1. Introduction

Eukaryotic cells have different membrane-bound compartments or organelle with specific biochemical and biological functions. In recent days, the crosstalk between different organelles is studied to elucidate their impact on various human diseases ^[1][2][3]. Inter-organellar connections are achieved by direct physical contact of the membranes or via membrane-bound proteins. These connections are highly regulated and are dynamic in nature^[4]. Among different organelles, one of the most studied interactions is between mitochondria and the endoplasmic reticulum (ER). The first evidence of this particular interaction was discovered in rat liver cells in 1952 ^[5] and 1956^[6][7] through electron microscopy. The isolation of ER-mitochondria contacts was performed ~45 years ago by density gradient differential centrifugation from rat liver^[8][9]. Vance subsequently coined the term mitochondria-associated ER membranes [MAM] ^[10]. These functional sites are involved in lipid metabolism, calcium signaling, mitochondrial fission and fusion, ER stress, apoptosis, and autophagy ^[3]. The molecular foundation of this relationship is complex and involves not only ion homeostasis through the shuttling of calcium but also many structural and apoptotic proteins. The important proteins that are involved in the tethering are mitofusin (MFN), inositol triphosphate receptor (IP₃R), voltage-dependent anion channel (VDAC), glucose-regulated protein 75 (Grp75), mitochondrial fission 1 protein (Fis1), B-cell receptor-associated protein 31 (BAP31), protein tyrosine phosphatase interacting protein 51 (PTPIP51) and vesicle-associated membrane protein-associated protein B (VAPB)^[11]. Studies showed different mitochondrial proteins can regulate the ER stress and Unfolded Protein Response (UPR) pathways. The outer mitochondrial membrane GTPase mitofusin 2 (Mfn2) is known to regulate the shape of the endoplasmic reticulum (ER) controlling ER-mitochondrial contacts. Interestingly deletion of Mfn2 causes activation of ER stress ^[12].

This review will primarily focus on the ER stress sensors and their relation to ER-mitochondrial communication, MAM proteins, and their involvement in various cellular functions. A special effort has been taken to discuss recent insights/developments about the non-canonical role of ER stress sensors in different physiological processes beyond its canonical ER stress signaling.

2. ER Stress and ER Stress Sensors

Endoplasmic reticulum is an organelle that spans a large area in the cytoplasm in the shape of elongated tubules and flat discs. . ER plays a crucial role in maintaining the cellular protein homeostasis or proteostasis. Before the export of proteins from ER to Golgi apparatus, the proteins undergo post-translational modifications and folding with the help of chaperons and folding enzymes/foldases such as Protein Disulphide Isomerases (PDI) present in the ER. Proteins such as calnexin and calreticulin act as a quality control for the proteins to ensure if they are properly folded ^[13][14][15].

Many exogenous and endogenous factors such as UV radiation, reactive oxygen species, hypoxia, protein mutations, lipid homeostasis, deletion of genes and nutrient starvation can cause accumulation of misfolded proteins resulting in ER stress. Also, the membrane phospholipid synthesis is important in maintaining the function of the organelles, hence impairment of phospholipid biosynthesis can upregulate the protein quality control pathway such as the Unfolded Protein Response (UPR) of ER or ER-associated protein degradation (ERAD) ^[16]. Major perturbations to PC or PE can prematurely degrade certain transmembrane proteins such as sarco/endoplasmic reticulum Calcium-ATPase (SERCA) ion pump, which leads to disturbance in the calcium homeostasis ultimately resulting in ER stress. Eukaryotic organisms switch on the UPR of ER that initiates a cascade of cellular signaling to restore homeostasis and regular ER function ^[17].

UPR of ER was first studied on yeast, where it is solely regulated by IRE1, while in mammals there are three major proteins involved in controlling ER stress response: IRE1, protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) ^[17] (Figure 1, bottom). These ER stress sensors get activated upon the presence of misfolded proteins and their activation mechanism is still unclear ^[18][17]. If UPR fails to rescue the cellular protein homeostasis, the cells undergo apoptosis.

IRE1(also known as ERN1, for ER to nucleus transducer 1) is a highly conserved UPR sensor protein with kinase and RNase activity ^[19](Table 1). IRE1 and its downstream transcription factor HAC1/IRE2 (the yeast ortholog of the metazoan XBP1) were first identified as a factor required for growth in the medium deprived of inositol ^[19][20]. IRE1, a type 1 transmembrane protein, has both serine/threonine (S/T) kinase domain and an RNAse domain. In the search for the mammalian counterpart two groups independently identified two homologs of IRE1, named IRE1 alpha and IRE1 beta ^[21][22][23]. IRE1α is found ubiquitously while IRE1β is restricted to the intestinal epithelium. During ER stress the IRE1 is activated through direct or indirect activation by the misfolded proteins^[24][25]. A subsequent oligomerization of the cytoplasmic domain of IRE1 leads to the activation of the RNase domain which splices introns from mRNA encoding the XBP1^[26]. The transcriptionally active XBP1 induces expression on genes such as Glucose regulated protein (GRP78), Protein disulphide isomerase (PDI) and other translocation proteins to regulate the protein folding during ER stress ^[27]. However, in the case of prolonged or unresolved ER stress, IRE1α cleaves various mRNAs localized in the ER through a process called Regulated IRE1α dependent decay (RIDD) ^[28][29].

PERK is a major sensor protein identified from rat pancreatic islets, which identifies the imbalances in the ER during the stress conditions and resolves them by reducing the overall translation. The activation of PERK during ER stress is still unclear but after its activation, it can selectively bind to the misfolded proteins and not the native proteins ^[17][30](Table 1). The ubiquitously expressed PERK has luminal and cytosolic serine/threonine domains. The BiP removal from the luminal domain is involved in the oligomerization and trans-autophosphorylation step which results in the activation of PERK ^[30]. The activated PERK phosphorylates serine 51 (Ser 51) of the alpha subunit in the eukaryotic translation initiation factor 2 alpha (eIF2α). The phosphorylation of eIF2α results in the inhibition of eIF2B, thus it eventually reduces the initiation of the global translation process and the subsequent protein load in the ER. The phosphorylation also results in translation of mRNAs encoding for several factors such as activating transcription factor 4 (ATF4), BiP, GRP94, XBP1, ATF6. During ER stress, all the above-mentioned proteins are required to improve cellular homeostasis. Some of the PERK associated pathways that are involved in ER-Stress induced apoptosis are PERK/eIF2α/ATF4, PERK/CaN, PERK/eIF2α/TDAG51, PERK/eIF2α/IAP2 and PERK/NRF2^[31][32]. One of the main downstream targets of eIF2α-ATF4 is CCAAT/enhancer-binding protein homologous protein (CHOP). CHOP induces apoptosis by activating pro-apoptotic factors such as death receptor 5 (DR5), Bim and telomere repeat binding factor 3 (TRB3) and inhibits anti-apoptotic factors such as BCL-2 ^[33]. The negative feedback loop in the mechanism is played by growth arrest and DNA damage-inducible 34 (GADD34), as it can dephosphorylate eIF2α and restart the protein synthesis^[30].Role of ER Stress Sensor Proteins in ER-Mitochondrial Communication and Beyond.

ATF-6 is a leucine zipper protein, which is encoded by ATF6A for ATF6 alpha and ATF6B for ATF6 beta (Table 1). It is activated in the ER by misfolded proteins and exported to the Golgi where it gets cleaved by a protease; membrane-bound transcription factor peptidase, site1 (S1P). Approximately 400 amino acids from its N-terminal region are cleaved off and now it activates different UPR gene expression in the nucleus. Combining IRE1 and PERK activity, ATF6 also activates XBP1 and CHOP to enhance the UPR signaling pathway^[30][34][35].

3. Non-Canonical Role of Stress Sensors

3.1. IRE1

The role of IRE1 is extremely diverse due to its broad-spectrum role in various cellular processes; however, it has been majorly implicated in ER stress. Few recent studies have identified its new role specifically in the context of ER-mitochondrial communication. It has been found that the AKT-mTOR signaling axis modulates the dynamics of IRE1 RNAse activity by regulating ER-mitochondria contact. The study demonstrated a two-step mechanism of IRE1 attenuation. The auto-phosphorylation first initiates the termination of IRE1 RNAse activity but the complete cessation of RNAse activity only occurs if ER-mitochondria contacts are reformed after their initial uncoupling by ER stress^[36]. IRE1 controls the expression of GRP78 upon ER stress. During ER stress GRP78 plays a significant role in the stabilization of WASF3 on mitochondrial membranes. Interestingly another MAM protein ATAD3 forms a ternary complex involving ATAD3A, WASF3, and MAM associates GRP78. ATAD3A acts as a crucial mediator to promote cell invasion in breast and colon cancer via regulating GPR78-mediated stabilization of WASF3^[37].

Besides its canonical role in ER stress several new reports establish the involvement of IRE1 in different physiological processes. Interestingly a recent study on yeast identified iRE1/hac1 splicing pathway is essential for cellular adaptation upon diauxic shift via mitochondrial enlargement. They demonstrated IRE1 dependent increase of mitochondrial gene expression upon diauxic shift ^[38]. Similarly, in higher organisms, the non-canonical function of IRE1α also determines the mitochondrial calcium uptake. IRE1 α deficiency alters mitochondrial metabolism in vivo. Through mutagenesis analysis, Carreras-Sureda et al. successfully dissected out the housekeeping gene property of IRE1 α and uncovered a contribution of IRE1α to the maintenance of MAM composition and function even in the absence of ER stress. IRE1 α, itself, determines the distribution of inositol-1,4,5-trisphosphate receptors at MAMs. This study proposed a new model that establishes the fact that IRE1α operates as a scaffold that stabilizes InsP3Rs at MAMs ^[39]. Not only controlling ER-mitochondrial dynamics and mitochondrial calcium uptake IRE1α plays a very crucial role in terms of cell survivability. Mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). This is the first study that shows the regulation of IRE1α activity by ubiquitylation through a mitochondrial protein. MITOL promotes K63-linked chain ubiquitination of IRE1α and subsequently prevents hyper-oligomerization of IRE1alpha and regulates IRE1α -dependent decay (RIDD) ^[40][36]. Additionally, MITOL also ubiquitylates another MAM protein mitofusin 2 (Mfn2) ^[41]. It enhances the GTPase activity of Mfn2, resulting in the tethering between the ER and mitochondria rather than mitochondrial fusion (Sugiura et al., 2013). Interestingly, it is already known that Mfn2 can also modulate the UPR and mitochondrial function via repression of PERK, another ER stress sensor, and a member of MAM ^[42]. All these indicate a complex interrelation of ER stress sensors in controlling ER mitochondrial communication beyond its canonical role.

3.2. PERK

Accumulation of misfolded proteins inside ER activates PERK through its dimerization and auto-phosphorylation. The serine/threonine (S/T) kinase PERK relieves folding pressure through eIF2α phosphorylation mediated translation shutdown and increases ER proteostasis mainly through the transcriptional activation of ATF4 ^[43]. Beyond this canonical role, PERK is also involved in various UPR-independent signaling functions. Cullinan et al. 2003 identified PERK mediated activation of nuclear factor-erythroid-2-related factor 2 (NRF2) transcription factor for cell survival^[44]. Later Agostinis group demonstrated that PERK is absolutely essential in ER mitochondrial sites to convey apoptosis during ROS-induced ER stress^[45]. Later several other studies also establish that PERK facilitates the propagation of different signaling routes from the ER to juxtaposed mitochondria by tethering these organelles. Munoz et al. found repression of PERK can control mitochondrial function. Mfn2 is a novel PERK modulator and its deficiency causes mitochondrial dysfunction through sustained activation of PERK^[42]. It was also observed that PERK-regulated translational attenuation reduces mitochondrial protein import through the degradation of the TIM23 subunit TIM17A^[46]. The Mfn2 -PERK signaling might also be linked through microRNA-mediated signaling. During ER stress, miR-106b-25 cluster is repressed by the Perk-dependent transcription factors Atf4 and Nrf2 ^[47]. Conversely, another independent study demonstrated that Mir106b mediated Mfn2 suppression is absolutely required for mitochondrial fusion via PKM2 ^[48]. Taken together it can be concluded that during ER stress while PERK leads to Mir106b repression it may subsequently modulate mitochondrial fission. A very recent study has shown that during ER stress there is a dynamic remodeling of mitochondrial morphology by promoting protective stress-induced mitochondrial hyper fusion (SIMH) through PERK by depleting Yme1l ^[49]. A recent study identified a direct link between the PERK mediated ubiquitination pathway and another MAM component, Leucine-rich repeat kinase 2 (LRRK2). It was found that LRRK2 regulates endoplasmic reticulum–mitochondrial tethering and mitochondrial bioenergetics. LRRK2 regulates the activities of E3 ubiquitin ligases MARCH5, MULAN, and Parkin via kinase-dependent protein–protein interactions. When the Kinase-active LRRK2(G2019S) becomes dissociated from these ligases, it leads to their PERK-mediated phosphorylation and activation. PERK-mediated phosphorylation of those ligases, in turn, induces ubiquitin-mediated degradation of ER–mitochondrial tethering proteins ^[50].

Not only PERK-mediated ER mitochondrial connection but also PERK-dependent ER plasma membrane connection is crucial in maintaining cellular homeostasis during ER stress. Using proximity-dependent biotin identification (BioID), Vliet et al. identified the actin-binding protein Filamin A (FLNA) is a key PERK interactor. Loss of PERK results in disturbed actin cytoskeleton and increased cortical F-actin^[51].

^[52]. Nutrient stress due to glucose starvation demands a cellular energetic shift from cytosolic glycolysis to mitochondrial oxidative phosphorylation (OXPHOS) system in order to maintain cellular growth and survival^[53]. Moreover, nutrient starvation can also enable ER stress by disrupting protein folding and glycosylation in the ER. In a recent study, Balsa et al. demonstrated how ER communicates with OXOPHOS system to increase ATP production and promote proteostasis in the cell through a previously unknown mechanism controlled by PERK. The study found that PERK activation during ER stress and glucose deprivation stimulates mitochondrial bioenergetics through the formation of respiratory supercomplexes (SCs). PERK/eIF2α/ATF4 axis transcriptionally controls SC assembly factor 1 (SCAF1) levels to maintain the formation of respiratory supercomplexes. Interestingly, the PERK-dependent mechanism can restore the defects in patients with complex I mutations, thus proving PERK activation can be future targets for mitochondrial diseases^[54].

A direct link between PERK-mediated control on insulin resistance has recently been demonstrated by Biddinger group^[55]. The authors found that trimethylamine N-oxide (TMAO) binds to PERK at physiologically relevant concentrations and selectively activates the PERK mediated induction of the transcription factor FoxO1, a key driver of metabolic disease. TMAO which is derived from gut microbiota is increased by insulin resistance and associated with several metabolic syndromes in humans. At pathologically relevant concentrations, TMAO binds and activates PERK and subsequently promotes hyperglycemia. Inhibition of TMAO synthesizing enzyme, flavin-containing monooxygenase 3 (FMO3), with 3,30 -diindolylmethane reduces PERK Activation and Insulin resistance in vivo suggesting a potential route of the therapeutic intervention for metabolic syndrome.

Brown adipose tissue (BAT) is one of the major tissues involved in thermogenesis and plays an important role in metabolic function that contributes to energy consumption (Cannon & Nedergaard, 2004). During brown adipose tissue differentiation, phosphorylation of PERK happens without any ER stress. This PERK phosphorylation induces transcriptional activation by GA-binding protein transcription factor α subunit (GABPα), which is required for mitochondrial inner membrane protein biogenesis.

3.3. ATF6

ATF6 is the third sensor that plays a significant role in the activation of ER stress pathways. Unlike IRE1 and PERK, Atf6 is unique for its specialized activation process. Upon ER stress, the ATF6 traffics from the ER to the Golgi apparatus followed by a sequential cleavage. The cleaved active form acts as a transcription factor of various genes to restore ER homeostasis. Though it is not a MAM component, it is also involved in different non-canonical processes controlling organelle homeostasis beyond ER stress.^[56][57]. Stress independent activation of ATF6 transcription factor in various in vitro cellular models selectively reduces secretion and extracellular aggregation of destabilized amyloid disease-associated proteins. No significant changes have been observed after selective overexpression of ATF6 on the secretion of the endogenous proteome^[58][59].

Recently Burkewitz et al. 2020 demonstrated the loss of ATF6 promotes longevity in C elegans. Interestingly, the life span extension is not through canonical proteostasis pathways but through the modulation of ER-mitochondrial calcium homeostasis.^[60]. Not only in C elegans, but another study conducted by Wang et al. 2018 with human mesenchymal stem cells (hMSCs) also showed that ATF6 can control aging through maintaining organelle homeostasis. Inactivation of ATF6 led to organelles’ dysfunction and accelerated cellular senescence, a process in which FOS functioned as one of the mediators ^[61]. Not only stem cells, Druelle et al. (2018) found significant changes in ER morphology during cellular senescence in normal human dermal fibroblasts (NHDFs). Senescent NHDFs also exhibited activation of UPR along with ER expansion. Knockdown of Atf6α inhibited ER expansion, the modification of senescence-associated cell shape, and decreased senescence-associated β-galactosidase activity ^[62]. Besides this non-canonical regulation, maintenance of mitochondrial biogenesis and function are also controlled during ER stress via Atf6 and PGC1α ^[63].

Earlier ATF6 got its attention when it was found to be extremely important for an adaptive response during Ischemia/Reperfusion (I/R). The canonical ATF6-dependent ER stress response genes conferred protection from I/R damage in ex vivo isolated perfused heart preparations and maintained contractile function^[64]. ^[65]. Another subsequent study using the same mouse model determined ATF6 specific regulation of particular microRNA that can control the activity of ER luminal calcium-binding protein, calreticulin (Belmont et al., 2012). Overexpression of the active ATF6 transcription factor in the heart also has been shown to improve cardiac performance in mouse models of ischemic heart disease, through a mechanism involving ATF6-dependent regulation of the antioxidant gene, catalase^[66]. Very recently using conditionally deleted cardiac myocyte-specific ATF6 knockout mouse (ATF6 cKO) showed exacerbated myocardial damage in comparison to wild type (Blackwood et al., 2019b). The study also found ATF6 was required to induce the expression of a small GTP-binding protein, Rheb, and, thus, control the mTORC1-dependent growth in pathological hypertrophy. Similarly, another tissue-specific role of ATF6 has been observed in the liver. Insulin sensitivity has been improved significantly after overexpression of the active ATF6α transcription factor in the liver of obese mice^[67].

Although UPR and its sensors (IRE1, PERK, and ATF6) have classically been linked to ER stress, increasing evidence suggests that the sensors have various non-canonical functions in various cellular processes beyond the secretory pathway surveillance during ER stress. Specifically, IRE1 and PERK as a member of MAM are directly involved in various signaling events that establish coordinated function with mitochondria and other cellular organelles.

Conclusively, the MAM site is a hot spot for the transfer of signals between the ER and mitochondria. Along with other MAM proteins, IRE1 and PERK, two membrane-bound ER stress sensors, regulate signaling events not only during stress but also in several other physiological processes. An enhanced understanding of these players can lead to targeted therapeutic interventions as these two molecules are highly involved in the development of different diseases including neurodegenerative disease through different pathways. Table 1. Unfolded Protein Response (UPR) sensors from yeast to mammals.