- Eukaryotic translation initiation factor 2 subunit alpha

InteractiveFly: GeneBrief

eukaryotic translation initiation factor 2 subunit alpha: Biological Overview | References |

Gene name - eukaryotic translation initiation factor 2 subunit alpha

Synonyms -

Cytological map position - 14C6-14C6

Function - translation factor

Keywords - mediates the binding of tRNAiMet to the ribosome in a GTP-dependent manner - eIF2 is a heterotrimer consisting of an alpha, a beta (eIF2β), and a gamma (eIF2γ) subunit - activation of the integrated stress response (ISR) signaling includes endoplasmic reticulum stress, and amino acid deprivation, sensed by specialized kinases PERK and GCN2 that converge on phosphorylation of eIF2α

Symbol - eIF2α

FlyBase ID: FBgn0261609

Genetic map position - chrX:16,445,176-16,446,993

Classification - eukaryotic translation initiation factor 2 subunit 1

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein

GENE orthologs: Biolitmine
Recent literature
Lewis, S. A., Bakhtiari, S., Forstrom, J., Bayat, A., Bilan, F., Le Guyader, G., Alkhunaizi, E., Vernon, H., Padilla-Lopez, S. R. and Kruer, M. C. (2023). AGAP1-associated endolysosomal trafficking abnormalities link gene-environment interactions in a neurodevelopmental disorder. Dis Model Mech. PubMed ID: 37470098
AGAP1 is an Arf1 GAP that regulates endolysosomal trafficking. Damaging variants have been linked to cerebral palsy and autism. This study reports three new individuals with microdeletion variants in AGAP1. Affected individuals have intellectual disability (3/3), autism (3/3), dystonia with axial hypotonia (1/3), abnormalities of brain maturation (1/3), growth impairment (2/3) and facial dysmorphism (2/3). Mechanisms potentially underlying AGAP1 neurodevelopmental impairments were investigated using the Drosophila ortholog, CenG1a. Reduced axon terminal size, increased neuronal endosome abundance, and elevated autophagy were discovered at baseline. Given potential incomplete penetrance, gene-environment interactions were assessed. Basal elevation was found in phosphorylation of the integrated stress-response protein eIF2α and inability to further increase eIF2α-P with subsequent cytotoxic stressors. CenG1a-mutant flies have increased lethality from exposure to environmental insults. A model is proposed wherein disruption ofa endolysosomal trafficking, chronically activating the integrated stress response, and leaving AGAP1-deficient cells susceptible to a variety of second hit cytotoxic stressors. This model may have broader applicability beyond AGAP1 in instances where both genetic and environmental insults co-occur in individuals with neurodevelopmental disorders.
Xue, M., Cong, F., Zheng, W., Xu, R., Liu, X., Bao, H., Sung, Y. Y., Xi, Y., He, F., Ma, J., Yang, X. and Ge, W. (2023). Loss of Paip1 causes translation reduction and induces apoptotic cell death through ISR activation and Xrp1. Cell Death Discov 9(1): 288. PubMed ID: 37543696
Regulation of protein translation initiation is tightly associated with cell growth and survival. This study identified Paip1, the Drosophila homolog of the translation initiation factor PAIP1 and analyzed its role during development. Through genetic analysis, this study found that loss of Paip1 causes reduced protein translation and pupal lethality. Furthermore, tissue specific knockdown of Paip1 results in apoptotic cell death in the wing imaginal disc. Paip1 depletion leads to increased proteotoxic stress and activation of the integrated stress response (ISR) pathway. Mechanistically, it was shown that loss of Paip1 promotes phosphorylation of eIF2α via the kinase PERK, leading to apoptotic cell death. Moreover, Paip1 depletion upregulates the transcription factor gene Xrp1, which contributes to apoptotic cell death and eIF2α phosphorylation. It was further shown that loss of Paip1 leads to an increase in Xrp1 translation mediated by its 5'UTR. These findings uncover a novel mechanism that links translation impairment to tissue homeostasis and establish a role of ISR activation and Xrp1 in promoting cell death.
Kiparaki, M. and Baker, N. E. (2023). Ribosomal protein mutations and cell competition: autonomous and nonautonomous effects on a stress response. Genetics 224(3). PubMed ID: 37267156
Ribosomal proteins (Rps) are essential for viability. Genetic mutations affecting Rp genes were first discovered in Drosophila, where they represent a major class of haploinsufficient mutations. One mutant copy gives rise to the dominant "Minute" phenotype, characterized by slow growth and small, thin bristles. Wild-type (WT) and Minute cells compete in mosaics, that is, Rp+/- are preferentially lost when their neighbors are of the wild-type genotype. Many features of Rp gene haploinsufficiency (i.e. Rp+/- phenotypes) are mediated by a transcriptional program. In Drosophila, reduced translation and slow growth are under the control of Xrp1, a bZip-domain transcription factor induced in Rp mutant cells that leads ultimately to the phosphorylation of eIF2α; and consequently inhibition of most translation. Rp mutant phenotypes are also mediated transcriptionally in yeast and in mammals. In mammals, the Impaired Ribosome Biogenesis Checkpoint activates p53. Recent findings link Rp mutant phenotypes to other cellular stresses, including the DNA damage response and endoplasmic reticulum stress. It is suggested that cell competition results from nonautonomous inputs to stress responses, bringing decisions between adaptive and apoptotic outcomes under the influence of nearby cells. In Drosophila, cell competition eliminates aneuploid cells in which loss of chromosome leads to Rp gene haploinsufficiency. The effects of Rp gene mutations on the whole organism, in Minute flies or in humans with Diamond-Blackfan Anemia, may be inevitable consequences of pathways that are useful in eliminating individual cells from mosaics. Alternatively, apparently deleterious whole organism phenotypes might be adaptive, preventing even more detrimental outcomes. In mammals, for example, p53 activation appears to supress oncogenic effects of Rp gene haploinsufficiency.
Lidsky, P. V., Yuan, J., Lashkevich, K. A., Dmitriev, S. E. and Andino, R. (2023). Monitoring integrated stress response in live Drosophila. bioRxiv. PubMed ID: 37502856
Cells exhibit stress responses to various environmental changes. Among these responses, the integrated stress response (ISR) plays a pivotal role as a crucial stress signaling pathway. While extensive ISR research has been conducted on cultured cells, understanding of its implications in multicellular organisms remains limited, largely due to the constraints of current techniques that hinder the ability to track and manipulate the ISR in vivo. To overcome these limitations, this study has successfully developed an internal ribosome entry site (IRES)-based fluorescent reporter system. This innovative reporter enables labelling of Drosophila cells, within the context of a living organism, that exhibit eIF2 phosphorylation-dependent translational shutoff - a characteristic feature of the ISR and viral infections. Through this methodology, this study has unveiled tissue- and cell-specific regulation of stress response in Drosophila flies and have even been able to detect stressed tissues in vivo during virus and bacterial infections. To further validate the specificity of the reporter, this study has engineered ISR-null eIF2αS50A mutant flies for stress response analysis. The results shed light on the tremendous potential of this technique for investigating a broad range of developmental, stress, and infection-related experimental conditions. Combining the reporter tool with ISR-null mutants establishes Drosophila as an exceptionally powerful model for studying the ISR in the context of multicellular organisms.

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. (Harding, 2000; Kang, 2015; Vattem, 2004). 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 (Baumgartner, 2021; Recasens-Alvarez, 2021). 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).

Cell competition is driven by Xrp1-mediated phosphorylation of eukaryotic initiation factor 2alpha

Cell competition is a context-dependent cell elimination via cell-cell interaction whereby unfit cells ('losers') are eliminated from the tissue when confronted with fitter cells ('winners'). Despite extensive studies, the mechanism that drives loser's death and its physiological triggers remained elusive. Through a genetic screen in Drosophila, this study found that endoplasmic reticulum (ER) stress causes cell competition. Mechanistically, ER stress upregulates the bZIP transcription factor Xrp1, which promotes phosphorylation of the eukaryotic translation initiation factor eIF2α via the kinase PERK, leading to cell elimination. Surprisingly, the genetic data show that different cell competition triggers such as ribosomal protein mutations or RNA helicase Hel25E mutations converge on upregulation of Xrp1, which leads to phosphorylation of eIF2α and thus causes reduction in global protein synthesis and apoptosis when confronted with wild-type cells. These findings not only uncover a core pathway of cell competition but also open the way to understanding the physiological triggers of cell competition (Ochi, 2021).

Cell competition is an evolutionarily conserved quality control process that selectively eliminates viable unfit cells ('losers') when coexisting with fitter cells ('winners') within a growing tissue. For instance, cells with heterozygous mutations in the ribosomal protein genes, called Minute/+ (M/+) mutations, are viable on their own but are eliminated from Drosophila imaginal epithelium when surrounded by wild-type cells. Similarly, Drosophila cells homozygously mutant for Mahjong/VprBP (Mahj) or the RNA helicase Helicase25E (Hel25E) are viable on their own but are eliminated by apoptosis when confronted with wild-type cells. Several other factors also cause cell competition in Drosophila, which include high-level expression of the oncogene Myc, elevated activity of JAK-STAT or Wnt/Wg signaling, inactivation of the Hippo pathway, and loss of apico-basal cell polarity. However, the physiological triggers of cell competition have still remained unclear (Ochi, 2021).

A genetic study in Drosophila has identified a basic leucine zipper domain (bZIP) transcription factor Xrp1 as essential for driving M/+ cell competition. Xrp1 is upregulated in M/+ cell clones and contributes to their cell death. Intriguingly, Xrp1 upregulation is also required for M/+ cells to reduce protein synthesis levels. However, the mechanisms of how Xrp1 reduces protein synthesis and how it contributes to loser's death remained unknown. It has been found that, similarly to M/+ clones, loser clones such as Hel25E or Mahj mutant clones reduce protein synthesis levels compared to neighboring wild-type winners, which suggests a potential mechanistic link between the reduction of protein synthesis and induction of loser's death (Ochi, 2021).

Under various stress conditions, cells adapt to the environment via activation of the integrated stress response (ISR) signaling, an evolutionarily conserved intracellular signaling network that restore cellular homeostasis. These stresses include endoplasmic reticulum (ER) stress, nutrient deprivation, viral infection, and oxidative stress. The stresses are sensed by four specialized kinases (PERK, GCN2, PKR, and HRI) that converge on phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α). For instance, upon accumulation of unfolded proteins in the ER, the ER-resident chaperone BiP/Hsc70-3 is released from PERK, leading to homodimerization and activation of the eIF2α kinase PERK. eIF2α phosphorylation results in a reduction in global protein synthesis, while allowing the translation of selected genes including activating transcription factor 4 (ATF4). Two of four eIF2α kinases, PERK and GCN2, are conserved in Drosophila, which are activated by ER stress and amino acid deprivation, respectively. ER stress is induced by various intracellular factors that cause the accumulation of unfolded proteins in the ER, which leads to activation of the unfolded protein response (UPR) signaling to recover ER function. In the Drosophila UPR pathway, PERK phosphorylates eIF2α and thus inhibits global protein synthesis to decrease the burden of ER capacity, while the endoribonuclease inositol-requiring enzyme-1 (Ire1) activates the transcription factor Xbp1 via a specific mRNA splicing which leads to upregulation of various genes helping the recovery of ER function (Ochi, 2021).

Through a genetic screen in Drosophila, this study found that mutations that cause ER stress make cells to be losers of cell competition when surrounded by wild-type cells. Mechanistically, ER stress, as well as other cell competition triggers such as M/+ and Hel25E mutations, upregulate Xrp1, which promotes phosphorylation of eIF2α via PERK, thereby causing reduction in protein synthesis and induction of cell death. The data suggest that ER stress or other environmental stresses activating ISR signaling, which converge on the phosphorylation of eIF2α, could be a physiological trigger of cell competition (Ochi, 2021).

Importantly, the data show that eIF2α phosphorylation is also required for the induction of loser's death. Similarly, recent studies have shown that M/+ cells experience proteotoxic stress and thus induce phosphorylation of eIF2α, which acts as a driver of M/+ cell competition. Whether the global inhibition of protein synthesis or other downstream event(s) of eIF2α phosphorylation such as upregulation of UPR-activating transcription factor ATF4 is linked to their apoptosis is an outstanding important question. Notably, Xrp1 has been implicated to be a functional homolog of mammalian CHOP, a transcription factor that is induced by ATF4. Consistently, this study found that overexpression of PERK leads to upregulation of Xrp1 expression. In addition, recent studies have shown that overexpression of PERK or ATF4 upregulates Xrp1. These observations suggest that Xrp1 acts both upstream and downstream of the PERK-eIF2α axis in a positive feedback loop. The upstream Xrp1 may activate the PERK-eIF2α axis via upregulation of PERK expression. Alternatively, Xrp1 upregulation may cause ER stress, which induces PERK activation. These are also important issues that should be addressed in the future studies (Ochi, 2021).

It would also be important to clarify the mechanistic relationship between the Xrp1-PERK-eIF2α axis and other cell competition regulators so far reported, which include autophagy, Toll-related receptor signaling, Flower, and Azot. In addition, it is crucial to understand in the future studies how Xrp1 is commonly upregulated by different cell competition triggers. Nonetheless, the current study identified a critical signaling axis that converge a variety of cellular stress signaling to a common cell competition pathway via upregulation of Xrp1 (Ochi, 2021).

While studies in Drosophila have uncovered several triggers of cell competition and the downstream molecules essential for cell elimination, the physiological triggers of cell competition within animals have remained unknown. The genetic screen identified a series of mutations that cause ER stress as triggers of cell competition. ER stress is induced by the accumulation of unfolded or misfolded proteins in the ER via a variety of intracellular factors under both physiological and pathological conditions, leading to activation of the evolutionarily conserved PERK-eIF2α pathway. The PERK-eIF2α pathway is also activated by the conserved ISR signaling triggered by cell extrinsic factors such as amino acid deprivation, glucose deprivation, hypoxia, and viral infection. Moreover, Xrp1 expression is induced by genotoxic stresses such as irradiation. Thus, the current finding that the Xrp1-PERK-eIF2α axis commonly drives cell competition has opened the way to understanding the physiological and pathological role of cell competition. Intriguingly, mutations in the fused in sarcoma (FUS) gene, which are linked to amyotrophic lateral sclerosis (ALS), cause ER stress and the Drosophila FUS orthologue cabeza genetically interacts with Xrp1. In addition, it has been shown that cell competition plays a role in neurodegenerative diseases, which are thought to be driven by ER stress. Further studies on the physiological and pathological regulations of Xrp1-PERK-eIF2α signaling would unveil the in vivo role of cell competition (Ochi, 2021).

PERK-mediated eIF2α phosphorylation contributes to the protection of dopaminergic neurons from chronic heat stress in Drosophila

Environmental high-temperature heat exposure is linked to physiological stress such as disturbed protein homeostasis caused by endoplasmic reticulum (ER) stress. Abnormal proteostasis in neuronal cells is a common pathological factor of Parkinson's disease (PD). Chronic heat stress is thought to induce neuronal cell death during the onset and progression of PD, but the exact role and mechanism of ER stress and the activation of the unfolded protein response (UPR) remains unclear. This study showed that chronic heat exposure induces ER stress mediated by the PKR-like eukaryotic initiation factor 2alpha kinase (PERK)/eIF2α phosphorylation signaling pathway in Drosophila neurons. Chronic heat-induced eIF2α phosphorylation was regulated by PERK activation and required for neuroprotection from chronic heat stress. Moreover, the attenuated protein synthesis by eIF2α phosphorylation was a critical factor for neuronal cell survival during chronic heat stress. Genetic downregulation of PERK, specifically in dopaminergic (DA) neurons, impaired motor activity and led to DA neuron loss. Therefore, these findings provide in vivo evidence demonstrating that chronic heat exposure may be a critical risk factor in the onset of PD, and eIF2α phosphorylation mediated by PERK may contribute to the protection of DA neurons against chronic heat stress in Drosophila (Elvira, 2020).

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 (DlOE), 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 DlOE, 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, DlOE, 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 DlOE, 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 DlOE, 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).

Mitochondrial dysfunction induces dendritic loss via eIF2alpha phosphorylation

Mitochondria are key contributors to the etiology of diseases associated with neuromuscular defects or neurodegeneration. How changes in cellular metabolism specifically impact neuronal intracellular processes and cause neuropathological events is still unclear. This study dissected the molecular mechanism by which mitochondrial dysfunction induced by Prel aberrant function mediates selective dendritic loss in Drosophila melanogaster class IV dendritic arborization neurons. Using in vivo ATP imaging, it was found that neuronal cellular ATP levels during development are not correlated with the progression of dendritic loss. Mitochondrial stress signaling pathways were sought that induce dendritic loss; mitochondrial dysfunction was found associated with increased eIF2α phosphorylation, which is sufficient to induce dendritic pathology in class IV arborization neurons. It was also observed that eIF2α phosphorylation mediates dendritic loss when mitochondrial dysfunction results from other genetic perturbations. Furthermore, mitochondrial dysfunction induces translation repression in class IV neurons in an eIF2alpha phosphorylation-dependent manner, suggesting that differential translation attenuation among neuron subtypes is a determinant of preferential vulnerability (Tsuyama, 2016).

Pur-α functionally interacts with FUS carrying ALS-associated mutations
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to motor neuron loss. Fused in sarcoma (FUS) protein carrying ALS-associated mutations localizes to stress granules and causes their coalescence into larger aggregates. This study shows that Pur-α physically interacts with mutated FUS in an RNA-dependent manner. Pur-α colocalizes with FUS carrying mutations in stress granules of motoneuronal cells differentiated from induced pluripotent stem cells and that are derived from ALS patients. Both Pur-α and mutated FUS upregulate phosphorylation of the translation initiation factor EIF-2α and consistently inhibit global protein synthesis. In vivo expression of Pur-α in different Drosophila tissues significantly exacerbates the neurodegeneration caused by mutated FUS. Conversely, the downregulation of Pur-α in neurons expressing mutated FUS significatively improves fly climbing activity. All these findings suggest that Pur-α, through the control of mRNA translation, might be involved in the pathogenesis of ALS associated with the mutation of FUS, and that an alteration of protein synthesis may be directly implicated in the disease. Finally, in vivo RNAi-mediated ablation of Pur-α produced locomotion defects in Drosophila, indicating a pivotal role for this protein in the motoneuronal function (Di Salvio, 2015).

Coordinate regulation of eIF2alpha phosphorylation by PPP1R15 and GCN2 is required during Drosophila development

Phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α) by the kinase GCN2 attenuates protein synthesis during amino acid starvation in yeast, whereas in mammals a family of related eIF2α kinases regulate translation in response to a variety of stresses. Unlike single-celled eukaryotes, mammals also possess two specific eIF2α phosphatases, PPP1R15a and PPP1R15b, whose combined deletion leads to a poorly understood early embryonic lethality. This study reports the characterisation of the first non-mammalian eIF2α phosphatase (PPP1R15) and the use of Drosophila to dissect its role during development. The Drosophila protein demonstrates features of both mammalian proteins, including limited sequence homology and association with the endoplasmic reticulum. Of note, although this protein is not transcriptionally regulated, its expression is controlled by the presence of upstream open reading frames in its 5'UTR, enabling induction in response to eIF2α phosphorylation. Moreover, this study showed that its expression is necessary for embryonic and larval development and that this is to oppose the inhibitory effects of GCN2 on anabolic growth (Malzer, 2013).

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 αalpha subunit of eukaryotic translation initiation factor 2 (eIF2α) at serine-51, a modification that leads to global inhibition of mRNA translation. This study demonstrates induction of the phosphorylation of eIF2α 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 eIF2α 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 eIF2α (eIF2αP) 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-eIF2αP pathway mediates survival and facilitates adaptation to the deleterious effects of the inactivation of PI3K or Akt. Inactivation of the PERK-eIF2αP pathway increases the susceptibility of tumor cells to death by pharmacological inhibitors of PI3K or Akt. Thus, it is suggested that the PERK-eIF2αP pathway provides a link between Akt signaling and translational control, which has implications for tumor formation and treatment (Mounir, 2011).

Functions of eIF2α orthologs in other species

mRNA translation in astrocytes controls hippocampal long-term synaptic plasticity and memory

Activation of neuronal protein synthesis upon learning is critical for the formation of long-term memory. This study report sthat learning in the contextual fear conditioning paradigm engenders a decrease in eIF2&α (eukaryotic translation initiation factor 2) phosphorylation in astrocytes in the hippocampal CA1 region, which promotes protein synthesis. Genetic reduction of eIF2α phosphorylation in hippocampal astrocytes enhanced contextual and spatial memory and lowered the threshold for the induction of long-lasting plasticity by modulating synaptic transmission. Thus, learning-induced dephosphorylation of eIF2α in astrocytes bolsters hippocampal synaptic plasticity and consolidation of long-term memories (Sharma, 2023).

Activation of the integrated stress response by inhibitors of its kinases

Phosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. This study found that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases (Szaruga, 2023).

Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1(-/y)). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1(-/y) mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS (Hooshmandi, 2023).

Nuclear translocation of an aminoacyl-tRNA synthetase may mediate a chronic "integrated stress response"

Various stress conditions are signaled through phosphorylation of translation initiation factor eukaryotic initiation factor 2α (eIF2α) to inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. However, this integrated stress response is acute and cannot resolve lasting stress. This study reports that tyrosyl-tRNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase family that responds to diverse stress conditions through cytosol-nucleus translocation to activate stress-response genes, also inhibits global translation. However, it occurs at a later stage than eIF2α/ATF4 and mammalian target of rapamycin (mTOR) responses. Excluding TyrRS from the nucleus over-activates translation and increases apoptosis in cells under prolonged oxidative stress. Nuclear TyrRS transcriptionally represses translation genes by recruiting TRIM28 and/or NuRD complex. It is proposed that TyrRS, possibly along with other family members, can sense a variety of stress signals through intrinsic properties of this enzyme and strategically located nuclear localization signal and integrate them by nucleus translocation to effect protective responses against chronic stress (Jones, 2023).


Search PubMed for articles about Drosophila Eif2α

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

Baumgartner, M. E., Dinan, M. P., Langton, P. F., Kucinski, I., Piddini, E. (2021). Proteotoxic stress is a driver of the loser status and cell competition. Nat Cell Biol23(2):136-146. PubMed ID: 33495633

Di Salvio, M., et al. (2015). Pur-α functionally interacts with FUS carrying ALS-associated mutations. Cell Death Dis 6: e1943. PubMed ID: 26492376.

Elvira, R., Cha, S. J., Noh, G. M., Kim, K. and Han, J. (2020). PERK-mediated eIF2α phosphorylation contributes to the protection of dopaminergic neurons from chronic heat stress in Drosophila. Int J Mol Sci 21(3). PubMed ID: 32013014

Harding H. P., Novoa, I., Zhang, Y., Zeng, H., Wek, R., Schapira, M., Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell6(5):1099-1108. PubMed ID: 11106749

Hooshmandi, M., Sharma, V., Thorn Perez, C., Sood, R., Krimbacher, K., Wong, C., Lister, K. C., Urena Guzman, A., Bartley, T. D., Rocha, C., Maussion, G., Nadler, E., Roque, P. M., Gantois, I., Popic, J., Levesque, M., Kaufman, R. J., Avoli, M., Sanz, E., Nader, K., Hagerman, R. J., Durcan, T. M., Costa-Mattioli, M., Prager-Khoutorsky, M., Lacaille, J. C., Martinez-Cerdeno, V., Gibson, J. R., Huber, K. M., Sonenberg, N., Gkogkas, C. G. and Khoutorsky, A. (2023). Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome. Neuron 111(19):3028-3040. PubMed ID: 25978358

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., Szajewska-Skuta, M., Dalton, L. E., Thomas, S. E., Hu, N., Skaer, H., Lomas, D. A., Crowther, D. C., Marciniak, S. J. (2013). Coordinate regulation of eIF2alpha phosphorylation by PPP1R15 and GCN2 is required during Drosophila development. J Cell Sci126(Pt 6):1406-1415. PubMed ID: 21954288

Ochi, N., Nakamura, M., Nagata, R., Wakasa, N., Nakano, R. and Igaki, T. (2021). Cell competition is driven by Xrp1-mediated phosphorylation of eukaryotic initiation factor 2alpha. PLoS Genet 17(12): e1009958. PubMed ID: 34871307

Recasens-Alvarez, C., Alexandre, C., Kirkpatrick, J., Nojima, H., Huels, D. J., Snijders, A. P., Vincent, J. P. (2021). Ribosomopathy-associated mutations cause proteotoxic stress that is alleviated by TOR inhibition. Nat Cell Biol23(2):127-135. PubMed ID: 33495632

Sharma, V., Oliveira, M. M., Sood, R., Khlaifia, A., Lou, D., Hooshmandi, M., Hung, T. Y., Mahmood, N., Reeves, M., Ho-Tieng, D., Cohen, N., Cheng, P. C., Rahim, M. M. A., Prager-Khoutorsky, M., Kaufman, R. J., Rosenblum, K., Lacaille, J. C., Khoutorsky, A., Klann, E. and Sonenberg, N. (2023). mRNA translation in astrocytes controls hippocampal long-term synaptic plasticity and memory. Proc Natl Acad Sci U S A 120(49):e2308671120. PubMed ID: 32433968

Szaruga, M., Janssen, D. A., de Miguel, C., Hodgson, G., Fatalska, A., Pitera, A. P., Andreeva, A. and Bertolotti, A. (2023). Activation of the integrated stress response by inhibitors of its kinases. Nat Commun 14(1):5535. PubMed ID: 28209644

Vattem K. M., Wek, R. C. (2004). Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci U S A101(31):11269-11274. PubMed ID: 15277680

Biological Overview

date revised: 18 February 2024

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