Unfolded protein response

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[edit] The Unfolded Protein Response (UPR)

The unfolded protein response is a cellular stress response unique to the endoplasmic reticulum, the intracellular organism central to performing post-translational modifications to proteins (termed protein folding) produced by the cell. It is a stress response that has been found to be conserved between all mammalian species, including yeast and worm models. This article focuses on the mammalian response. The UPR is activated in response to a change in various cellular circumstances that cause a disruption to the processes involved in protein folding, disruptions which can subsequently cause the protein to not function correctly. In this respect, the UPR has 2 primary aims: initially to restore normal function of the cell by halting protein translation, whilst signalling to increase production of the molecular chaperones involved in removing malfolded proteins. Where this is not achieved within a certain time or the disruption is prolonged, the UPR aims to initiate the programmed cell death (apoptosis) of the cell.

[edit] Protein Folding and the Endoplasmic Reticulum

The term protein folding incorporates all of the processes involved in the production of the final protein. Almost all proteins involved in producing secretory proteins pass through the ER. Protein production begins by genetic transcription within the nucleus into messenger RNA (mRNA) strands, which pass into the cytosol through pores. Those proteins destined to become part of the secretory mechanism of the cell carry an N-terminal signal sequence that directs it to specifically bind to a signal receptor protein (SRP) on the ER membrane, in close proximity to membrane-bound ribosomes. Once the sequence has “docked”, the protein begins translation, with the resultant strand being fed through a nearby translocator directly into the ER. Protein folding commences as soon as the strand enters the intraluminal environment, even as translation of the remaining strand continues.

Protein folding steps involve a range of enzyme catalysts 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 disulphide 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) and calnexin (CNX)¹. Favoured by the highly oxidising environment of the ER, CRT and CNX facilitate formation of disulphide 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 recognising malfolding 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 malfolding protein becomes characteristically devoid of glucose residues, targeting it for identification and re-glycosylation by the enzyme UGGT (UGT-glucose:glycoprotein glucosyltransferase)¹. If this fails to restore the normal folding process, exposed hydrophobic residues of the malfolded protein are bound by the protein glucose regulate protein 78 (Grp78), a heat shock protein 70kDa family member² that prevents the protein from further transit and secretion³. Where circumstances continue to cause a particular protein to malfold, the protein is recognised 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 malfolded protein back into the cytosol in transient complexes with PDI and Grp78⁴. Here it enters the ubiquitin-proteasome pathway, as it is tagged by multiple ubiquitin molecules, targeting it for degradation by cytosolic proteasomes.

A simplified diagram of the processes involved in protein folding. The polypeptide is translated from its ribosome directly into the ER, where it is glycosylated and guided through modification steps to reach its desired conformation. It is then endocytosed and passes on to the Golgi for final modifications. Where malfolding proteins continually breach quality control, chaperones including Grp78 facilitate its removal from the ER through retrotranslocation, where it is broken down by the ubiquitin-proteasome pathway as part of the ERAD system.

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 oxidising environment required for disulphide bond formation⁵.

However where circumstances cause a more global disruption to protein folding that overwhelms the ER’s coping mechanisms, the UPR is activated.

[edit] Initiation of the UPR

The molecular chaperone Grp78 has a range of functions within the ER. It maintains specific transmembrane receptor proteins involved in initiating downstream signalling of the UPR in an inactive state by binding to their luminal domains. An overwhelming load of malfolded proteins requires more of the available cellular Grp78 to bind to the exposed hydrophobic regions of these proteins, and consequently Grp78 dissociates from these receptor sites to meet this requirement. Dissociation from the intracellular receptor domains allows them to become active.

[edit] Functions of the UPR

The initial phases of UPR activation have 2 key roles:

Translation Attenuation and Cell Cycle Arrest by the PERK Receptor This occurs within minutes to hours of UPR activation to prevent further translational loading of the ER. PERK ((protein kinase-like endoplasmic reticulum kinase) activates itself by oligomerization and autophosphorylation of the free luminal domain. The activated cytosolic domain causes translational attenuation by directly phosphorylating the α subunit of the regulating initiator of the mRNA translation machinery, eIF2⁶. This also produces translational attenuation of the protein machinery involved in running the cell cycle, producing cell cycle arrest in the G1 phase⁷.

A simplified diagram of the initiation of the UPR by prolonged and overwhelming protein malfolding. Grp78 recruitment to chaperone the malfolded proteins results in Grp78 dissociation from its conformational binding state to the transmembrane receptor proteins PERK, IRE1 and ATF6. Dissociation results in receptor homodimerisation and oligomerisation to an active state. The activated cytosolic domain of PERK phosphorylates the eIF2alpha, inhibiting translation and resulting in cell cycle arrest. The activated cytosolic domain of IRE1 cleaves the 252bp intron from its substrate XBP1, facilitating its translation to form the transcription factor XBP1. Activated ATF6 translocates to the Golgi, cleaved by proteases to form an active 50kDa fragment (ATF6 p50). ATF6 p50 and XBP1 bind ERSE promoters in the nucleus to produce upregulation of the proteins involved in the unfolded protein response.

Increased Production of Proteins Involved in the Functions of the UPR UPR activation also results in upregulation of proteins involved in chaperoning malfolding proteins, protein folding and ERAD, including further production of Grp78. Ultimately this increases the cell’s molecular mechanisms by which it can deal with the malfolded protein load. These receptor proteins have been identified as:
• Inositol-requiring kinase 1⁸, whose free luminal domain activates itself by homodimerisation and transautophosphorylation⁹. The activated domain is able to activate the transcription factor XBP1 (X-box binding protein) mRNA (the mammalian equivalent of the yeast Hac1 mRNA by cleavage and removal of a 252bp intron. The activated transcription factor upregulates UPR ‘stress genes’ by directly binding to stress element promoters in the nucleus¹⁰.
• ATF6 (activating transcription factor 6) is a basic leucine zipper transcription factor¹¹.Upon Grp78 dissociation the entire 90kDa protein translocates to the Golgi, where it is cleaved by proteases to form an active 50kDa transcription factor¹² that translocates to the nucleus. It binds to stress element promoters upstream of genes that are upregulated in the UPR ¹³.

The aim of these responses is to remove the accumulated protein load whilst preventing any further addition to the stress, so that normal function of the ER can be restored as soon as possible.

[edit] Initiating Apoptosis

In conditions of prolonged stress, the goal of the UPR changes from being one that promotes cellular survival to one that commits the cell to a pathway of programmed cell death (or apoptosis). Proteins downstream of all 3 UPR receptor pathways have been identified as having pro-apoptotic roles. However, the point at which the ‘apoptotic switch’ is activated has not yet been determined, but it is a logical consideration that his should be beyond a certain time period in which resolution of the stress has not been achieved. The 2 principal UPR receptors involved are Ire1 and PERK.
By binding with the protein TRAF2, Ire1 activates a JNK signaling pathway¹⁴, at which point human procaspase 4 is believed to cause apoptosis by activating downstream caspases. Although PERK is recognised to produce a translational block, certain genes can bypass this block. An important example is the the proapoptotic protein CHOP (CCAAT/-enhancer-binding protein homologous protein), upregulated downstream of the bZIP transcription factor ATF4 (activating transcription factor 4) and uniquely responsive to ER stress¹⁵. CHOP causes downregulation of the anti-apoptotic mitochondrial protein Bcl-2¹⁶, favouring a pro-apoptotic drive at the mitochondria by proteins that cause mitochondrial damage, cytochrome c release and caspase 3 activation.

[edit] References

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