InteractiveFly: GeneBrief

pancreatic eIF-2α kinase: Biological Overview | References

BIOLOGICAL OVERVIEW

Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. This study shows that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK was also specifically activates in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response became deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. These studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPRER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan (Wang, 2015).

Progressive decline of proliferative homeostasis in high-turnover tissues is a hallmark of aging, resulting in cancers and degenerative diseases. This is of particular relevance in barrier epithelia, such as the intestinal epithelium, where homeostatic tissue renewal has to be balanced with acute regenerative episodes in response to acute damage or infection. Accordingly, the control of intestinal stem cell (ISC) proliferation has to integrate endogenous control mechanisms with stress and inflammatory signals that promote mitogenic activity of these cells. How cellular stress responses of intestinal epithelial cells (IECs) and intestinal stem cells (ISCs) coordinate and maintain such regenerative processes is a critical question that will provide insight into the etiology of pathologies ranging from inflammatory bowel diseases (IBDs) to colorectal cancers (Wang, 2015).

Long-term homeostasis of the intestinal epithelium is significantly impacted by ER stress. In mouse models for IBDs, ER stress is increased in the intestinal epithelium, and genetic conditions that impair protein folding capacity in the ER of IECs result in complex cell-autonomous and non-autonomous activation of stress signaling pathways, triggering inflammatory conditions similar to IBDs. Recent studies in mice suggest that the UPR^ER may also influence regenerative processes in the gut directly, as it is engaged in cells transitioning from a stem-like state into the transit amplifying state in the small intestine of mice. In flies, ER stress promotes ISC proliferation, and increased ER stress across the intestinal epithelium is associated with age-related dysplasia in this tissue (Wang, 2014). The downstream signaling mechanisms promoting ISC proliferation in response to ER stress remain unclear (Wang, 2015).

Three highly conserved UPR^ER sensors coordinate the cell-autonomous response to ER stress: PERK, the transcription factor ATF6, and the endoribonuclease IRE1. IRE1 promotes splicing of the mRNA encoding the transcription factor Xbp1 (see Drosophila Xbp1), PERK phosphorylates and inhibits the translation initiation factor 2 alpha (eIF2α) (Heijmans, 2013; Shi, 1998; Harding, 1999), and ER stress-induced cleavage of ATF6 promotes its nuclear translocation and activation of stress response genes, including Xbp1 (Schroder, 2005). The activation of Xbp1 and ATF6 results in transcriptional induction of ER chaperones, of genes encoding ER components, and of factors required to degrade un/misfolded proteins through ER-associated degradation (ERAD), thus enhancing ER folding capacity and proteostatic tolerance (Wang, 2015 and references therein).

Studies in worms have shown that, in addition to these cell-autonomous responses to ER stress, local activation of the UPR^ER can trigger UPR^ER responses in distant tissues, indicating that endocrine processes exist that coordinate such stress responses across cells and tissues. The mechanism(s) regulating and mediating these non-autonomous responses remain elusive (Wang, 2015).

By regulating eIF2α, ATF4 and Nrf2, PERK activation integrates the response to both protein misfolding in the ER and to misfolding-associated oxidative stress. Accumulation of un/misfolded proteins in the ER results in the production of reactive oxygen species (ROS), most likely due to the generation of hydrogen peroxide as a byproduct of protein disulfide bond formation by protein disulfide isomerase (PDI) and ER oxidoreductin 1 (Ero1) (Wang, 2015).

The coordinated control of cellular protein and redox homeostasis by the UPR^ER and other stress signaling pathways is likely critical to maintain SC function, as the intracellular redox state significantly impacts SC pluripotency, proliferative activity, and differentiation. Recent studies shown that this coordination is achieved in Drosophila ISCs by integration of Nrf2/CncC-mediated responses and Xbp1-mediated ER stress responses (Wang, 2014). The fly orthologue of Nrf2, CncC (Cap 'n' collar isoform-C), counteracts intracellular oxidants and limits proliferative activity of ISCs (Hochmuth, 2011). In ISCs, CncC is inhibited in response to high ER stress (as in Xbp1 loss-of-function conditions), resulting in increased oxidative stress and activation of ISC proliferation (Wang, 2015).

The Drosophila ISC lineage exhibits a high degree of functional and morphological similarities with the ISC lineage in the mammalian small intestine. ISCs self-renew and give rise to transient, non-dividing progenitor cells called EnteroBlasts (EBs) that are lineage-restricted (by Robo/Slit signaling and differential Notch signaling) to differentiate into either absorptive EnteroCytes (ECs) or secretory EnteroEndocrine (EEs) cells. ISCs are the only dividing cells in the posterior midgut of Drosophila and their entry into a highly proliferative state is regulated by multiple stress and mitogenic signaling pathways, including Jun-N-terminal Kinase (JNK), Jak/Stat, Insulin, Wnt, and EGFR signaling (Wang, 2015).

During aging, flies develop epithelial dysplasia in the intestine, caused by excessive ISC proliferation and deficient differentiation of EBs (Biteau, 2008; Choi, 2008). This phenotype is a consequence of an inflammatory condition initiated by immune senescence and dysbiosis of the commensal bacteria, and causes metabolic decline, loss of epithelial barrier function, and increased mortality (Rera, 2012; Biteau, 2010; Guo, 2014), and is associated with a strong tissue-wide increase in ER stress (Wang, 2014). Increasing ER proteostasis in ISCs (by over-expressing Xbp1 or the ERAD-associated factor Hrd1) prevents the age-related over-proliferation of ISCs, suggesting that limiting ER stress-associated signaling in ISCs may be beneficial for tissue homeostasis (Wang, 2015).

This study has tested this hypothesis. The regulation of ISC proliferation by cell-autonomous and non-autonomous UPR^ER responses was explored in detail, and the consequences of limiting ER stress responses in ISCs for longevity were explored. By analyzing loss of function conditions for Ero1L this study finds that the induction of ISC proliferation by ER stress can be uncoupled from the production of ROS, but that ISC-specific activation of PERK is critical for the proliferative response. Interestingly, PERK activation in ISCs is triggered both by ER stress within ISCs and non-autonomously by ER stress in other cells of the intestinal epithelium, which activate PERK in ISCs through the secretion of Unpaired ligands and activation of JAK/Stat signaling in ISCs. PERK thus integrates epithelial stress responses to control ISC proliferation under challenging proteostatic conditions. Strikingly, PERK is also essential for normal cell proliferation in the ISC lineage, and excessive or chronic PERK activity in ISCs is a cause for the development of epithelial dysplasia in aging flies. Accordingly, this study demonstrates that limiting PERK expression in ISCs is sufficient to extend lifespan (Wang, 2015).

This study identifies the PERK branch of the UPRER as a central node in the control of proliferative homeostasis in the intestinal epithelium, and establishes a previously unrecognized role for PERK in promoting regenerative responses to both tissue-wide and cell-autonomous ER stress. This critical function of PERK in tissue regeneration, however, also results in the aging-associated loss of proliferative homeostasis in the intestinal epithelium, limiting organismal lifespan. The unique and specific increase in eIF2α phosphorylation in ISCs in stressed and aging conditions suggests a differential activation of the PERK-eIF2α branch of the UPRER between ISCs and their daughter cells. It remains unclear whether this differential regulation reflects different strategies in combating ER stress between these cell populations, and additional studies are necessary to address this interesting question (Wang, 2015).

Drosophila ISCs, as many other stem cell types, are controlled extensively by redox signals. Previous work, as well as the results shown in this study, suggests that ER-induced oxidative stress plays a central role in the control of ISC proliferation after a proteostatic challenge. The results support the notion that ER-induced ROS is a consequence of the PDI/Ero1L system, as has been proposed in mammalian cells (Harding, 2003). However, Ero1L, as a thiol oxidase, may also affect the proper folding and maturation of Notch directly (as described previously), inhibiting ISC differentiation, and resulting in stem cell tumors. The phenotype of Ero1L-deficient ISC lineages supports a role for Ero1L in Notch signaling (tumors with elevated numbers of Dl+ cells). At the same time, this study's results also support a role for Ero1L in limiting ISC proliferation directly through the UPRER (and independently of Notch signaling or oxidative signals), as loss of Ero1L induces PERK activity without promoting ROS production in these cells. PERK itself is required for the induction of cell cycle and DNA replication genes in ISCs responding to TM treatment, yet it also induces antioxidant genes under these conditions, suggesting complex crosstalk between PERK-mediated control of mitotic activity of ISCs and the control of redox homeostasis in these cells (Wang, 2015).

The fact that loss of Ero1L activates PERK while not inducing Xbp1 in ISCs suggests selective activation mechanisms for these two branches of the UPRER. The study proposes that this selectivity is associated with the production of ROS and that ER protein stress activates the Xbp1 branch when associated with a ROS signal, while PERK can be activated by unfolded proteins independently of ROS production. Further studies are needed to dissect the relative contribution of ROS production, PERK activation and Notch perturbation in the control of ISC proliferation in Ero1L loss of function conditions (Wang, 2015).

This study highlights the interaction between cell-autonomous and non-autonomous events in the ER stress response of ISCs and support the notion that improving proteostasis by boosting ER folding capacity in stem cells improves long-term tissue homeostasis and can impact lifespan. The regulation of PERK activity in ISCs by the JAK/Stat signaling pathway provides a tentative mechanism for the interaction between IECs experiencing ER stress and ISCs: the study proposes that JNK-mediated release of JAK/Stat ligands from stressed IECs results in JAK/Stat mediated activation of PERK in ISCs, and that this activation is required for the proliferative response of ISCs to epithelial dysfunction. The activation of JAK/Stat signaling in the intestinal epithelium of animals in which Xbp1 is knocked down in ECs, the requirement for JNK activation and Upd expression in ECs for ISC proliferation in response to stress, and the requirement for Stat (and Hop and Dome) in ISCs for the activation of eIF2α phosphorylation and stress-induced ISC proliferation, support this model. The mechanisms by which Stat mediates activation of PERK remain unclear, and will be interesting topics of further study (Wang, 2015).

Studies in worms have established the UPRER as a critical determinant of longevity, and Xbp1 extends lifespan by improving ER stress resistance. This study's data further support the notion that regulating ER stress response pathways is critical to increase health- and lifespan. Here, chronic PERK activation can be considered a downstream readout of the buildup of proteotoxic stress in the intestinal epithelium during aging, which then perturbs proliferative homeostasis by continuously providing pro-mitotic signals to ISCs. Knocking down PERK in ISCs limits these pro-mitotic signals, improving homeostasis and barrier function, and extending lifespan. Lifespan is generally extended when ISC proliferation is limited in older flies, but not when it is completely inhibited. Accordingly, they observe lifespan extension when PERK is knocked down using an RNAi approach that does not completely ablate PERK function.

ER stress has been documented as tightly associated with intestinal inflammation and the development of IBDs in mice and humans. Genetic variants in Xbp1 are associated with higher susceptibility to IBD and a recent study indicates that Xbp1 can act as a tumor suppressor in the intestinal epithelium, by limiting intestinal proliferative responses and tumor development through the control of local inflammation. In this context, the specific role of PERK in the control of ISC proliferation in the fly gut is consistent with the function of PERK in the intestinal epithelium of mice, where activation of PERK can promote transition of ISCs into the transient amplifying cell population. While the Drosophila midgut epithelium does not contain a transit amplifying cell population, this study's data suggest that a role for PERK in the proliferative response of the ISC lineage to ER stress is conserved (Wang, 2015).

Due to the importance of the UPRER in the maintenance of tissue homeostasis in aging organisms, therapies targeting the UPRER are promising strategies to delay the aging process. Accordingly, pharmaceuticals that can limit ER stress (such as Tauroursodeoxycholic acid, TUDCA and 4-phenylbutyrate, PBA) have had therapeutic success in various human disorders. Interestingly, flies fed PBA show increased lifespan, yet the effects of PBA on intestinal homeostasis have not yet been explored. Based on this work, it is likely that further characterization of the effects of UPRER-targeting drugs on ISC function and intestinal homeostasis will help develop clinically relevant strategies to limit human aging and extend healthspan (Wang, 2015).

The PERK pathway independently triggers apoptosis and a Rac1/Slpr/JNK/Dilp8 signaling favoring tissue homeostasis in a chronic ER stress Drosophila model

The endoplasmic reticulum (ER) has a major role in protein folding. The accumulation of unfolded proteins in the ER induces a stress, which can be resolved by the unfolded protein response (UPR). Chronicity of ER stress leads to UPR-induced apoptosis and in turn to an unbalance of tissue homeostasis. Although ER stress-dependent apoptosis is observed in a great number of devastating human diseases, how cells activate apoptosis and promote tissue homeostasis after chronic ER stress remains poorly understood. This study used the Drosophila wing imaginal disc as a model system. Presenilin overexpression induces chronic ER stress in vivo. In this novel model of chronic ER-stress, a PERK/ATF4-dependent apoptosis required downregulation of the antiapoptotic diap1 gene. PERK/ATF4 also activated the JNK pathway through Rac1 and Slpr activation in apoptotic cells, leading to the expression of Dilp8. This insulin-like peptide caused a developmental delay, which partially allowed the replacement of apoptotic cells. Thanks to a novel chronic ER stress model, these results establish a new pathway that both participates in tissue homeostasis and triggers apoptosis through an original regulation (Demay, 2014).

As has been reported in mammalian cells, this study hase validated that Psn overexpression can provoke chronic ER stress in Drosophila. In mammalian models, the UPR branches can display opposite roles depending on the model. For example, Perk can be either anti or proapoptotic (Hamanaka, 2009; Verfaillie, 2013; Oomen, 2013) Thanks to a new model of chronic ER stress, this study has demonstrated that the PERK/ATF4 pathway has a fundamental role in Drosophila tissue homeostasis. So far, the autosomal dominant retinitis pigmentosa (ADRP) model was the only model of strong chronic ER stress reported in Drosophila. This study has validated that Psn overexpression can also provoke a chronic ER stress in Drosophila, as previously reported in mammalian cells. In both Drosophila models, apoptosis is induced by UPR in response to ER stress. Nevertheless, this induction involves totally different pathways. In this study's chronic ER stress model, cell death induction is PERK/ATF4 dependent and JNK independent, contrarily to the ADRP model in which CDK5 activates JNK signaling that triggers apoptosis. These differences show that the complexity of ER stress-induced signaling found in mammals is conserved in Drosophila, thus highlighting the usefulness of ER stress models plurality (Demay, 2014).

This study has shown that the PERK/ATF4 pathway induces a caspase-dependent apoptosis by repressing diap1 transcription. However, PERK has been described to exert some antiapoptotic activity by inducing IAP gene expression in mammals. This effect does not seem to rely on direct targets of PERK, ATF4 and CHOP. Similarly, this study did not find any ATF4 consensus binding sequence (5'-RTTRCRTCA-3') in the diap1 promoter region, and no CHOP homolog has been found in Drosophila. Therefore, the mechanisms involved in PERK regulation of IAPs remain to be clarified (Demay, 2014).

In the chronic ER stress model, the JNK pathway is activated in apoptotic cells to favor tissue homeostasis without stimulating cell proliferation. This is in contrast to a JNK activation in cells neighboring apoptotic cells, which results in an increase of the proliferation rate. Similar to this observation, JNK activation in apoptotic cells has been observed in 'undead cell' models. In these models, the JNK pathway could be activated by DIAP1 or DRONC, whereas JNK signaling seems to be primarily independent from DIAP1/DRONC in the current model. In a mammalian model of chronic ER stress, the IRE1 branch of the UPR activated the JNK pathway to trigger apoptosis thanks to TRAF2/ASK1. In the ER stress model, depletion of traf2 or ask1 had no effect. Instead, this study has shown that JNK pathway activation mainly depends on the PERK/ATF4 pathway. Interestingly, this particular JNK pathway is not mainly activated by apoptosis and does not modulate cell death or proliferation (Demay, 2014).

It was also shown that PERK/ATF4 regulates an ER stress-induced developmental delay. As previously reported, it was observed that Dilp8 is a major contributor to developmental delay. An obvious candidate for a Dilp8-independent developmental delay regulation was the retinoic acid signaling that has been reported to modulate an irradiation-induced developmental delay. This study tested if this pathway could also regulate the developmental delay caused by Psn overexpression. No wing phenotype modification was detected upon the downregulation of this pathway (Demay, 2014).

This study characterized the components of the JNK signaling that is activated in response to chronic ER stress in Drosophila wing imaginal discs (see Model of tissue homeostasis maintenance after an ER stress). The small GTPase Rac1 would activate the JNKKK Slpr, which in turn would activate JNK signaling core to regulate dilp8 expression and ultimately favor development delay and tissue homeostasis maintenance. How the ATF4/PERK branch activates Rac1 remains to be elucidated. The results also suggest the existence of a negative feedback loop regulating the JNK pathway, which would involve the JNKKKK, Msn. This is in agreement with a genetic and phosphoproteomic study showing that Msn is able to inhibit the phosphorylation of Jun. Considering that the JNK pathway induces dilp8 expression in abnormally growing imaginal discs in other stress models, one may wonder whether the same JNK pathway is implicated in these models. Moreover, one may wonder whether dilp8 control during tissue homeostasis-associated developmental delay is always JNK-dependent and relies on a Rac1/Slpr pathway (Demay, 2014).

To summarize, this study has shown that in response to an ER stress induced by Psn overexpression, the PERK pathway is activated resulting in a Janus-faced ATF4 role. On one hand, ATF4 induces caspase-dependent apoptosis by repressing diap1 expression and on the other hand, it favors tissue homeostasis maintenance through the induction of a Rac1/Slpr/JNK pathway and the resulting dilp8 expression. More investigations on this new Drosophila chronic ER-stress model should allow the identification of novel regulators of UPR-dependent tissue and organism homeostasis that may be conserved in mammals (Demay, 2014).

ATF4-induced Warburg metabolism drives over-proliferation in Drosophila

The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. This study shows that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. This stress adaptation is found to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. These results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity (Sorge, 2020).

Controlling cell proliferation is one of the major challenges of multicellular life, both during phases of growth in developing organisms and phases of homeostatic cell replenishment essential in adult animals. Lack of appropriate control can lead to severe disorders, including cancer, at any stage of life. While over-proliferation of transformed, cancerous cells is usually caused by inactivation of tumor suppressors and/or activation of oncogenes, it has long been noted that tumors exhibit altered cellular characteristics such as a glycolytic metabolism. While this metabolic switch has been shown to be caused by oncogene signaling, cellular metabolism is also controlled at multiple levels under normal physiological (non-transformed) conditions, including at the transcriptional level through diverse stress-response pathways. One of these is the activating transcription factor 4 (ATF4), which is known to activate a transcriptional (integrated) stress response (ISR) under various stress conditions that trigger phosphorylation of eIF2α. The ATF4 transcriptional program consists of a diverse set of genes with cytoprotective function, but chronic activation induces apoptosis indirectly through transcription of the mammalian ATF4 target CHOP. Yet, ATF4 activation has been detected in several human tumors, especially in hypoxic or nutrient-deprived regions, where ATF4 has been attributed with pro-survival and pro-proliferative effects. Interestingly, a recent study showed that melanoma cells respond to inhibition of their glycolytic metabolism by activating an ATF4 response, whose metabolic reconfiguration allows these cells to continue oncogenic growth, together arguing that ATF4 can provide cancer cells with a metabolic flexibility that allows them to tolerate hypoxic and nutritional stress or cancer therapy aimed at metabolism. Among the many conditions activating ATF4, recent work with cultured cells showed that inhibition of mitochondrial function is linked to ATF4 translation and activity. However, from these and other studies, both the mechanistic basis and the in vivo implications of this response remain to be elucidated (Sorge, 2020).

This study shows that in the fruit fly, Drosophila melanogaster, genetic perturbation of the electron transport chain (ETC), which induces a Warburg-like metabolism, activates a transcriptional stress response mediated through the eIF2α-kinase PERK and ATF4 in eye progenitor cells of Drosophila larvae. Importantly, this in vivo stress response is activated under ETC knockdown conditions, in the absence of obvious mitochondrial dysfunction. Interestingly, these results show that the ATF4 transcriptional response, which by itself causes reduced fitness of progenitor cells, is hijacked by growth-promoting pathways like Notch or Ras, leading to increased cellular fitness and enhanced proliferation. The data furthermore suggest that the pH changes associated with ETC impairment resulting in a switch of the metabolism to aerobic glycolysis play an important role in progenitor over-proliferation. In sum, this study shows that ATF4-mediated transcriptional adaptation provides a cell-autonomous response to ETC defects, altering cellular behavior through metabolic adaptation (Sorge, 2020).

Genetically induced disturbance of ETC complex assembly resultrd in a metabolic shift typical for mitochondrial impairment and activated an ATF4-dependent stress response. The in vivo transcriptional adaptation presented in this study confirmed the regulation of LDH and glycolytic enzymes, as shown in Drosophila cultured cells, and further includes several targets shown to be ATF4 target genes in mammalian models. The results showed that the eIF2α-kinase PERK, so far only described for its role in mediating one branch of the unfolded protein response of the endoplasmic reticulum (UPRER), is the upstream kinase phosphorylating eIF2α, thereby inducing ATF4 translation in response to mitochondrial ETC disturbance. Mitochondrial ETC disturbance specifically activated PERK, while other branches of the UPRER were non-responsive. PERK activation upon mitochondrial defects was recently observed in Drosophila models of Parkinson's disease and was explained by the authors by its preferential localization to mitochondria-associated ER membranes, which might make PERK more susceptible to a local stress signal. ROS (reactive oxygen species) released by mitochondria have been suggested to mediate mitochondrial retrograde signaling. While this study observed an attenuation of Delta overexpression (Dl^OE), COX7RNAi-induced over-proliferation upon overexpression of either cytoplasmic catalase or GPx (but not mitochondrial catalase), this study failed to detect increased ROS levels in the larval eye disc. A possible scenario to explain these observations is that ROS are generated locally in the cytoplasm or ER in response to ETC disturbance, thereby triggering PERK activation. Importantly, Drosophila PERK isoform B contains a potential mitochondrial signal peptide, which is not found in mammalian PERK isoforms. Although no evidence for this has been found, Drosophila PERK could reside in the mitochondrial membrane and sense the folding status of mitochondrial complexes. This hypothesis could explain the evolutionary difference between mitochondrial defects and ATF4 induction, as this appears to require GCN2 but not PERK in mammals or to be triggered independently of a single eIF2α-kinase. In addition to canonical ATF4 target genes, ATF4-dependent upregulation of mitochondrial chaperones, a response classically referred to as the mitochondrial UPR (UPRmt) was observed. In C. elegans, mitochondrial chaperone induction upon stress is mediated by ATF4-like transcription factor Atfs-1, while the mammalian UPRmt has been shown to be regulated by another evolutionary-related transcription factor, ATF5. The current data now showed that Drosophila ATF4 is required cell autonomously for the induction of mitochondrial chaperones upon ETC subunit knockdown, implying that Drosophila might represent the evolutionary ancestral ISR-UPRmt regulation through a single ATF4-like transcription factor (Sorge, 2020).

The cooperation between ATF4 target genes and the Notch or Ras pathways in Drosophila imaginal progenitors raised the intriguing possibility that these or other oncogenic pathways could benefit from ATF4 activity in human cancers. Over the last decades, it had been demonstrated that human cancer cells are exposed to several stresses, including hypoxia, ROS, or limitations in nutrient availability. In order to survive these conditions and maintain their growth capacity, tumor cells activate responses like the HIF1α transcription factor axis. Though less well studied, an involvement of ATF4 in cancer has been suggested mostly through work with cultured cells. This study analyzed gene expression in human cancer samples of The Cancer Genome Atlas (TCGA) datasets using Cancer-RNaseq-Nexus and the human protein pathology atlas and found that many of the well-characterized direct ATF4 targets are upregulated in a variety of cancer types. Most strikingly, transcriptomes of kidney renal clear cell carcinoma showed progressive induction of ATF4 and many of its direct targets (EIF4EBP1, ASNS, TRIB3, and VEGFA) on the transcriptional level, which strongly correlated with a poor prognosis in this type of cancer. These data suggest that the ATF4-mediated ISR is used by cancer cells to adapt their metabolic repertoire, thereby sustaining fast growth under increasingly unfavorable conditions (Sorge, 2020).

A novel finding presented in this study was the discovery that ATF4-mediated transcriptional adaptation due to ETC impairment allowed eye progenitors to increase their proliferation in response to signals from the Notch and Ras pathways. The primary questions arising from this genetic interaction is how these signaling pathways can overcome the apparent cellular stress and reduction in proliferation and induce the opposite effect, a massively increased rate of proliferation. Several lines of evidence suggest that over-proliferation in Dl^OE, COX7RNAi eye imaginal discs is controlled by pH changes induced by LDH that modify the activity of Notch downstream effectors. First, in COX7a-depleted cells, the metabolism is switched to aerobic glycolysis, leading to an increased production of lactate due to the activity of LDH. And, consistent with an accumulation of this metabolic acid, this study found the intracellular pH to be reduced in COX7RNAi cells. Second, Dl^OE, COX7RNAi-mediated over-proliferation was rescued by ATF4 knockdown and, to a lesser extent, pH buffering, showing that intracellular pH changes (downstream of ATF4 and LDH) play an important role in proliferation control. Third, LDH phenocopies COX7RNAi, indicating that most of the cooperative effects of Dl overexpression and COX7a knockdown are mediated by the ATF4 target LDH. In the same line, expression of LDH as one of the many ATF4 targets was sufficient to drive Dl-expressing cells into over-proliferation, strongly suggesting that the processes downstream of LDH-in particular, the changes in intracellular pH-lead to a modification of the Notch pathway. Finally, a cooperation of Notch and the COX7a nuclear effector ATF4 on the transcriptional level was not observed, showing that the Notch pathway is not hyper-activated, but arguing that over-proliferation is due to changes in the activity of Notch downstream effectors. The next obvious question is how changes in intracellular pH can modify the activity of signaling pathways. It is known that the intracellular pH can control the protonation of specific histidine residues in proteins acting as pH sensors, leading to changes in protein properties. Importantly, it has been shown recently in chicken embryos that intracellular pH changes induced by a Warburg-like metabolism control the acetylation of the Wnt effector &betsa;-catenin, thereby mediating Wnt signaling activation. Thus, this study envisions that pH changes induced by LDH expression lead to a (non-enzymatic) modification of Notch effectors, thereby increasing fitness and proliferation rates of eye progenitor cells (Sorge, 2020).

This is a very attractive model; however, one result was puzzling. Although LDH is sufficient to induce over-proliferation when combined with the Notch pathway, no rescue (but an increase in the severity) of the Dl^OE, COX7RNAi phenotype was observed when LDH was selectively depleted. This obvious discrepancy can be explained by different hypotheses. One of them is based on a recent study showing that LDHA inhibition in melanoma cell lines also failed to impact cell proliferation, survival, or tumor growth. In this context, LDHA inhibition engaged the GCN2-ATF4 signaling axis to initiate an expansive pro-survival response, including the upregulation of the glutamine transporter SLC1A5 and glutamine uptake, as well as mTORC1 activation. Another hypothesis is based on the finding that a major driver of over-proliferation is the intracellular pH. Since LDH catalyzes the conversion of pyruvate to lactate (and back), reducing LDH levels will affect the ratio of lactate to pyruvate, leading to an increase of the even stronger metabolic acid pyruvate. It has been shown that pyruvate as lactate induces a concentration-dependent intracellular acidification. Thus, it could be envisioned that an enhancement of proliferation rates beyond those observed in Dl^OE, COX7RNAi cells is a consequence of pyruvate accumulation in the absence of LDH, which enhances the decrease in the intracellular pH, resulting in the increase in proliferation rates (Sorge, 2020).

Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. This study addresses which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt (Zhang, 2021).

Cell compartmentalization is not only mediated by membrane-bound organelles. It also relies on non-membrane bound biomolecular condensates (so-called membraneless organelles) that populate the nucleus and the cytoplasm (Zhang, 2021).

The formation of membraneless organelles has been shown to occur through phase separation, which can be driven by stress (such as ER, oxidative, proteostatic or nutrient stress), resulting in the formation of stress assemblies. Those are mesoscale coalescence of specific and defined components that phase separate. For instance, nutrient stress leads to the formation of many biocondensates. Most of them are RNA based, such as stress granules and P-bodies, but some are not. This is the case for glucose-starved yeast where metabolic enzymes foci and proteasome storage granules form, as well as Drosophila S2 cells that form Sec bodies under conditions of amino acid starvation (Zhang, 2021).

Sec bodies are related to the inhibition of protein secretion in the early secretory pathway. The early secretory pathway comprises the endoplasmic reticulum (ER), where newly synthesized proteins destined to the plasma membrane and the extracellular medium are synthesized. Proteins exit the ER at the ER exit sites (ERES) to reach the Golgi. The ERES are characterized by the concentration of COPI-coated vesicles whose formation requires six proteins, including Sec12 and Sar1, the inner coat proteins Sec23 and Sec24, and the outer coat proteins Sec13 and Sec31 (Gomez-Navarro, 2016). In addition, a larger hydrophilic protein called Sec16, has been identified as a key regulator of the ERES organization and COPII vesicle budding. Many additional lines of evidence support the role of Sec16 in optimizing COPII-coated vesicle formation and export from the ER (Zhang, 2021).

Upon the stress of amino acid starvation in Krebs Ringer bicarbonate buffer (KRB), the ERES of Drosophila S2 cells are remodeled into large round non-membrane bound phase-separated Sec bodies. They are typically observed by immunofluorescence after staining of endogenous Sec16, Sec23 and expressed Sec24-GFP. Importantly, Sec bodies are very quickly resolved upon stress relief (addition of growth medium). Finally, they appear to protect the components of the ERES from degradation and they help cells to survive under conditions of amino acid shortage (Zhang, 2021).

Phase separation has been shown to be driven by specific components, the so-called drivers, either RNAs or proteins harboring structural features that become exposed or modified under certain conditions. In the case of Sec bodies, Sec24AB and Sec16 have been shown to drive Sec body coalescence in a manner that depends on a small stretch of 44 residues in Sec16 and on the mono-ADP-ribosylation enzyme by PARP16. This illustrates the critical role of post-translational modifications in phase separation (Zhang, 2021).

In parallel, changes in cytoplasmic biophysical properties have also been shown to be important in phase separation, such as a drop of cytoplasmic pH within minutes, without post-translational modifications (Zhang, 2021).

This study sought out to (1) identify the pathways elicited in S2 cells upon incubation in the starvation medium KRB that lead to Sec body formation, and (2) to assess whether changes in the cytoplasmic biophysical properties play a role in the phase transition leading to Sec body formation. Amino acid starvation in KRB is shown to stimulate ER stress and activation of two downstream kinases, IRE1 and PERK (also known as PEK in flies) leading to the stimulation of the unfolded protein response (UPR). However, the sole activation of the IRE1 and PERK does not lead to Sec body formation. To form Sec bodies in KRB, IRE1 and PERK activation needs to be combined with a moderate salt stress. Accordingly, KRB incubation is faithfully mimicked by cell incubation with dithiothreitol (DTT) and addition of 100 mM NaCl. Interestingly, a high-salt stress addition of 150 mM NaCl, which activates the salt-inducible kinases (SIKs; SIK2 and SIK3), is sufficient to efficiently drive Sec body formation. Importantly, it was found that a decrease in the cytoplasmic ATP concentration, a general RNA degradation and the stimulation of the UPR are factors strongly correlated to Sec body formation (Zhang, 2021).

This study shows that the Sec bodies that form in Drosophila S2 cells incubated in KRB are fully recapitulated by activation of SIKs, IRE1 and PERK (through SCH100 plus DTT), leading to the activation of a downstream UPR. Strikingly, the strong activation of SIKs in (SCH150) also induces the UPR and leads to Sec body formation. The resulting structures in each condition appear to be similar in size and number, and their formation is reversible. Whether their content is strictly similar has not been addressed in this study (Zhang, 2021).

Taken together, the results show that Sec body formation requires the stimulation of two main signaling pathways. The first is the salt stress pathway (addition of 150 mM NaCl), which activates the SIKs in a necessary and sufficient manner. It also does not lead to a change in the cytoplasmic ATP concentration. It does induce RNA degradation and it stimulates the UPR in an unexpected manner, given that PERK and IRE1 inhibitors do not alter SCH150 driven Sec body formation (Zhang, 2021).

The second pathway is the activation of IRE1 and PERK (but not ATF6), downstream of ER stress, which is partly induced by the absence of amino acids in KRB. Activation of either IRE1 or PERK is necessary but not sufficient. To form Sec bodies, this activation needs to be combined with a moderate salt stress. It is proposed that IRE1 and PERK activation combined with SIK activation occur in KRB, which is recapitulated by SCH100 plus DTT. This is associated with a decrease in the cytoplasmic concentration of ATP, with RNA degradation and with a stimulation of the UPR (Zhang, 2021).

Interestingly, both strong salt stress (SCH150) and KRB lead to the activation of the UPR (measured by the increase in Bip protein level), leading to the possibility that SIKs, IRE1 and PERK interact with and/or activate, each other. Either IRE1 and/or PERK activate the SIKs, or SIK activation activates IRE1 and/or PERK. This still needs to be refined. The prominent role of salt stress and SIKs in remodeling the cytoplasm Strong salt stress is induced by a 4-fold increase of Na+ in the medium combined with bicarbonate. This triggers an increase of Na+ in the cytoplasm that activates one or more SIK (as shown by the phosphorylation of the SIK target HDAC4). Accordingly, SIK inhibition decreases Sec body formation (Zhang, 2021).

Keeping intracellular Na+ as near as possible to physiological concentrations (5 mM) is critical for cellular life, and the cell spends 40% of its available ATP to extrude Na+ against K+ with the NaK ATPase. It is therefore not surprising that Na+ stress would elicit a cytoprotective response, such as prominent as Sec body formation (and stress granule formation in mammalian cells). This will need to be further elucidated, as many organisms and tissues are subjected to increased circulating Na+. This study shows, however, that it is not equivalent to an osmotic shock and that this addition of salt does not lead to a decrease in a cell volume. In contrast to P-bodies in yeast, osmotic stress does not induce Sec bodies. Interestingly, Na+/salt stress has recently been shown to induce the biogenesis of the lysosomal pathway (i.e. more endo/lysosomes as well as an increase in its activity) via TFEB and TOR (Zhang, 2021).

Increased Na+ activates the intracellular Na+-sensor network revolving around the SIKs. The SIKs belong to the family of AMPKs, and have been shown to be part of a nutrient-sensing mechanism so far revolving around glucose and unbalance of the ATP-to-ADP ratio. In mammals, there are three genes encoding SIK (SIK1-SIK3) but only 2 in Drosophila. Drosophila SIK2 is the ortholog of human SIK1 and SIK2, and has been shown to have a link to the fly Hippo pathway, possibly linking nutrient to growth. Drosophila SIK3 is required for glucose sensing in the fly. Which SIK is involved in Sec body formation has not been clarified, as overexpression of each SIK individually has not proven enough to trigger Sec body formation, even when combined to some excess salt (SCH84 or SCH100). However, at least two SIKs appear to change their intracellular localization in KRB, that is, SIK2 and the long SIK3 isoform, which appear to cluster near the plasma membrane and localize to the nuclear envelope. The role of SIKs in the formation of stress assemblies (here the Sec bodies) appears important and novel, and needs to be investigated further. Other members of the AMPK family do not appear to be involved and changing the ratio ADP-to-ATP did not alter Sec body formation in the SIC system (Zhang, 2021).

Although a high salt stress is sufficient to trigger Sec body formation, the Sec body formation observed during incubation in the amino acid starvation buffer KRB elicits another pathway, the ER stress pathway, leading to the activation of both IRE1 and PERK. Indeed, KRB-induced Sec body formation is entirely mimicked by a moderate salt stress (SCH100) combined with activation of IRE1 and PERK induced by DTT (SCH100 plus DTT) (Zhang, 2021).

Surprisingly, this study found that salt stress as well as KRB induces the UPR, which this study found is a common downstream event in all conditions inducing Sec body formation. How salt stress activates the UPR and the exact role of IRE1 and PERK is still not fully understood. It does not appear to be via SIKs, as HG does not modulate the UPR, yet strongly reduces Sec body formation. Conversely, IRE1 and PERK inhibitors do not influence SCH150-induced Sec body formation, so the exact link between IRE1, PERK, SIKs and the UPR remains to be further investigated (Zhang, 2021).

How UPR stimulation induces Sec body formation is not completely understood. It could occur through the clustering of IRE1 and PERK, two membrane kinases, and them forming a template in the plane of the ER membrane. In this regard, UPR stimulation has been linked to the MARylation enzyme Drosophila PARP16, which is also a transmembrane protein of the ER that undergoes a remodeling in KRB. What other roles are played by events downstream of the UPR, for example, Bip upregulation itself, and cytoplasmic changes, remains to be elucidated in detail. One interesting aspect is whether the activation of UPR might affect biophysical properties, membrane association dynamics, and conformation and post-translational modifications of Sec16 and COPII subunits, which would lead to their enhanced phase separation properties under stress (Zhang, 2021).

Lowering the cytoplasmic ATP concentration is one parameter that induces Sec body formation The lowering of the cytoplasmic ATP concentration occurs in KRB and SCH100 plus DTT. Forcing this lowering or preventing it has a strong incidence on Sec body formation. This finding is consistent with the hydrotropic property of ATP, that is, it being able to prevent phase separation in the cytoplasm. At least in vitro, addition of 8 mM ATP dissolves or prevents the phase separation of purified FUS and TAF15. This is a concentration matching physiological range of ATP level in mammalian cells (1-10 mM) and that is also compatible with the S2 cell ATP concentration (1.7 mM) (Zhang, 2021).

One possibility to explain the decrease of ATP concentration is the activation of kinases (among them, IRE1, PERK and SIK) that would consume the ATP pool. However, it is proposed that the decrease in the intracellular ATP concentration is upstream of the kinase activation. Indeed, incubating cells in KRB induces a ROS shock (as this study showed experimentally), which could damage mitochondrial respiration, resulting, in turn, in ER stress, and UPR activation. Taken together, although this study unraveled a strong causality between low ATP and Sec body formation, the noticeable exception of such formation in SCH150 suggests other possibilities (Zhang, 2021).

In conclusion, this work illustrates the complexity of amino acid starvation, the number of pathways it activates and how they interact with each other, as well as the different cellular cytoplasmic biophysical parameters it affects. It is proposed that the formation of Sec bodies depends on the activation of signaling pathways leading to activation of SIKs, IRE1 and PERK, altogether leading to the activation of the UPR, one of the common features of all pathways leading to Sec body formation (Zhang, 2021).

The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function

Ribosomal Protein (Rp) gene haploinsufficiency affects translation rate, can lead to protein aggregation, and causes cell elimination by competition with wild type cells in mosaic tissues. This study finds that the modest changes in ribosomal subunit levels observed were insufficient for these effects, which all depended on the AT-hook, bZip domain protein Xrp1. Xrp1 reduced global translation through PERK-dependent phosphorylation of eIF2α. eIF2α phosphorylation was itself suficient to enable cell competition of otherwise wild type cells, but through Xrp1 expression, not as the downstream effector of Xrp1. Unexpectedly, many other defects reducing ribosome biogenesis or function (depletion of TAF1B, eIF2, eIF4G, eIF6, eEF2, eEF1alpha1, or eIF5A), also increased eIF2α phosphorylation and enabled cell competition. This was also through the Xrp1 expression that was induced in these depletions. In the absence of Xrp1, translation differences between cells were not themselves sufficient to trigger cell competition. Xrp1 is shown here to be a sequence-specific transcription factor that regulates transposable elements as well as single-copy genes. Thus, Xrp1 is the master regulator that triggers multiple consequences of ribosomal stresses and is the key instigator of cell competition (Kiparaki, 2022).

This study has explored the mechanisms by which Rp mutations affect Drosophila imaginal disc cells, causing reduced translation and elimination by competition with wild-type cells in mosaics. The findings reinforced the key role played by the AT-hook bZip protein Xrp1, which is a sequence-specific transcription factor responsible for multiple aspects of not only the Rp phenotype, but also other ribosomal stresses. It was Xrp1, rather than the reduced levels of ribosomal subunits, that affected overall translation rate, primarily through PERK-dependent phosphorylation of eIF2α. Phosphorylation of eIF2α, as well as other disruptions to ribosome biogenesis and function such as reduction in rRNA synthesis or depletion of translation factors, were all sufficient to cause cell competition with nearby wild type cells, but this occurred because all these perturbations activated Xrp1, not because differences in translation levels between cells were sufficient to cause cell competition directly. In fact, the data show that differences in translation are neither sufficient nor necessary to trigger cell competition, which therefore depends on other Xrp1-dependent processes. Protein aggregation and activation of 'oxidative stress response' genes were also downstream effects of Xrp1 activity. While this paper was in preparation, other groups have also reported relationships between eIF2α phosphorylation, cell competition, and Xrp1, but none have reached the same overall conclusions as this study (Kiparaki, 2022).

Our findings lead to a picture of Xrp1 as the key instigator of cell competition in response to multiple genetic triggers. Failure to appreciate the role of Xrp1 may have led to questionable conclusions in some previous studies. The current findings confirm the central importance of the transcriptional response to Rp mutations, and to other disruptions of ribosome biogenesis and function. They suggest therapeutic approaches to ribosomopathies, and have implications for the surveillance of aneuploid cells (Kiparaki, 2022).

Rp gene haploinsufficiency has been proposed to affect ribosome concentrations, and hence translation, lead to the accumulation of ribosome components and assembly intermediates, and cause proteotoxic stress. Any of these could have been responsible for activating Xrp1 in Rp^+/- cells (Kiparaki, 2022).

The current data show that in fact ribosome subunit concentration is only moderately affected by Rp haploinsufficiency. 15-20% reduction in LSU concentrations in several RpL mutants, and 20-25% reduction in SSU (small subunit) concentrations in several RpS mutants. RpL14^+/- also reduced SSU ~ 25%. Ribosomal subunit levels were unaffected by Xrp1. Broadly similar results have been reported in yeast, and by mass spec quantification of ribosomal proteins in RpS3^+/- and RpS23^+/- Drosophila wing discs (Kiparaki, 2022).

Multiple explanations for the modest effects on ribosome subunit number are possible. It is particularly pointed out that, even if expression of a particular Rp is reduced in proportion to a 50% reduction in mRNA level, the respective protein concentration (i.e. number of molecules/cell volume) is unlikely to fall to 50%, because ribosomes are required for cellular growth, so that an Rp mutation affects the denominator in the concentration equation, as well as the numerator. It is even possible that a 50% reduction in its rate of Rp synthesis could leave steady state ribosome subunit concentration unaffected, if cellular growth rate was slowed by the same amount (Kiparaki, 2022).

Modest changes in SSU and LSU levels could still affect ribosome function, which may depend more on the concentrations of free subunits than on total subunits. The data suggests, however, that cellular and animal models of ribosomopathy Diamond Blackfan Anemia (DBA) that have generally sought to achieve a 50% reduction in Rp protein expression could be significantly more severe than occurs in DBA patients, and that actual ribosome subunit concentrations should be measured in DBA patient cells to guide future models (Kiparaki, 2022).

This study confirmed that ribosome assembly intermediates accumulate in Drosophila wing discs following Rp haploinsufficiency. In yeast, aggregates of unused Rp rapidly trigger transcriptional changes. It has been suggested proteotoxic stress might lead to eIF2α phosphorylation in Drosophila, with Xrp1 amplifying this effect, but this study found that while Perk was responsible for eIf2α phosphorylation, it was not required for Xrp1 expression in Rp mutants, placing Perk and eIF2α phosphorylation downstream. Consistent with this, it was shown that the protein aggregates reported in Rp^+/- cells were only seen in some Rp mutants, all affecting the SSU, and were also a downstream consequence of Xrp1 activity, as also now seen by others. It remains plausible that unused ribosomal components are the initial trigger for cellular responses in Drosophila as in yeast, but in Drosophila the species involved have not yet been identified. Because Xrp1 expression depends particularly on RpS12, an RpS12-containing signaling species is one possibility (Kiparaki, 2022).

PERK-dependent phosphorylation of eIF2α was the mechanism by which Xrp1 suppresses global translation in Rp^+/- mutants (Kiparaki, 2022).

It is interesting that Xrp1 protein levels increase under conditions of reduced global translation. Perhaps Xrp1 is one of the few genes whose translation is enhanced when eIF2α is phosphorylated. Although PERK is known to be activated by ER stress, the IRE/Xbp1 branch of the UPR was not unequivocally detected in Rp^+/- mutants. It is suspected that the UPR might be suppressed in Rp^+/- mutants by Xrp1-dependent changes in transcription of Perk, BiP, and other UPR genes. Perhaps in proliferative tissues it is preferable to replace stressed cells than to repair them (Kiparaki, 2022).

It will be interesting to determine whether eIF2α phosphorylation occurs in human ribosomopathies. Notably, knock-out of CReP, one of the two mouse PPP1R15 homologs, causes anemia, similar to DBA, and PERK-dependent eIF2α phosphorylation occurs in RpL22-deficient mouse αβ T-cells and activates p53 there. Thus, inhibitors of eIF2α phosphorylation could be explored as potential DBA drugs. TAF1B depletion, which also acted through Xrp1 and eIF2α phosphorylation in Drosophila, is a model of Treacher Collins Syndrome, and failure to release eIF6, leading to defective LSU maturation and 80 S ribosome formation, causes Schwachman Diamond syndrome, two other ribosomopathies where potential contributions of eIF2α phosphorylation are possible (Kiparaki, 2022).

Because eIF2α phosphorylation alone was sufficient to target cells for competitive elimination, at first it seemed that eIF2α phosphorylation was the mechanism by which Xrp1 caused cell competition, which often correlates with differences in cellular translation levels. One group has suggested this. Another group concluded that eIF2α phosphorylation in Rp^+/- cells did not lead to cell competition, but the opposite conclusion is corroborated by the independent finding that haploinsufficiency for the γ subunit of eIF2 also causes cell competition. It is concluded that eIF2α phosphorylation can cause cell competition but not directly. Instead, phosphorylation of eIF2α is itself sufficient to activate Xrp1 expression, as found by this and several other groups. Crucially, Perk inactivation restored eIF2α phosphorylation and global translation to normal in Rp^+/- cells, without preventing cell competition, which must therefore depend on other Xrp1 targets. Elimination of eIF2γ haploinsufficient cells is also Xrp1-dependent, as expected if Xrp1 is downstream of eIF2 activity in cell competition (Kiparaki, 2022).

Knock-down of factors directly involved in the translation mechanism further distinguished cell competition from differential translation levels. Different factors affected translation in diverse ways. In Rp^+/- mutants, PERK-dependent phosphorylation of eIF2α suppressed global translation, which was normalized by Perk or Xrp1 depletion. PERK-dependent phosphorylation of eIF2α also contributed to the translation deficits of cells depleted for TAF1B, eIF6, and possibly eEF1α1, which were all partially restored by eIF2α dephosphorylation and fully by Xrp1 depletion, suggesting that Xrp1 can also affect translation by additional mechanisms. By contrast, translation deficits caused by eIF4G, eIF5A, or eEF2 depletion were restored little by eIF2α dephosphorylation or Xrp1 depletion, indicating Xrp1-independent effects of these factors on translation (Kiparaki, 2022).

Several conclusions follow from studies of these factors. As noted above, reduced translation cannot be required for cell competition, because perk^-/- Rp^+/- mutant cells are eliminated by perk+/- Rp+/+ cells. Secondly, lower translation is not sufficient for competitive elimination, because no competitive cell death was observed in eIF4G Xrp1-depleted, eIF5A Xrp1-depleted, and eEF2 Xrp1-depleted cells, even though their translation was lower than the nearby wild type cells. Another group also concluded that lower translation alone was not sufficient for cell competition, based on different data (Kiparaki, 2022).

The current findings focus attention on Xrp1 activity as the key factor marking cells for competition, distinct from its effects on global translation, which only trigger cell competition when Xrp1 is induced (Kiparaki, 2022).

It was confirmed that Xrp1 is a sequence-specific transcriptional activator, and it is proposed that direct transcriptional targets of Xrp1 predispose Rp^+/- cells, and other genotypes, to elimination by wild-type cells. Expression of several hundred single copy genes is regulated by Xrp1 in Rp mutant cells, and this study reports that expression of some transposable elements is affected in addition, whose potential contribution to cell competition might also be interesting. One or more of these transcriptional targets may lead to competitive interactions with wild-type cells (Kiparaki, 2022).

These Xrp1 targets include genes that also contribute to oxidative stress responses, such as GstD genes, which has previously led to the suggestion that an oxidative stress response is responsible for cell competition. Because the oxidative stress reporter used in previous studies is probably activated in Rp^+/- cells by direct Xrp1-binding, and not by the Nrf2-dependent ARE site, it is not now certain whether Rp^+/- cells experience oxidative stress or Nrf2 activity. An alternative explanation of cell competition in response to Nrf2 over-expression could be induction of Xrp1 expression by Nrf2 (Kiparaki, 2022).

These results reveal the central importance of Xrp1 as the driver of cell competition. Far from being expressed specifically in Rp mutants, this study now finds that Xrp1 is induced by multiple challenges, not only to ribosome biogenesis, such as by depletion of the polI cofactor TAF1B or LSU maturation factor eIF6, but also challenges to ribosome function, both at the levels of initiation or elongation, all leading to cell competition and to Xrp1-dependent eIF2α phosphorylation (Kiparaki, 2022).

Had Xrp1 expression and function not been evaluated in PPP1R15-depleted cells, it would have been concluded that eIF2α phosphorylation was the likely downstream effector of competition in Rp mutant cells, rather than an example of another upstream stress that induces Xrp1. It is becoming apparent that other triggers of cell competition, including depletion for Helicase at 25E (Hel25E), a helicase that plays roles in mRNA splicing and in mRNA nuclear export, over-expression of Nrf2, the transcriptional master regulator of the oxidative stress response, and loss of mahjong, a ubiquitin ligase implicated in planar cell polarity, all lead to Xrp1 expression. Earlier models regarding these cell competition mechanisms, in which the role of Xrp1 was not recognized, may be questionable. It would be important now to check for possible activation of Xrp1 in cells with other defects affecting translation, including mutations of an eIF5A-modifying enzyme and mutations of a pre-rRNA processing enzyme. It would not be surprising if other conditions that lead to eIF2α phosphorylation, such as ER stress, nutrient deprivation, or viral infection, also activate Xrp1 and are thereby marked for elimination by more normal neighbors. It will be particularly interesting to determine whether any of these environmental perturbations could interfere with surveillance and removal of aneuploid cells, given the potential importance for tumor surveillance (Kiparaki, 2022).

The transcription factor Xrp1 is required for PERK-mediated antioxidant gene induction in Drosophila

PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2α to initiate the Unfolded Protein Response (UPR). eIF2α phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5' leader. Possible PERK effectors other than ATF4 remain poorly understood. This study reports that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell-type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1(G69D). Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2α phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5' leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1 (Brown, 2021).

The endoplasmic reticulum (ER) is the site where most membrane and secretory proteins undergo folding and maturation. This organelle contains an elaborate network of chaperones, redox buffers, and signaling mediators, which work together to maintain ER homeostasis. When the amount of misfolded or nascent proteins exceeds the folding capacity of a given cell, the ER initiates a gene expression regulatory program that is referred to as the Unfolded Protein Response (UPR) (Brown, 2021).

The ER also represents an important nexus between protein folding and oxidative stress. The ER maintains an oxidizing environment for the formation of intra- and intermolecular disulfide bonds that contribute to the oxidative folding of client proteins. A product of this reaction is hydrogen peroxide, and excessive protein misfolding in the ER can cause the accumulation of reactive oxygen species (ROS). Consistently, genes involved in redox homeostasis are induced in response to ER stress (Brown, 2021).

In metazoans, there are three evolutionarily conserved branches of the UPR initiated by the ER transmembrane proteins IRE1, PERK (PKR-like ER Kinase, also known as Pancreatic ER Kinase (PEK)), and ATF6. The best studied downstream effectors of IRE1 and PERK signaling are the bZIP family transcription factors XBP1 and ATF4, respectively. Once activated in response to ER stress, these transcription factors induce the expression of genes involved in ER quality control, antioxidant response, and amino acid transport. The Drosophila genome encodes mediators of all three branches of the UPR, and the roles of the IRE1-XBP1 and PERK-ATF4 branches in Drosophila development and tissue homeostasis have been established (Brown, 2021).

The PERK branch of UPR draws considerable interest in part because its abnormal regulation underlies many metabolic and neurodegenerative diseases. Stress-activated PERK is best known to initiate downstream signaling by phospho-inhibiting the translation initiation factor eIF2α. While most mRNA translation becomes attenuated under these conditions, ATF4 protein synthesis increases to mediate a signaling response. Such ATF4 induction requires ATF4's regulatory 5' leader sequence that has an upstream Open Reading Frame (uORF) that overlaps with the main ORF in a different reading frame. This overlapping uORF interferes with the main ORF translation in unstressed cells. But eIF2α phosphorylation causes the scanning ribosomes to bypass this uORF, ultimately allowing the translation of the main ORF assisted by the noncanonical translation initiation factors eIF2D and DENR. The literature also reports PERK effectors that may be independent of ATF4. These include a small number of factors that are translationally induced in parallel to ATF4 in stressed mammalian cells. Compared to the ATF4 axis, the roles of these ATF4-independent PERK effectors remain poorly understood (Brown, 2021).

This study reports that a previously uncharacterized UPR signaling axis is required for the expression of the most significantly induced UPR targets in the larval eye disc of Drosophila melanogaster. Specifically, glutathione-S-transferases (gstDs) were among the most significantly induced UPR target genes in Drosophila. It was further shown that such gstD induction was dependent on Perk, but did not require crc, the Drosophila ortholog of ATF4. Instead, this response required Xrp1, which encodes a bZIP transcription factor with no previously established connections to the UPR. Together, these findings suggest that PERK-Xrp1 forms a previously unrecognized signaling axis that mediates the induction of the most highly upregulated UPR targets in Drosophila (Brown, 2021).

This study reports that ER stress activates a previously unrecognized UPR axis mediated by PERK and Xrp1. Specifically, it was shown that gstD family genes are among the most highly induced UPR targets in Drosophila, and that such induction requires Perk, one of the three established ER stress sensors in metazoans. Surprisingly, the induction of gstD genes in this context did not require crc, the ATF4 ortholog. Instead, it was found that a poorly characterized transcription factor Xrp1 is induced downstream of Perk to promote the expression of gstDs and other antioxidant genes (Brown, 2021).

These findings are surprising given that ATF4 is considered a major effector of PERK-mediated transcription response. ATF4 was the first PERK downstream transcription factor to be identified in part based on the similarity of its regulatory mechanisms with that of yeast GCN4. But more recent studies have shown there could be parallel effectors downstream of PERK activation. The functional significance of these alternative factors had remained poorly understood. This study has led to the conclusion that an ATF4-independent branch of PERK signaling is required for the expression of the most highly induced UPR target in Drosophila (Brown, 2021).

As a potential mediator of this ATF4-independent PERK signaling, cncC was first considered as a prime candidate for a few reasons: cncC is an established regulator of gstD-GFP induction, and previous studies had reported that Nrf2 is activated by PERK in cultured mammalian cells and in zebrafish. However, the results reported in this study do not support the simple idea that gstD-GFP is induced by CncC, which in turn is activated by PERK. Specifically, it was found that the loss of Perk blocked gstD-GFP induction in this experimental setup, but the loss of cncC did not. While Nrf2/CncC clearly regulates antioxidant gene expression in response to paraquat, the results indicate that PERK mediates an independent antioxidant response in Drosophila (Brown, 2021).

The data indicates that this ATF4-independent PERK signaling response requires the AT-hook bZIP transcription factor Xrp1. Several pieces of evidence support the idea that Xrp1 is translationally induced, analogous to the mechanism reported for ATF4 induction. First, RNA-seq and qRT-PCR results indicate that Xrp1 transcript levels do not change significantly in Rh1G69D expressing eye discs. These results argue against the idea that Xrp1 is induced at the transcriptional level. Second, it was found that PERK's kinase domain is required for Xrp1 protein induction. Third, knockdown of gadd34 (Protein phosphatase 1 regulatory subunit 15), which increases phospho-eIF2α levels downstream of Perk, is sufficient to induce Xrp1 protein and gstD-GFP expression. Finally, this study find that Xrp1's 5' leader has a uORF that overlaps with the main ORF, similar to what is found in ATF4's regulatory 5' leader sequence. Moreover, Xrp1's uORF2 encodes a peptide sequence that is phylogenetically conserved in other Drosophila species. High-sequence conservation at the peptide level enhances confidence that uORF2 is a peptide coding sequence (Brown, 2021).

Xrp1 is known to respond to ionizing radiation, motor neuron-degeneration in a Drosophila model for amyotrophic lateral sclerosis (ALS), and in cell competition caused by Minute mutations that cause haplo-insufficiency of ribosomal protein genes. Interestingly, two recent studies reported that these Minute cells induce gstD-GFP, and also show signs of proteotoxic stress as evidenced by enhanced eIF2α phosphorylation. Although these studies did not examine the relationships between Xrp1, gstD-GFP and eIF2α kinases such as Perk, the current findings make it plausible that the PERK-Xrp1 signaling axis regulates cell competition caused by Minute mutations (Brown, 2021).

Despite the rising levels of interest in Xrp1 as a stress response factor, the identity of its mammalian equivalent remains unresolved. Xrp1 is well conserved in the Dipteran insects, but neither NCBI Blast searches nor Hidden Markov Model-based analyses identify clear orthologs in other orders. Such evolutionary divergence is not unprecedented in UPR signaling: GCN4 is considered a yeast equivalent of ATF4, but they are not the closest homologs in terms of their peptide sequences. Likewise, the yeast equivalent of XBP1 (IRE1 effector, not to be confused with Xrp1 in this study) is Hac1, but there is little sequence conservation between the two genes. Yet, the UPR signaling mechanisms are considered to be conserved due to the shared regulatory mechanisms. Along these lines, mammalian cells may have functional equivalents of Xrp1. Among the candidate equivalent factors those with regulatory 5' leader sequences that respond to eIF2α phosphorylation were considered. Based on the emerging roles of Xrp1 in Drosophila models of human diseases, it is speculated that those ATF4-independent PERK signaling effectors may play more significant roles in diseases associated with UPR than had been generally assumed (Brown, 2021).

It is noted that genes encoding cytoplasmic glutathione S-transferases (GSTs) such as gstD1 and gstD9 are among the most prominently induced UPR targets in the eye imaginal disc-based gene expression profiling analysis. Previous studies also reported these as ER stress-inducible genes in Drosophila S2 cells. GSTs are cytoplasmic proteins that participate in the detoxification of harmful, often lipophilic intracellular compounds damaged by ROS. These enzymes catalyze the formation of water-soluble glutathione conjugates that can be more easily eliminated from the cell. It is noteworthy that ROS is generated as a byproduct of Ero-1-mediated oxidative protein folding, and such ROS generation increases when mutant proteins undergo repeated futile cycles of protein oxidation. Therefore, it is speculated that cytoplasmic GSTs evolved as UPR targets as they have the ability to detoxify lipid peroxides or oxidized ER proteins that increase in response to ER stress (Brown, 2021).

In conclusion, these findings support the idea that an ATF4-independent branch of PERK signaling mediates the expression of the most highly induced UPR targets in eye disc cells. This axis of the UPR requires Xrp1, a gene that had not previously been associated with ER stress response. The identification of this new axis of UPR signaling may pave the way for a better mechanistic understanding of various physiological and pathological processes associated with abnormal UPR signaling in metazoans (Brown, 2021).

The Hedgehog signalling pathway regulates autophagy

Autophagy is a highly conserved degradative process that removes damaged or unnecessary proteins and organelles, and recycles cytoplasmic contents during starvation. Autophagy is essential in physiological processes such as embryonic development but how autophagy is regulated by canonical developmental pathways is unclear. This study shows that the Hedgehog signalling pathway inhibits autophagosome synthesis, both in basal and in autophagy-induced conditions. This mechanism is conserved in mammalian cells and in Drosophila, and requires the orthologous transcription factors Gli2 and Ci, respectively. Furthermore, it was demonstrated activation of the Hedgehog pathway reduces PERK levels, concomitant with a decrease in phosphorylation of the translation initiation factor eukaryotic initiation factor 2alpha, suggesting a novel target of this pathway and providing a possible link between Hedgehog signalling and autophagy (Jimenez-Sanchez, 2012).

In Drosophila, a single Ptch receptor responds to Hh molecules, whereas in mammalian cells Ptch1 and Ptch2 share this function. Although knockdown of Ptch1 did not inhibit autophagy in basal conditions, activation of the Hh pathway was not as efficient with Ptch1 compared with Ptch2 knockdown. Also, Ptch1 is a transcriptional target of the Hh pathway and its expression is expected to increase when the pathway is activated by Ptch1 siRNA treatment. This feedback implies that Ptch1 siRNA may not be efficient enough to completely counteract its own transcriptional activation. Although more detailed experiments would be needed to rule out a differential contribution of these receptors, the data suggest that Shh modulates autophagy through Ptch1 and/or Ptch2 (Jimenez-Sanchez, 2012).

Gli transcription factors have distinct activator and repressor functions and their roles differ during embryonic development. Although a contribution of Gli1 and Gli3 to autophagy regulation cannot be completely excluded, it was confirmed that Gli2 is necessary for the inhibition of autophagy by Hh signalling. The increased LC3-II levels in Gli2-knockout embryos further confirmed the data in a biologically relevant context. These data do not explain the developmental defects in these mice but might suggest that some of their phenotypes could be secondary to autophagy dysregulation. However, almost certainly, factors other than autophagy inhibition will have major contributions to the developmental defect in these Gli2-knockout mice. Equally, it cannot be excluded that other pathways that increase autophagy could be aberrantly activated in these embryos (Jimenez-Sanchez, 2012).

Despite the effects of Gli2 and Ci on autophagy, the role of their upstream activator Smo was less clear-cut. This is not surprising, as it has been shown that Smo levels are not sufficient to stimulate the pathway, but an activation process is required, with Ptch influencing the transition to an active state of Smo in response to Shh. In Drosophila, null mutations in Smo did not influence autophagy whereas Costal2 inhibition did, and it is therefore also possible that the Hh/Ptch complex and Ci may partially bypass Smo in Drosophila, consistent with the effects of this pathway on Costal2 localization to endomembranes. Consequently, the data suggest that canonical and non-canonical Hh mechanisms might be involved in regulating autophagy, or that other factors might be necessary in Hh-mediated regulation of autophagy (Jimenez-Sanchez, 2012).

Hh inhibition has been largely pursued as a therapeutic strategy for types of cancer resulting from a hyperactivation of the pathway, such as basal cell carcinomas or medulloblastoma. Although the roles of autophagy in cancer are controversial and complex, it is interesting to consider whether inhibition of autophagy by Hh would exacerbate or prevent the progression of these tumours when searching for pharmacological strategies. Cyclopamine, which binds and inhibits Smo, is one of the best characterized Hh signalling inhibitors and it has been shown to be protective in Hh-related cancers. As expected, cyclopamine caused LC3-II (microtubule-associated protein 1 light chain 3 lipidated form, a product of autophagy). In the presence of bafA1, the increase was not as large as in non-treated cells. This result was unexpected because bafA1 should exacerbate the changes on LC3-II if cyclopamine is triggering autophagosome synthesis, suggesting that cyclopamine might also affect autophagosome maturation. Consistent with an impairment in autophagosome degradation, cyclopamine and bafA1 both similarly reduced the number of acidic vesicles in mRFP-GFP-LC3 cells. These data suggest that cyclopamine affects autophagosome degradation but that this effect is independent of Hh inhibition, as cyclopamine could increase LC3-II levels even in Gli2-null MEFs. These data suggest that cyclopamine has two opposite effects on autophagy. It increases autophagosome synthesis by inhibiting Smo activity, but it simultaneously impairs autophagosome maturation through an unknown mechanism that is independent of Gli2. Effects of cyclopamine independent of Smo inhibition have been reported in human breast cancer cell lines and in zebrafish, suggesting that this drug has additional molecular targets (Jimenez-Sanchez, 2012).

PRK-like ER kinase (PERK or EIF2AK3) is an ER-resident protein responsible for the phosphorylation of eukaryotic initiation factor 2α (eIF2α), which inhibits protein translation in response to ER stress, In search of potential Hh transcriptional targets that could modulate autophagy, PERK was identified as a consistent potential target and, in agreement with previous observation, PERK is repressed upon Gli1 and Gli2 induction. It has also been suggested that expression of Gli2 repressed a considerably larger number of genes than Gli1, an observation that may be of relevance when considering the more dominant effect of Gli2 on autophagy. Although Gli2 acts mainly as a transcriptional activator upon Hh activation, it can be proteolytically processed into a transcriptional repressor, similar to the dual function of Ci in Drosophila. The autophagy inhibitory action of Gli2 can be explained by either a gain of repressor function towards some genes (such as PERK) in its active form, or by the fact that when activated it leads to increased expression of a second transcriptional repressor. It remains to be elucidated which of these mechanisms is in action in the case of PERK transcription (Jimenez-Sanchez, 2012).

In response to unfolded proteins in the ER lumen, PERK is activated and phosphorylates eIF2α, increasing translation of the transcription factor ATF4. The exact mechanism by which eIF22α controls autophagy is unknown, eIF22α regulates Atg12 levels in the presence of expanded polyglutamines and ATF4 has been suggested to increase transcription of Atg5 and LC3. Indeed PCR Array data show that levels of >Atg5 upon Shh ligand are decreased to 0.8-fold compared with control cells. However, it is unlikely that this is the complete mechanism, as it has yet to be conclusively demonstrated that transcriptional induction of LC3 and Atg5 upregulate autophagy (Jimenez-Sanchez, 2012).

It is also possible that PERK interacts more directly with other signalling pathways involved in autophagy regulation. As an example, the transcription factor Nrf2, known to be involved in autophagy regulation, is phosphorylated by PERK independently of eIF22α in response to ER stress, promoting its import to the nucleus and activation of transcription (Jimenez-Sanchez, 2012).

In conclusion, these data not only provide insights into the connection between Hh and autophagy but also into the potential implications that Hh has in controlling protein homeostasis in physiological and disease conditions (Jimenez-Sanchez, 2012).

The defender against apoptotic cell death 1 gene is required for tissue growth and efficient N-glycosylation in Drosophila melanogaster

How organ growth is regulated in multicellular organisms is a long-standing question in developmental biology. It is known that coordination of cell apoptosis and proliferation is critical in cell number and overall organ size control, while how these processes are regulated is still under investigation. This study found that functional loss of a gene in Drosophila, named Drosophila Defender against apoptotic cell death 1 (dDad1), leads to a reduction of tissue growth due to increased apoptosis and lack of cell proliferation. The dDad1 protein, an orthologue of mammalian Dad1, was found to be crucial for protein N-glycosylation in developing tissues. Loss of dDad1 function activates JNK signaling and blocking the JNK pathway in dDad1 knock-down tissues suppresses cell apoptosis and partially restores organ size. In addition, reduction of dDad1 triggers ER stress and activates unfolded protein response (UPR) signaling, prior to the activation of JNK signaling. Furthermore, Perk-Atf4 signaling, one branch of UPR pathways, appears to play a dual role in inducing cell apoptosis and mediating compensatory cell proliferation in this dDad1 knock-down model (Zhang, 2016).

Cell apoptosis, or programmed cell death (PCD), is one of the most fundamental processes essential for normal development in multicellular organisms. Cell apoptosis is tightly controlled to eliminate excess or damaged cells during development, while dysregulation can lead to pathological diseases, such as neurodegenerative diseases and cancer. Although regulation of cell apoptosis involves a sophisticated and complex molecular cascade, the core machinery that initiates, mediates and executes programmed cell death as well as related regulatory pathways are evolutionary conserved among species. Drosophila melanogaster, with its well-annotated genome and established genetic tools, is commonly used to study mechanism of cell apoptosis and discover novel regulatory pathways and genes for regulating development and tissue homeostasis (Zhang, 2016).

The defender against apoptotic cell death 1 (Dad1) gene was first identified and considered as a negative regulator of cell apoptosis from a temperature-sensitive hamster cell line, tsBN7. With the temperature sensitive mutation in Dad1 gene, cells are normal at permissive temperature, while shifting to restrictive temperature led to programmed cell death. Homologous proteins of hamster Dad1 from other species were found to be highly conserved in sequence and function. The yeast homologue of Dad1 is called Ost2 with 40% identity compared to hamster Dad1 and ost2 mutant induces yeast cell apoptosis. The function of Dad1 has been studied in multicellular organisms as well. Mouse Dad1 mutants exhibited developmental delay, aberrant morphology and increased cell apoptosis during embryogenesis and only survived up to stage of midgestation. In Caenorhabditis elegans, overexpression of either Caenorhabditis Dad1 or human Dad1 induced by a heat-shock promoter inhibited developmental cell death during embryogenesis. While ectopic expression of Dad1 in mouse thymocytes did not increase survival of T cells under apoptotic stimuli, peripheral T cells from spleen and lymph nodes in Dad1 transgenic mice increased cell proliferation. These results indicate that Dad1 plays a critical role in regulating cell viability and apoptosis (Zhang, 2016).

Dad1 is identified as a subunit of oligosaccharyltransferase complex (OST), which acts at the first step for protein N-linked glycosylation. N-linked glycosylation is an important protein modification process, which transfers oligosaccharide to selected asparagine of target nascent proteins to ensure their proper folding and maturation in the endoplasmic reticulum (ER). In eukaryotes, OST complex has a catalytic subunit Stt3 as a core and other six non-catalytic subunits, including Ribophorin I, Ribophorin II, Ost48, Dad1/Ost2, N33/Ost3, and Ost4. There are two OST catalytic subunit genes in vertebrates and insects, referred to as Stt3A and Stt3B. Non-catalytic subunits facilitate the catalytic function of Stt3A and Stt3B. Among them, Ost48 and Dad1 are critical for assembly and stability of Stt3 as well as the OST complex, and loss of them can lead to hypoglycosylation. Ribophorin I escorts the N-glycosylation of OST complex on specific substrates, especially some membrane proteins. Because loss of any one non-catalytic subunit does not fully abolish the function of OST, it is possible that they may have redundant roles or different non-catalytic subunits assist the glycosylation of specific targets (Zhang, 2016).

Dad1, as an N-glycosylation regulatory protein, plays an important role in cell survival, but up to now no detailed mechanism is known about how loss of Dad1 induces cell apoptosis. Based on the evidence from studies in different organisms, Dad1 may facilitate the OST complex to target specific proteins that directly maintain cell survival. Another possibility is that accumulation of unfolded or misfolded proteins of hypoglycosylation at ER triggers stress signaling and initiates programmed cell death. It is also possible that Dad1 affects cell viability in an OST-independent manner. For instance, Dad1 interacts with a Bcl2 family protein, Mcl1, which might lead to apoptosis inhibition (Zhang, 2016).

This study used Drosophila Dad1 (dDad1), which shows more than 70% identity with human DAD1, to study how loss of Dad1 function affects cell viability and tissue growth. Loss of Dad1 in Drosophila was found to decreased tissue growth due to induction of apoptosis and absence of cell proliferation. dDad1 is required for efficient N-glycosylation in developing tissues and the small wing phenotype induced by dDad1 knock-down can be enhanced by reducing gene dosage of the OST catalytic subunit. In addition, the c-Jun N-terminal kinase (JNK) pathway was found to mediate dDad1 knockdown-induced cell apoptosis in wing discs through mitogen-activated protein kinase kinase kinase 1 (Mekk1) and wallenda (wnd). Moreover, in dDad1 knock-down tissues, ER stress is induced and Perk-Atf4 pathway functions upstream of the JNK pathway. Intriguingly, unfolded protein response (UPR) signaling appears to play a dual role in inducing cell death and stimulating compensatory proliferation of neighboring cells for tissue homeostasis (Zhang, 2016).

This study found that loss of dDad1 in the wing interferes with N-linked protein glycosylation, which triggers ER stress and activates the downstream JNK pathway to cause apoptosis. Interestingly, although this study suggests that dDad1 regulates tissue growth through the OST complex, only one of two catalytic subunits, Stt3A (CG1518), genetically interacts with dDad1 in growth control. With the association of dDad1, Stt3A may become the major functioning complex modifying crucial proteins or more nascent proteins in developing tissues, therefore inducing severe or prolonged ER stress if dDad1 activity is absent. Different tissues or regions of tissues may have different levels of sensitivity to the loss of dDad1, which could explain why cell apoptosis was more frequently observed in wing pouch than other region of the wing disc in these experiments. Similar phenomena were also reported in other studies. For instance, Dad1 mutant mice exhibit cell death which leads to developmental defect and embryonic lethality, but the death of cells only happens in some tissues of the embryo (Zhang, 2016).

One study reports that dDad1 homozygous mutant clones in larval fact body induced cell autophagy. However, the current study did not observe cell autophagy using lyso-tracker staining in dDad1 mutant clones in larval wing discs. The different response may be due to different tissue-specific context. Therefore, apoptosis induced by loss of dDad1 in the wing is unlikely to be dependent of autophagy process (Zhang, 2016).

As an N-glycosylation regulator, dDad1 may have other target proteins essential for animal viability. In this study, although blocking Perk-Atf4 or JNK pathway suppresses apoptosis induced by loss of dDad1, the lethality of en-Gal4/UAS-dDad1 RNAi individuals cannot be rescued. Thus, cell apoptosis induced by loss of dDad1 may not be the major reason for causing lethality of the mutant organism. It would be interesting to identify potential targets of dDad1 that are essential for normal development of Drosophila (Zhang, 2016).

The Perk-Atf4 pathway may play both protective and destructive roles in growth control in loss of dDad1 model: on one hand, it activates JNK signaling to cause cell apoptosis; on the other, it may send out signals to neighboring cells to increase cell proliferation (Zhang, 2016).

In Drosophila, it is known that prolonged ER stress induces cell apoptosis, however the mechanism of ER stress activates apoptosis remains elusive. A few Drosophila ER stress models have been established and their connection with apoptosis has been validated. Nevertheless, they suggested different pathways leading to cell death and none of them is the same as what was found in the current study. In a chronic ER stress model due to Presenilin (Psn) overexpression, cell apoptosis is induced in a Perk-Atf4 dependent but JNK-independent manner. Although the study found that the JNK pathway is activated by Perk-Atf4, it induces dilp8 expression which extends developmental stage to keep tissue homeostasis, rather than stimulates cell death. In the current study, induction of cell apoptosis in the absence of dDad1 function was found to be dependent on both Perk-Atf4 and JNK pathways. The JNK pathway acts downstream of Perk-Atf4 and causes cell apoptosis. It was also found that Mekk1 and Wnd are two major JNKKK's that activate JNK pathway in response to loss-of-dDad1 induced ER stress. This is consistent with the observations from another ER stress model, autosomal dominant retinitis pigmentosa (ADRP) model. However, ADRP model suggests that activation of JNK pathway and cell apoptosis does not rely on UPR signaling. In mammalian models of ER stress, cell apoptosis is induced through activation of IRE1-XBP1 branch, followed by JNK pathway activation via TRAF2/ASK1. In the current study, it was not possible to test the role of Ire1-Xbp1 branch due to the embryonic lethality caused by expression of Ire1 RNAi or Xbp1 RNAi driven by en-Gal4, although its activation was observed. Ask1 does not seem to play a role in mediating cell apoptosis in the loss-of-dDad1 model, since knocking down of Ask1 cannot restore loss-of-dDad1 induced wing size deduction (Zhang, 2016).

Compensatory proliferation is a self-rescue mechanism to keep tissue homeostasis when some cells are under stress or apoptosis. Mechanism of compensatory proliferation was mostly studied in Drosophila. In fly wing discs, Dronc in the apoptotic cells stimulates JNK pathway, inducing expression of mitogens, Dpp and Wg, to presumably promote proliferation of neighboring cells. Interestingly, in this study, activation of Perk-Atf4 pathway appears to play a role in inducing extra cell proliferation but does not rely on activation of the JNK pathway nor cell death. It suggests that Atf4 transcription factor may induce transcription of mitogenic genes to stimulate cell proliferation, in addition to the genes that are related to stress response (Zhang, 2016).

N-glycosylation of Wg was affected in dDad1 mutant cells. However, it turned out that N-glycosylation of Wg is unimportant for its secretion and signaling in fly tissues. Regardless, it is unlikely that Wg was responsible for mediating extra cell proliferation in this loss-of-dDad1 model based on the following observations. Firstly, compensatory cell proliferation can still occur in the absence of wg and dpp in tissues undergoing massive cell apoptosis. Moreover, this study found that proliferation of extra cells can still occur when JNK signaling was blocked, while the JNK pathway is presumably responsible for Wg induction. Therefore, further studies are needed to address how Perk-Atf4 signaling acts to induce compensatory cell proliferation to maintain tissue homeostasis (Zhang, 2016).

Some studies suggest that Dad1 may have a direct effect on cell death suppression. Overexpression of hDAD1 or ceDAD1 has been shown to be sufficient to inhibit developmental cell apoptosis in C. elegans. Overexpression of DAD1 in mouse thymocyte did not increase survival of T cells under apoptosis stimuli, however peripheral T cells from spleen and lymph node in Dad1 transgenic mice have increased cell proliferation. This work examined the phenotypes of dDad1 transgenic flies in different tissues. Drosophila pupa eye is a good model to study developmental apoptosis because wild-type flies have fixed number of interommatidial cells in mid-pupal eye discs due to the elimination of excess cells by apoptosis. This study found that overexpression of dDad1 in the eye did not cause the accumulation of extra interommatidial cells in mid-pupal eye discs, which indicates that dDad1 is not sufficient to block normal apoptosis during eye development. In addition, this study overexpressed apoptosis-inducing genes in Drosophila wing and eye to test if dDad1 is able to suppress induced apoptosis. The cell apoptosis in the wing induced by overexpression of UAS-eiger failed to be suppressed by dDad1 overexpression. However, overexpression of dDad1 could moderately rescue cell death induced by GMR-hid in adult eyes. These observations indicate that Drosophila dDad1 inhibits induced cell apoptosis moderately and conditionally, but exhibits no effect on normal developmental apoptosis. Therefore, these observations suggest that dDad1 may not function as an active defender of cell apoptosis in Drosophila (Zhang, 2016).

Akt determines cell fate through inhibition of the PERK-eIF2alpha phosphorylation pathway

Metazoans respond to various forms of environmental stress by inducing the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) at serine-51, a modification that leads to global inhibition of mRNA translation. This study demonstrates induction of the phosphorylation of eIF2alpha in mammalian cells after either pharmacological inhibition of the phosphoinositide 3-kinase (PI3K)-Akt pathway or genetic or small interfering RNA-mediated ablation of Akt. This increase in the extent of eIF2alpha phosphorylation also occurred in Drosophila cells and depends on the endoplasmic reticulum (ER)-resident protein kinase PERK, which is inhibited by Akt-dependent phosphorylation at threonine-799. The activity of PERK and the abundance of phosphorylated eIF2alpha (eIF2alphaP) were reduced in mouse mammary gland tumors that contained activated Akt, as well as in cells exposed to ER stress or oxidative stress. In unstressed cells, the PERK-eIF2alphaP pathway mediates survival and facilitates adaptation to the deleterious effects of the inactivation of PI3K or Akt. Inactivation of the PERK-eIF2alphaP pathway increases the susceptibility of tumor cells to death by pharmacological inhibitors of PI3K or Akt. Thus, it is suggested that the PERK-eIF2alphaP pathway provides a link between Akt signaling and translational control, which has implications for tumor formation and treatment (Mounir, 2011).

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. This study reports the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA (N-methyl-D-aspartate receptor-associated protein) synergies with TMBIM6/BI-1 (Bax Inhibitor-1) in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane (Rojas-Rivera, 2012).

Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. This study has developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2alpha phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest (Malzer, 2010).

Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2alpha (eIF2alpha) kinase

Four distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases phosphorylate eIF2alpha at S51 and regulate protein synthesis in response to various environmental stresses. These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticulum (ER)-resident kinase (PERK) and the GCN2 protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes. This study has characterized the second eIF2alpha kinase found in Drosophila, a PERK homologue (DPERK). Expression of DPERK is developmentally regulated. During embryogenesis, DPERK expression becomes concentrated in the endodermal cells of the gut and in the germ line precursor cells. Recombinant wild-type DPERK, but not the inactive DPERK-K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2alpha at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2. The full length protein, when expressed in 293T cells, located in the ER-enriched fraction, and its subcellular localization changed with deletion of different N-terminal fragments. Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function. Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress. Furthermore, the stable complexes between wild-type DPERK and DPERK-K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2alpha kinase activity (Pomar, 2003).

Pancreatic eukaryotic initiation factor-2alpha kinase (PEK) homologues in humans, Drosophila melanogaster and Caenorhabditis elegans that mediate translational control in response to endoplasmic reticulum stress

In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). Recently, a new family member, pancreatic eIF-2alpha kinase (PEK) has been identified from rat pancreas. PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER. This study identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans. Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues. In mammalian cells subjected to ER stress, elevated eIF-2alpha phosphorylation was found to be coincident with increased PEK autophosphorylation and eIF-2alpha kinase activity. Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen. To address the role of C. elegans PEK in translational control, this kinase was expressed in yeast, and it was found to inhibit growth by hyperphosphorylation of eIF-2alpha and inhibition of eIF-2B. Furthermore, vaccinia virus K3L protein, an inhibitor of the eIF-2alpha kinase PKR involved in an anti-viral defence pathway, was also found to reduce PEK activity. These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway (Sood, 2000). Sorge, S., J. Theelke, K. Yildirim, H. Hertenstein, E. McMullen, S. Muller, C. Altburger, S. Schirmeier and I. Lohmann (2020). ATF4-Induced Warburg Metabolism Drives Over-Proliferation in Drosophila. Cell Rep 31(7): 107659. PubMed ID: 32433968

ATF4-induced Warburg metabolism drives over-proliferation in Drosophila

The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. This study shows that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. This stress adaptation is found to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. These results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity (Sorge, 2020).

Controlling cell proliferation is one of the major challenges of multicellular life, both during phases of growth in developing organisms and phases of homeostatic cell replenishment essential in adult animals. Lack of appropriate control can lead to severe disorders, including cancer, at any stage of life. While over-proliferation of transformed, cancerous cells is usually caused by inactivation of tumor suppressors and/or activation of oncogenes, it has long been noted that tumors exhibit altered cellular characteristics such as a glycolytic metabolism. While this metabolic switch has been shown to be caused by oncogene signaling, cellular metabolism is also controlled at multiple levels under normal physiological (non-transformed) conditions, including at the transcriptional level through diverse stress-response pathways. One of these is the activating transcription factor 4 (ATF4), which is known to activate a transcriptional (integrated) stress response (ISR) under various stress conditions that trigger phosphorylation of eIF2α. The ATF4 transcriptional program consists of a diverse set of genes with cytoprotective function, but chronic activation induces apoptosis indirectly through transcription of the mammalian ATF4 target CHOP. Yet, ATF4 activation has been detected in several human tumors, especially in hypoxic or nutrient-deprived regions, where ATF4 has been attributed with pro-survival and pro-proliferative effects. Interestingly, a recent study showed that melanoma cells respond to inhibition of their glycolytic metabolism by activating an ATF4 response, whose metabolic reconfiguration allows these cells to continue oncogenic growth, together arguing that ATF4 can provide cancer cells with a metabolic flexibility that allows them to tolerate hypoxic and nutritional stress or cancer therapy aimed at metabolism. Among the many conditions activating ATF4, recent work with cultured cells showed that inhibition of mitochondrial function is linked to ATF4 translation and activity. However, from these and other studies, both the mechanistic basis and the in vivo implications of this response remain to be elucidated (Sorge, 2020).

This study shows that in the fruit fly, Drosophila melanogaster, genetic perturbation of the electron transport chain (ETC), which induces a Warburg-like metabolism, activates a transcriptional stress response mediated through the eIF2α-kinase PERK and ATF4 in eye progenitor cells of Drosophila larvae. Importantly, this in vivo stress response is activated under ETC knockdown conditions, in the absence of obvious mitochondrial dysfunction. Interestingly, these results show that the ATF4 transcriptional response, which by itself causes reduced fitness of progenitor cells, is hijacked by growth-promoting pathways like Notch or Ras, leading to increased cellular fitness and enhanced proliferation. The data furthermore suggest that the pH changes associated with ETC impairment resulting in a switch of the metabolism to aerobic glycolysis play an important role in progenitor over-proliferation. In sum, this study shows that ATF4-mediated transcriptional adaptation provides a cell-autonomous response to ETC defects, altering cellular behavior through metabolic adaptation (Sorge, 2020).

Genetically induced disturbance of ETC complex assembly resultrd in a metabolic shift typical for mitochondrial impairment and activated an ATF4-dependent stress response. The in vivo transcriptional adaptation presented in this study confirmed the regulation of LDH and glycolytic enzymes, as shown in Drosophila cultured cells, and further includes several targets shown to be ATF4 target genes in mammalian models. The results showed that the eIF2α-kinase PERK, so far only described for its role in mediating one branch of the unfolded protein response of the endoplasmic reticulum (UPRER), is the upstream kinase phosphorylating eIF2α, thereby inducing ATF4 translation in response to mitochondrial ETC disturbance. Mitochondrial ETC disturbance specifically activated PERK, while other branches of the UPRER were non-responsive. PERK activation upon mitochondrial defects was recently observed in Drosophila models of Parkinson's disease and was explained by the authors by its preferential localization to mitochondria-associated ER membranes, which might make PERK more susceptible to a local stress signal. ROS (reactive oxygen species) released by mitochondria have been suggested to mediate mitochondrial retrograde signaling. While this study observed an attenuation of Delta overexpression (Dl^OE), COX7RNAi-induced over-proliferation upon overexpression of either cytoplasmic catalase or GPx (but not mitochondrial catalase), this study failed to detect increased ROS levels in the larval eye disc. A possible scenario to explain these observations is that ROS are generated locally in the cytoplasm or ER in response to ETC disturbance, thereby triggering PERK activation. Importantly, Drosophila PERK isoform B contains a potential mitochondrial signal peptide, which is not found in mammalian PERK isoforms. Although no evidence for this has been found, Drosophila PERK could reside in the mitochondrial membrane and sense the folding status of mitochondrial complexes. This hypothesis could explain the evolutionary difference between mitochondrial defects and ATF4 induction, as this appears to require GCN2 but not PERK in mammals or to be triggered independently of a single eIF2α-kinase. In addition to canonical ATF4 target genes, ATF4-dependent upregulation of mitochondrial chaperones, a response classically referred to as the mitochondrial UPR (UPRmt) was observed. In C. elegans, mitochondrial chaperone induction upon stress is mediated by ATF4-like transcription factor Atfs-1, while the mammalian UPRmt has been shown to be regulated by another evolutionary-related transcription factor, ATF5. The current data now showed that Drosophila ATF4 is required cell autonomously for the induction of mitochondrial chaperones upon ETC subunit knockdown, implying that Drosophila might represent the evolutionary ancestral ISR-UPRmt regulation through a single ATF4-like transcription factor (Sorge, 2020).

The cooperation between ATF4 target genes and the Notch or Ras pathways in Drosophila imaginal progenitors raised the intriguing possibility that these or other oncogenic pathways could benefit from ATF4 activity in human cancers. Over the last decades, it had been demonstrated that human cancer cells are exposed to several stresses, including hypoxia, ROS, or limitations in nutrient availability. In order to survive these conditions and maintain their growth capacity, tumor cells activate responses like the HIF1α transcription factor axis. Though less well studied, an involvement of ATF4 in cancer has been suggested mostly through work with cultured cells. This study analyzed gene expression in human cancer samples of The Cancer Genome Atlas (TCGA) datasets using Cancer-RNaseq-Nexus and the human protein pathology atlas and found that many of the well-characterized direct ATF4 targets are upregulated in a variety of cancer types. Most strikingly, transcriptomes of kidney renal clear cell carcinoma showed progressive induction of ATF4 and many of its direct targets (EIF4EBP1, ASNS, TRIB3, and VEGFA) on the transcriptional level, which strongly correlated with a poor prognosis in this type of cancer. These data suggest that the ATF4-mediated ISR is used by cancer cells to adapt their metabolic repertoire, thereby sustaining fast growth under increasingly unfavorable conditions (Sorge, 2020).

A novel finding presented in this study was the discovery that ATF4-mediated transcriptional adaptation due to ETC impairment allowed eye progenitors to increase their proliferation in response to signals from the Notch and Ras pathways. The primary questions arising from this genetic interaction is how these signaling pathways can overcome the apparent cellular stress and reduction in proliferation and induce the opposite effect, a massively increased rate of proliferation. Several lines of evidence suggest that over-proliferation in Dl^OE, COX7RNAi eye imaginal discs is controlled by pH changes induced by LDH that modify the activity of Notch downstream effectors. First, in COX7a-depleted cells, the metabolism is switched to aerobic glycolysis, leading to an increased production of lactate due to the activity of LDH. And, consistent with an accumulation of this metabolic acid, this study found the intracellular pH to be reduced in COX7RNAi cells. Second, Dl^OE, COX7RNAi-mediated over-proliferation was rescued by ATF4 knockdown and, to a lesser extent, pH buffering, showing that intracellular pH changes (downstream of ATF4 and LDH) play an important role in proliferation control. Third, LDH phenocopies COX7RNAi, indicating that most of the cooperative effects of Dl overexpression and COX7a knockdown are mediated by the ATF4 target LDH. In the same line, expression of LDH as one of the many ATF4 targets was sufficient to drive Dl-expressing cells into over-proliferation, strongly suggesting that the processes downstream of LDH-in particular, the changes in intracellular pH-lead to a modification of the Notch pathway. Finally, a cooperation of Notch and the COX7a nuclear effector ATF4 on the transcriptional level was not observed, showing that the Notch pathway is not hyper-activated, but arguing that over-proliferation is due to changes in the activity of Notch downstream effectors. The next obvious question is how changes in intracellular pH can modify the activity of signaling pathways. It is known that the intracellular pH can control the protonation of specific histidine residues in proteins acting as pH sensors, leading to changes in protein properties. Importantly, it has been shown recently in chicken embryos that intracellular pH changes induced by a Warburg-like metabolism control the acetylation of the Wnt effector &betsa;-catenin, thereby mediating Wnt signaling activation. Thus, this study envisions that pH changes induced by LDH expression lead to a (non-enzymatic) modification of Notch effectors, thereby increasing fitness and proliferation rates of eye progenitor cells (Sorge, 2020).

This is a very attractive model; however, one result was puzzling. Although LDH is sufficient to induce over-proliferation when combined with the Notch pathway, no rescue (but an increase in the severity) of the Dl^OE, COX7RNAi phenotype was observed when LDH was selectively depleted. This obvious discrepancy can be explained by different hypotheses. One of them is based on a recent study showing that LDHA inhibition in melanoma cell lines also failed to impact cell proliferation, survival, or tumor growth. In this context, LDHA inhibition engaged the GCN2-ATF4 signaling axis to initiate an expansive pro-survival response, including the upregulation of the glutamine transporter SLC1A5 and glutamine uptake, as well as mTORC1 activation. Another hypothesis is based on the finding that a major driver of over-proliferation is the intracellular pH. Since LDH catalyzes the conversion of pyruvate to lactate (and back), reducing LDH levels will affect the ratio of lactate to pyruvate, leading to an increase of the even stronger metabolic acid pyruvate. It has been shown that pyruvate as lactate induces a concentration-dependent intracellular acidification. Thus, it could be envisioned that an enhancement of proliferation rates beyond those observed in Dl^OE, COX7RNAi cells is a consequence of pyruvate accumulation in the absence of LDH, which enhances the decrease in the intracellular pH, resulting in the increase in proliferation rates (Sorge, 2020).

Search PubMed for articles about Drosophila Perk

Biteau, B., Hochmuth, C. E. and Jasper, H. (2008). JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut. Cell Stem Cell 3: 442-455. PubMed ID: 18940735

Biteau, B., Karpac, J., Supoyo, S., Degennaro, M., Lehmann, R. and Jasper, H. (2010). Lifespan extension by preserving proliferative homeostasis in Drosophila. PLoS Genet 6: e1001159. PubMed ID: 20976250

Brown, B., Mitra, S., Roach, F. D., Vasudevan, D. and Ryoo, H. D. (2021). The transcription factor Xrp1 is required for PERK-mediated antioxidant gene induction in Drosophila. Elife 10. PubMed ID: 34605405

Choi, N. H., Kim, J. G., Yang, D. J., Kim, Y. S. and Yoo, M. A. (2008). Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor. Aging Cell 7: 318-334. PubMed ID: 18284659

Demay, Y., Perochon, J., Szuplewski, S., Mignotte, B. and Gaumer, S. (2014). The PERK pathway independently triggers apoptosis and a Rac1/Slpr/JNK/Dilp8 signaling favoring tissue homeostasis in a chronic ER stress Drosophila model. Cell Death Dis 5: e1452. PubMed ID: 25299777

Gomez-Navarro, N. and Miller, E. (2016). Protein sorting at the ER-Golgi interface. J Cell Biol 215(6): 769-778. PubMed ID: 27903609

Guo, L., Karpac, J., Tran, S. L. and Jasper, H. (2014). PGRP-SC2 promotes gut immune homeostasis to limit commensal dysbiosis and extend lifespan. Cell 156: 109-122. PubMed ID: 24439372

Hamanaka, R. B., Bobrovnikova-Marjon, E., Ji, X., Liebhaber, S. A. and Diehl, J. A. (2009). PERK-dependent regulation of IAP translation during ER stress. Oncogene 28: 910-920. PubMed ID: 19029953

Harding, H. P., Zhang, Y. and Ron, D. (1999). Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397: 271-274. PubMed ID: 9930704

Harding, H. P., Zhang, Y., Zeng, H., Novoa, I., Lu, P. D., Calfon, M., Sadri, N., Yun, C., Popko, B., Paules, R., Stojdl, D. F., Bell, J. C., Hettmann, T., Leiden, J. M. and Ron, D. (2003). An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11: 619-633. PubMed ID: 12667446

Heijmans, J., van Lidth de Jeude, J. F., Koo, B. K., Rosekrans, S. L., Wielenga, M. C., van de Wetering, M., Ferrante, M., Lee, A. S., Onderwater, J. J., Paton, J. C., Paton, A. W., Mommaas, A. M., Kodach, L. L., Hardwick, J. C., Hommes, D. W., Clevers, H., Muncan, V. and van den Brink, G. R. (2013). ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response. Cell Rep 3: 1128-1139. PubMed ID: 23545496

Hochmuth, C. E., Biteau, B., Bohmann, D. and Jasper, H. (2011). Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila. Cell Stem Cell 8: 188-199. PubMed ID: 21295275

Jimenez-Sanchez, M., Menzies, F. M., Chang, Y. Y., Simecek, N., Neufeld, T. P. and Rubinsztein, D. C. (2012). The Hedgehog signalling pathway regulates autophagy. Nat Commun 3: 1200. PubMed ID: 23149744

Kiparaki, M., Khan, C., Folgado-Marco, V., Chuen, J., Moulos, P. and Baker, N. E. (2022). The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function. Elife 11. PubMed ID: 35179490

Malzer, E., Daly, M. L., Moloney, A., Sendall, T. J., Thomas, S. E., Ryder, E., Ryoo, H. D., Crowther, D. C., Lomas, D. A. and Marciniak, S. J. (2010). Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response. J Cell Sci 123: 2892-2900. PubMed ID: 20682638

Mounir, Z., Krishnamoorthy, J. L., Wang, S., Papadopoulou, B., Campbell, S., Muller, W. J., Hatzoglou, M. and Koromilas, A. E. (2011). Akt determines cell fate through inhibition of the PERK-eIF2alpha phosphorylation pathway. Sci Signal 4: ra62. PubMed ID: 21954288

Oommen, D. and Prise, K. M. (2013). Down-regulation of PERK enhances resistance to ionizing radiation. Biochem Biophys Res Commun 441: 31-35. PubMed ID: 24103755

Pomar, N., Berlanga, J. J., Campuzano, S., Hernandez, G., Elias, M. and de Haro, C. (2003). Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2alpha (eIF2alpha) kinase. Eur J Biochem 270: 293-306. PubMed ID: 12605680

Rera, M., Clark, R. I. and Walker, D. W. (2012). Intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in Drosophila. Proc Natl Acad Sci U S A 109: 21528-21533. PubMed ID: 23236133

Rojas-Rivera, D., Armisen, R., Colombo, A., Martinez, G., Eguiguren, A. L., Diaz, A., Kiviluoto, S., Rodriguez, D., Patron, M., Rizzuto, R., Bultynck, G., Concha, M. L., Sierralta, J., Stutzin, A. and Hetz, C. (2012). TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis. Cell Death Differ 19: 1013-1026. PubMed ID: 22240901

Schroder, M. and Kaufman, R. J. (2005). The mammalian unfolded protein response. Annu Rev Biochem 74: 739-789. PubMed ID: 15952902

Shi, Y., Vattem, K. M., Sood, R., An, J., Liang, J., Stramm, L. and Wek, R. C. (1998). Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol Cell Biol 18: 7499-7509. PubMed ID: 9819435

Sood, R., Porter, A. C., Ma, K., Quilliam, L. A. and Wek, R. C. (2000). Pancreatic eukaryotic initiation factor-2alpha kinase (PEK) homologues in humans, Drosophila melanogaster and Caenorhabditis elegans that mediate translational control in response to endoplasmic reticulum stress. Biochem J 346 Pt 2: 281-293. PubMed ID: 10677345

Sorge, S., Theelke, J., Yildirim, K., Hertenstein, H., McMullen, E., Muller, S., Altburger, C., Schirmeier, S. and Lohmann, I. (2020). ATF4-induced Warburg metabolism drives over-proliferation in Drosophila. Cell Rep 31(7): 107659. PubMed ID: 32433968

Verfaillie, T., van Vliet, A., Garg, A. D., Dewaele, M., Rubio, N., Gupta, S., de Witte, P., Samali, A. and Agostinis, P. (2013). Pro-apoptotic signaling induced by photo-oxidative ER stress is amplified by Noxa, not Bim. Biochem Biophys Res Commun 438: 500-506. PubMed ID: 23916707

Wang, L., Zeng, X., Ryoo, H. D. and Jasper, H. (2014). Integration of UPRER and oxidative stress signaling in the control of intestinal stem cell proliferation. PLoS Genet 10: e1004568. PubMed ID: 25166757

Wang, L., Ryoo, H.D., Qi, Y. and Jasper, H. (2015). PERK limits Drosophila lifespan by promoting intestinal stem cell proliferation in response to ER stress. PLoS Genet 11: e1005220. 25945494

Zhang, C., van Leeuwen, W., Blotenburg, M., Aguilera-Gomez, A., Brussee, S., Grond, R., Kampinga, H. H. and Rabouille, C. (2021). Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells. J Cell Sci 134(17). PubMed ID: 34350957

Zhang, Y., Cui, C. and Lai, Z. C. (2016). The defender against apoptotic cell death 1 gene is required for tissue growth and efficient N-glycosylation in Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 27693235

date revised: 18 February 2024

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