protein, BiP is released from PERK and IRE1 luminal domains. BiP dissociation from PERK and IRE1 is known to lead to their activation. Based on the work presented in this thesis a novel mechanism of ER stress sensing and UPR activation by PERK and IRE1 is proposed. BiP NBD normally interacts with the luminal domains of PERK and IRE1 and represses UPR signalling. During ER stress, binding of unfolded proteins to BiP's substrate binding domain leads to the dissociation of BiP-luminal domain complexes. As such, the luminal domains are free to intertwine, mediated by an extended α-helix, and form active tetramers competent for cytoplasmic UPR signalling.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:656595 |
| Date | January 2014 |
| Creators | Carrara, Marta |
| Contributors | Ali, Maruf; Haslam, Stuart |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/24953 |
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