The unfolded protein response (UPR) is a cellular stress response related to the endoplasmic reticulum (ER) stress. It is named for its primary function: sensing the accumulation of unfolded or misfolded proteins and triggering a coordinated "response" to restore balance or induce cell death. It has been found to be conserved between mammalian species, as well as yeast and worms. The UPR is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum. In this scenario, the UPR has three aims: initially to restore normal function of the cell by halting protein translation, degrading misfolded proteins, and activating the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these objectives are not achieved within a certain time span or the disruption is prolonged, the UPR aims towards apoptosis. Sustained overactivation of the UPR has been implicated in prion diseases as well as several other neurodegenerative diseases, including Wolfram syndrome, and inhibiting the UPR could become a treatment for those diseases. Diseases amenable to UPR inhibition include Creutzfeldt–Jakob disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, and Wolfram syndrome.
Protein folding in the endoplasmic reticulum
Protein synthesis The term protein folding incorporates all the processes involved in the production of a protein after the nascent polypeptides have become synthesized by the ribosomes. The proteins destined to be secreted or sorted to other cell organelles carry an N-terminal signal sequence that will interact with a signal recognition particle (SRP). The SRP will lead the whole complex (Ribosome, RNA, polypeptide) to the ER membrane. Once the sequence has "docked", the protein continues translation, with the resultant strand being fed through the polypeptide translocator directly into the ER. Protein folding commences as soon as the polypeptide enters to the luminal environment, even as translation of the remaining polypeptide continues.
Protein folding and quality control Protein folding steps involve a range of enzymes and molecular chaperones to coordinate and regulate reactions, in addition to a range of substrates required in order for the reactions to take place. The most important of these to note are N-linked glycosylation and disulfide bond formation. N-linked glycosylation occurs as soon as the protein sequence passes into the ER through the translocon, where it is glycosylated with a sugar molecule that forms the key ligand for the lectin molecules calreticulin (CRT; soluble in ER lumen) and calnexin (CNX; membrane bound). Favoured by the highly oxidizing environment of the ER, protein disulfide isomerases facilitate formation of disulfide bonds, which confer structural stability to the protein in order for it to withstand adverse conditions such as extremes of pH and degradative enzymes. The ER is capable of recognizing misfolding proteins without causing disruption to the functioning of the ER. The aforementioned sugar molecule remains the means by which the cell monitors protein folding, as the misfolding protein becomes characteristically devoid of glucose residues, targeting it for identification and re-glycosylation by the enzyme UGGT (UDP-glucose:glycoprotein glucosyltransferase). If this fails to restore the normal folding process, exposed hydrophobic residues of the misfolded protein are bound by the protein glucose regulate protein 78 (Grp78), a heat shock protein 70kDa family member that prevents the protein from further transit and secretion. Where circumstances continue to cause a particular protein to misfold, the protein is recognized as posing a threat to the proper functioning of the ER, as they can aggregate to one another and accumulate. In such circumstances the protein is guided through endoplasmic reticulum-associated degradation (ERAD). The chaperone EDEM guides the retrotranslocation of the misfolded protein back into the cytosol in transient complexes with PDI and Grp78. Here it enters the ubiquitin-proteasome pathway, as it is tagged by multiple ubiquitin molecules, targeting it for degradation by cytosolic proteasomes.
Successful protein folding requires a tightly controlled environment of substrates that include glucose to meet the metabolic energy requirements of the functioning molecular chaperones; calcium that is stored bound to resident molecular chaperones; and redox buffers that maintain the oxidizing environment required for disulfide bond formation. Unsuccessful protein folding can be caused by HLA-B27, disturbing balance of important (IL-10 and TNF) signaling proteins. At least some disturbances are reliant on correct HLA-B27 folding. However, where circumstances cause a more global disruption to protein folding that overwhelms the ER's coping mechanisms, the UPR is activated.
Molecular mechanism
Initiation The molecular chaperone BiP/Grp78 has a range of functions within the ER. It maintains specific transmembrane receptor proteins involved in initiation of the downstream signalling of the UPR in an inactive state by binding to their luminal domains. An overwhelming load of misfolded proteins or simply the over-expression of proteins (e.g. IgG) requires more of the available BiP/Grp78 to bind to the exposed hydrophobic regions of these proteins, and consequently BiP/Grp78 dissociates from these receptor sites to meet this requirement. Dissociation from the intracellular receptor domains allows them to become active. PERK dimerizes with BiP in resting cells and oligomerizes in ER-stressed cells. Although this is traditionally the accepted model, doubts have been raised over its validity. It has been argued that the genetic and structural evidence supporting the model simply shows BiP dissociation to be merely correlated with Ire1 activation, rather than specifically causing it. An alternative model has been proposed, whereby unfolded proteins interact directly with the ER-lumenal domain of Ire1, causing oligomerization and transautophosphorylation. However these models are not mutually exclusive, it is also possible that both direct interaction of Ire1 with unfolded proteins and dissociation of BiP from IRE1 contribute to the activation of the Ire1 pathway.
Functions The initial phases of UPR activation have two key roles:
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