The UPR branches

1.3.1.1 ATF6

ATF6 is a transcription factor that is synthesised as an ER TM protein. In homeostatic conditions, ATF6 forms dimers with GRP78, which maintains it in an inactive state. Upon GRP78 dissociation, ATF6 is translocated to the GA. In the GA, serine protease 1 (SP1) and SP2 perform ATF6 cleavage, generating a short ATF6 N-terminal cytosolic domain that translocates to the nucleus. In the nucleus, short ATF6 binds to cyclic adenosine monophosphate response element (CRE) and ERS response element-1 (ERSE-1) DNA regions, resulting in genes involved in cell fate transcription, such as CCAAT-enhancer-binding homologous protein (CHOP), or chaperone transcription, such as GRP78 and GRP94 (Figure 23) [322].

Figure 23. Endoplasmic reticulum stress (ERS) response induction and overactivation. a. The ERS

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response is mediated by three proteins located at the ER membrane: protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1α (IRE1α).

Under homeostatic conditions, these proteins are bound to the chaperone protein GRP78 by their ER lumen domains. GRP78 also binds caspase-7, maintaining this apoptosis effector in an inactive state at the ER membrane. b. Following unfolded protein accumulation in the ER lumen, GRP78 detaches from PERK, ATF6, IRE1α and caspase-7 to assist protein folding. PERK release induces its oligomerisation and autophosphorylation, which activates the transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2). Activated NRF2 binds to the DNA on anti-oxidant responsive element (ARE) regions to enhance the transcription of anti-oxidant genes. PERK also phosphorylates eukaryotic translation initiation factor 2α (eIF2α), which inhibits protein synthesis and stimulates transcription of genes involved in autophagy (ATGs) by activating the transcription factor ATF4. Upon dissociation of GRP78, ATF6 translocates to the Golgi apparatus where it is cleaved by serine protease 1 (SP1) and SP2. The short ATF6 N-terminal cytosolic domain translocates to the nucleus and binds to cyclic adenosine monophosphate response element (CRE) and ERS response element-1 (ERSE-1) regions of DNA to activate transcription of genes encoding chaperone proteins;

genes involved in autophagy, such as microtubule-associated proteins 1A/1B light chain 3B (LC3b); and genes involved in cell fate, such as C/EBP homologous protein (CHOP). Release of IRE1α by GRP78 induces its activation by oligomerisation and autophosphorylation. Active IRE1α is responsible for processing several mRNAs, including X-box binding protein-1 (XBP-1) mRNA. XBP-1 protein acts as a transcription factor and enhances the transcription of GRP78 and genes involved in ER-associated degradation (ERAD). IRE1α also induces autophagy through c-Jun N-terminal kinase (JNK) activation. c. In case of ERS overactivation, the IRE1α phosphorylated oligodimer activates tumour necrosis factor receptor-associated factor 2 (TRAF2).

TRAF2 activation promotes apoptosis signal-regulating kinase 1 (ASK1) activity, which induces both JNK and p38 mitogen-activated protein kinase (p38 MAPK). JNK is responsible for phosphorylation of the mitochondrial proteins B-cell lymphoma 2 (Bcl-2) (anti-apoptotic) and Bcl-2-interacting mediator of cell death (Bim) (pro-apoptotic), which are respectively activated and inhibited. p38 MAPK stimulates Bcl-2 homologous antagonist/killer (Bak) and Bcl-2 associated X (Bax), which are two mitochondrial pro-apoptotic proteins.

Activation of both PERK and ATF6 pathways following ER stress induction stimulates CHOP expression, which triggers different effects on cell fate. CHOP acts as a transcription factor to respectively enhance and inhibit Bcl-2 and Bim transcription. CHOP also promotes eIF2α dephosphorylation through growth arrest and DNA damage-inducible 34 (GADD34) activation, resulting in an increase in protein synthesis including pro-apoptotic proteins. Upon translocation into the nucleus, CHOP stimulates ER disulphide oxidase 1α (Ero1α) transcription (a cell death promoter), which induces Ca²⁺ transport from the ER to the mitochondria, through activation of inositol triphosphate receptor (IP3R). Ca²⁺ transport induces nitric oxide synthase (NOS) and reactive oxygen species (ROS), resulting in cytochrome c (cyt c) release from the mitochondria. Changes in the Ca²⁺ concentration initiate the caspase cascade by inducing both calpain and caspase-7 activation. These two proteins cleave pro-caspase-12, which in turn activates caspase-9. Caspase-9 forms a complex with apoptosis protease-activating factor-1 (Apaf-1) and cyt c, resulting in apoptosis. Adapted from [323].

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1.3.1.2 PERK

PERK is an ER TM kinase protein, which is complexed with GRP78 in homeostatic conditions. The separation of GRP78 and PERK under ERS induces PERK oligomerisation and autophosphorylation in its kinase domain, promoting activation of its intrinsic kinase activity [324]. Active PERK can phosphorylate several proteins and cause different effects in the cell. First, PERK can phosphorylate the nuclear factor erythroid 2-related factor 2 (NRF2), provoking its dissociation from the NRF2-kelch-related protein 1 complex.

Independent NRF2, which is a transcription factor, enters the nucleus and promotes the expression of anti-oxidant genes, promoting cell survival. Another target of active PERK is the eIF2α. PERK can phosphorylate eIF2α, preventing the exchange of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) and maintaining eIF2α in its GDP-bound inactive form. Inactivation of eIF2α reduces translational initiation and repression of global protein synthesis [325]. Nevertheless, eIF2α phosphorylation also favours the expression of specific proteins such as ATF4 and certain chaperones. ATF4 is a transcription factor that assists the transcription of many proteins, affecting several processes. It affects the cell fate by transcribing genes such as CHOP, growth arrestDNA damage-inducible 34 (GADD34) and ATF3. It also promotes autophagy by promoting the transcription of certain autophagy-related genes (ATGs) (Figure 23) [316].

1.3.1.3 IRE1α

IRE1α is a TM protein residing in the ER membrane that possesses endoribonuclease and kinase activity. When IRE1α is bound to GRP78, GRP78 inactivates it, blocking activation of its downstream pathway. Under ERS, GRP78 leaves the complex and the IRE1α gains the

66 capacity for activation and activates its signalling pathway [190]. Activation of IRE1α is a multistep process in which the IRE1α cytosolic domain oligomerises and is autophosphorylated [326]. IRE1α activation entails stimulation of the endoribonuclease activity that processes several mRNAs in the cytoplasm, among them microRNAs that control caspase expression and IRE1α mRNA [327,328]. The most well-known mRNA cleaved by IRE1α endoribonuclease activity is X-box binding protein-1 (XBP-1) mRNA.

XBP-1 mRNA cleavage is accompanied by XBP-1 protein production, a transcription factor that enhances ERAD-related and chaperone gene translation [329]. In this way, IRE1α counteracts high levels of unfolded proteins by guiding them for proteasomal degradation or assisting in correct folding, promoting cell survival [330]. With the same objective, IRE1α is involved in the promotion of autophagy, a cell survival signal, through a JNK-mediated signalling pathway [317].

Despite that, prolonged IRE1α activation shifts the cellular fate towards cell death by inducing apoptosis. IRE1α, together with other proteins that are synthesised during the first steps of the UPR, are capable of regulating cell fate [310]. More concretely, prolonged IRE1α activation mediates tumour necrosis factor (TNF) receptor-associated factor 2 (TRAF2) stimulation, which in turn promotes apoptosis signal-regulating kinase 1 (ASK1) activity. A downstream kinase cascade activation involving p38 MAPK and JNK is promoted as a result of the prolonged IRE1α stimulation, supporting apoptosis stimulation [311].

Finally, extended IRE1α activation triggers the expression of CHOP. CHOP is involved in several different processes that lead to apoptosis. One of these processes is due to its ability to be transported to the nucleus and act as a transcription factor. In the nucleus, CHOP can bind to different DNA regions, diminishing the expression of anti-apoptotic proteins such as

67 B-cell lymphoma 2 (Bcl-2), and enhancing the expression of pro-apoptotic proteins such as Bcl-2-interacting mediator of cell death (Bim) [315]. In its cytosolic location, CHOP activates the GADD34 protein, which in exchange activates eIF2α by dephosphorylating it, contrary to the effect of PERK on this initiation factor. Active eIF2α enhances the translation of pro-apoptotic proteins, while increasing the accumulation of unfolded proteins in the ER, thus magnifying the UPR [312]. The last CHOP apoptotic signal is achieved through the mitochondrial apoptotic pathway. CHOP targets ER oxidase 1α, which in turn activates the inositol phosphate receptor, generating Ca²⁺ transport from the ER to the mitochondria [313].

An increase in the intra-mitochondrial Ca²⁺ concentration provokes cytochrome c release from the mitochondria and activates the caspase pathway (Figure 23) [314].