genes associated with ER stress and Unfolded protein response
| Diseases often associated with disruption in ER stress and Unfolded protein response
Amyotrophic Lateral Sclerosis
X-linked mental retardation
of the disease
The endoplasmic reticulum (ER) is responsible for folding and processing proteins entering the secretory pathway. Because the flux of proteins through the ER varies considerably among cell types and in different conditions, cells maintain a balance between the load on the ER and its protein folding capacity. However, a number of biochemical, physiological, and pathological stimuli can disrupt this balance, resulting in ER stress. To re-establish ER homeostasis, the unfolded protein response (UPR) is activated. This network of pathways up-regulates genes encoding ER-specific chaperones and other proteins involved in protein secretion while also attenuating protein translation and degrading certain ER-associated mRNAs. The UPR is broadly conserved across eukaryotes and is essential for normal development in several model organisms, particularly for professional secretory cells, where it is thought to be important for the establishment and maintenance of high levels of protein secretion. It is also induced during many metabolic conditions, including diabetes, hyperlipidemia, and inflammation, and has been implicated in various cancers, especially in the growth of large tumors that rely on an effective response to hypoxia (Lee, 2015 and references therein).
The UPR is carried out by three main signaling branches. One of these is initiated by the ER transmembrane protein inositol-requiring enzyme 1 (Ire1). When activated by ER stress, the cytosolic endoribonuclease domain of Ire1 cleaves the mRNA encoding the transcription factor Xbp1, thereby initiating an unconventional splicing event that produces the mRNA template encoding a highly active form of Xbp1. Ire1 also cleaves other mRNAs associated with the ER membrane through a pathway that is particularly active in Drosophila cells and that may reduce the load on the ER. A second sensor of ER stress, activating transcription factor 6, is activated by proteolysis, which releases it from the ER membrane and allows it to travel to the nucleus and regulate gene expression. Finally, protein kinase RNA−like ER kinase (Perk) phosphorylates eukaryotic initiation factor 2 alpha, leading to a general attenuation of protein synthesis as well as the translational up-regulation of certain mRNAs that contain upstream open reading frames (ORFs) in their 5′ untranslated regions. In addition to its direct effects on the protein secretory pathway, the UPR influences several other cellular pathways, including apoptosis, inflammation, and lipid synthesis. Furthermore, the UPR (particularly the Perk/Atf4 branch) appears to have close ties to mitochondrial function (Lee, 2015 and references therein).
ER stress can be a primary cause and a modifier of many important human diseases. For example, diabetes is a common metabolic disease, caused by misregulation of blood glucose levels. Mutations in both PERK and XBP1 cause diabetes-like symptoms in mouse and humans. Similarly, adipose tissue from obese human individuals shows up-regulation of several key proteins involved in the UPR relative to levels of these proteins in adipose tissue of lean individuals. ER stress has also been implicated in the pathogenesis of several human neurological diseases, including Parkinson disease, polyglutamine diseases, amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease. Alteration of the UPR may worsen the disease. For example, in animal models of ALS, second-site mutations that increase ER stress result in earlier onset of disease and more severe symptoms. An individual’s ER stress response can be an important factor in determining disease severity. To understand the extent to which ER stress responses can act as a modifier of disease, it is critical to understand the extent and nature of the variation in ER stress responses (Chow, 2013 and references therein).
Relevant studies of ER stress and unfolded protein response
Lee, J.E., Oney, M., Frizzell, K., Phadnis, N. and Hollien, J. (2015). Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress. G3 (Bethesda) 5: 667-675. PubMed ID: 25681259
Despite a highly coordinated change in gene expression for
metabolic genes during ER stress, any changes in actual metabolism
in S2 cells were not detected. Because these cells have been in
culture for decades and have likely been selected for rapid
proliferation, it is possible that they are already undergoing
some version of aerobic glycolysis, such that the underlying gene
regulation during ER stress is preserved but any metabolic changes
are masked. Others have also noted that S2 cells are resistant to
hypoxia, and do not produce more lactate except in extreme
conditions. The increase in lactate observed through in vivo
studies in flies subjected to ER stress, however, suggests that in
a more physiological setting, the gene expression changes shown
here do mediate a metabolic shift toward aerobic glycolysis (Lee,
The link between ER stress and metabolism can be rationalized by the need to generate building blocks for biosynthesis of glycoproteins and lipids. Early intermediates of glycolysis are necessary for production of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), an important donor molecule for N-glycosylation of proteins in the ER. Both fructose-6-phosphate and dihydroxyacetone phosphate also are required for synthesis of glycolipids. An increased flux through glycolysis may therefore be important to support the increased production of glycerophospholipids and glycoproteins that are associated with the UPR. In support of this view, glucose deprivation or inhibition of glycolysis with 2-deoxy-D-glucose induces the UPR, which contributes to cell death, especially in cancer cells, and this effect can be rescued by UDP-GlcNAc. The hexosamine biosynthetic pathway generating UDP-GlcNAc is also directly activated by Xbp1, stimulates cardioprotection during ischemia/reperfusion injury, and increases longevity in worms (Lee, 2015).
A second, nonmutually exclusive explanation for a shift to glycolysis during ER stress is the need to limit production of ROS. Along with mitochondrial respiration, protein folding in the ER is one of the main sources of ROS, which are produced by the normal process of disulfide bond-coupled folding. If allowed to accumulate, these ROS can cause oxidative stress and damage to cells, eventually leading to apoptosis. Several studies have confirmed that ROS are produced during ER stress, when protein folding is inefficient and more rounds of oxidation and reduction are required to fold proteins. Limiting other sources of oxidative stress, such as by down-regulating the TCA cycle and thereby restricting the flux through OXPHOS (the main source of ROS in the mitochondria), may be a way to mitigate the damage and allow cells to recover more effectively (Lee, 2015).
Finally, the advantage of the Warburg effect for tumor growth may arise from the increased rate of ATP production by glycolysis compared to OXPHOS, despite its lower efficiency of conversion. By analogy, a metabolic shift during ER stress could rapidly supply ATP necessary for protein folding and processing. Indeed, cancer cells showing elevated levels of ENTPD5, an ER UDPase, promotes aerobic glycolysis to increase ATP for protein N-glycosylation and refolding (Lee, 2015).
Overall, results of this study identify Atf4 as a transcriptional regulator of glycolysis during ER stress. As Atf4 is expressed throughout fly development, it may regulate glycolysis in other situations as well: notably, Atf4 mutant flies are lean and have reduced circulating carbohydrates, suggesting a role in metabolism. Furthermore, because the Perk-Atf4 branch of UPR is activated during hypoxia, it will be interesting to see whether Atf4 contributes to regulation of glycolysis in other developmental, physiological (hypoxia), or pathological process during which glycolysis regulated. More broadly, because the UPR is activated in many types of cancer, its ability to regulate glucose metabolism may play a contributing role in the Warburg effect (Lee, 2015).
Weiss, S. and Minke, B. (2015). A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells. Channels (Austin) 9: 14-20. PLoS One 6: e27408. PubMed ID: 25664921
An illuminating example is the Drosophila model for class III autosomal dominant retinitis pigmentosa (ADRP), in which ER-stress has been shown to play a protective role against retinal degeneration. Moreover, it has been shown that when Drosophila cells (both photoreceptors and S2 cells) are pre-exposed to a mild ER-stress, they are protected from treatments with various cell death agents. It has also been shown recently that activation of mild ER stress is neuroprotective, both in Drosophila and mouse models of Parkinson disease, and this protection is a consequence of autophagy activation. Accordingly, it is well established that activation of the ER-stress response by accumulation of misfolded proteins (unfolded protein response, UPR) can trigger autophagy (Weiss, 2015).
It is well established that elevation of intracellular Ca2+ can trigger autophagy. The stimulation of autophagy by elevated cytosolic [Ca2+] has been studied mainly in tissue culture cells, in which mobilization of Ca2+ is triggered by application of various Ca2+ mobilizing agents (e.g. thapsigargin, ionomycin, vitamin D). However, since increased cytosolic [Ca2+] also activates ER-stress which induces autophagy, a question arises as to whether the activation of autophagy by Ca2+ is induced directly or indirectly via activation of ER-stress. The fact that thapsigargin-induced autophagy occurs in UPR-deficient cells, suggests a direct involvement of Ca2+ in the induction of autophagy. Although Ca2+ regulation of autophagy has been found to be mediated through the activation of Bcl-2 and calmodulin-dependent kinase kinase-β, the target by which Ca2+ activates autophagy directly is yet to be elucidated. These unsolved issues can be conveniently addressed in the new model of Ca2+ induced autophagy in Drosophila photoreceptor cells (Weiss, 2015).
Neurodegenerative diseases, such as Parkinson, Alzheimer and Huntington’s diseases are accompanied by the accumulation of large aggregates of mutant proteins and are autophagy associated diseases. These autophagy associated diseases, which are characterized by abnormal protein aggregations, have highly developed model systems of Drosophila. The increased autophagosome formation observed in these diseases, may play a pro- tective role by degrading misfolded proteins. Many of the above diseases, including Parkinson and Alzheimer diseases, are also associated with reduced cellular Ca2+ buffering and impairments in cellular Ca2+ homeostasis reminiscent of Drosophila photoreceptors with reduced calphotin. Therefore, combining the Drosophila models of autophagy associated diseases with calphotin hypomorph can be very useful for studying the possible link between reduced cellular Ca2+ buffering and neurodegeneration (Weiss, 2015).
In the genetic fly model of calphotin hypomorph, reduced calphotin levels, which trigger changes in cellular Ca2+ homeostasis allows induction of Ca2+ induced autophagy, simply by changing the illumination conditions of the flies’ environment. Accordingly, both the severity and progression of the observed degeneration can be highly controlled and manipulated. The degeneration process of this experimental model is sufficiently slow, allowing monitoring the progression of the degeneration process and elucidation of the involved proteins. Thus, the Drosophila Cpn hypomorph photoreceptor cells constitutes a powerful model, in which genetic manipulation combined with illumination determine the level of sustained cellular Ca2+ that trigger the induction of Ca2+ dependent autophagy, ER- stress and cell death. This model can provide a framework for further investigations into the link between cytosolic Ca2C, ER-stress and autophagy in human disorders and diseases (Weiss, 2015).
Ham, H., Woolery, A.R., Tracy, C., Stenesen, D., Krämer, H. and Orth, K. (2014). Unfolded protein response-regulated Drosophila Fic (dFic) protein reversibly AMPylates BiP chaperone during endoplasmic reticulum homeostasis. J Biol Chem 289: 36059-36069. PubMed ID: 25395623
AMPylation appears to be a promising posttranslational regulatory mechanism adopted by organisms of varying complexity due to the high conservation of Fic domains and its use of ATP as a substrate. Bacterial AMPylators have already been shown to play an essential role in hijacking host signaling pathways during pathogenesis, but the endogenous function of AMPylation in eukaryotes remained elusive. This study presents BiP, a well known ER chaperone and a major regulator of UPR, as the first substrate of AMPylation by a eukaryotic protein. BiP from the whole S2 cell lysate was predominantly labeled by dFic enzyme in the presence of divalent cations Mg2+, Mn2+, and Ca2+. ER is a major organelle for calcium storage with the concentration of calcium ions in the lumen in the millimolar range. Therefore, having calcium ions in the reaction may better mimic the chemical environment in the ER. It has also been reported that Hsp70 chaperones bind to calcium ions, which may stabilize the protein structure. The stable conformation of BiP achieved by calcium binding may render it a better substrate for dFic (Ham, 2014).
The essential role of BiP in ER stress response and many cellular and pathological processes supports the notion that there may be multiple mechanisms for its regulation. Upon ER stress, BiP is transcriptionally activated to assist in the folding of high levels of unfolded proteins. However, the dynamic fluctuation of unfolded protein loads in the ER due to stress response or alternating rates of protein synthesis may require a more rapid regulation of BiP. This is also supported by the discrepancy between the long half-life of BiP, which is up to 48 h, and its excess activity being detrimental to cells, as maturation and secretion of critical proteins can be significantly delayed. A post-translational modification provides an ideal mechanism to readily activate and deactivate BiP upon changes in unfolded protein loads in the ER. Previously, ADP-ribosylation has been suggested to inactivate BiP by attenuating substrate binding and interfering with the allosteric coupling between domains. In contrast to AMPylation, which was observed in the ATPase domain, ADP-ribosylation occurs in the substrate binding domain on arginine residues (Arg-470 and Arg-492). Therefore, this study speculates that BiP undergoes different modifications on both domains, ensuring its tight regulation at multiple levels. The exact molecular event triggering such modifications or the order in which they occur remains to be explored. Furthermore, identifying the enzyme that catalyzes the rapid deAMPylation of BiP during ER stress and understanding its functional consequence will be of great interest (Ham, 2014).
AMPylation occurs on a conserved threonine residue located near the ATP binding pocket, which suggests that AMPylation may affect the ATPase activity of BiP by either blocking nucleotide binding or inhibiting efficient ATP hydrolysis. Unfortunately, the inverse relationship of BiP suitability for AMPylation and its ATPase activity makes the measurement of activity differences caused by AMPylation technically challenging (Ham, 2014).
Another possible inhibitory mechanism of AMPylation on BiP is uncoupling of two-domain allostery, similar to the effect of ADP-ribosylation. Upon ATP binding, Hsp70 proteins undergo a structural change wherein the two domains come in close contact and form a compact structure. The addition of the bulky AMP moiety can potentially hinder the contact between the domains and thereby disrupt the functional cycle of BiP. From an intermolecular perspective, AMPylation of BiP may also alter its interaction with binding partners such as DnaJ co-chaperone or nucleotide exchange factors (Ham, 2014).
Induction of ER stress up-regulates not only BiP but also dFic, which is a surprising result considering that AMPylation of BiP decreases during ER stress. Nevertheless, it suggests that dFic is among many UPR genes that cells use to cope with the stress response. It is possible that dFic is induced along with other chaperones, but its activity or translocation is blocked until the unfolded protein load decreases and BiP has to be promptly inactivated. dFic could then be released from repression and subsequently inactivate BiP. Otherwise, excess levels of active BiP may prolong protein maturation and secretion, which would be deleterious to cells. How dFic is regulated upon ER stress is another interesting avenue to be explored. There might be another layer of posttranslational modification governing the function of dFic. It is also fascinating to speculate that autoAMPylation observed with dFic both in vitro and in vivo might be involved in its own regulation (Ham, 2014).
Previous studies have shown that flies without functional dFic in glial cells have impaired visual neurotransmission. This suggests that dFic substrate could be a component of visual signaling or a transporter of neurotransmitters. Alternatively, the blind phenotype could result from a loss of regulation on BiP. It can be speculated that the protein responsible for the visual signaling is not properly matured or secreted in the absence of tight regulation of BiP in dfic null flies, which thereby results in visual defect. Indeed, imbalance of protein homeostasis is a cause of many pathological processes due to accumulation of aberrant protein or impaired protein secretion (Ham, 2014).
Interestingly, dFic is mainly localized to the cell surface on glial cells and is particularly enriched in capitate projections in contrast to the ER localization in S2 cells. Therefore, dFic may target a different molecule on the cell surface that could directly impact neurotransmission. It is possible that in glial cells there might be cell type-specific factors that can induce the secretion of dFic from the ER to cell surface (Ham, 2014).
It was shown that human Fic also AMPylates BiP in vitro and that it is transcriptionally activated by ER stress. This suggests that AMPylation is a conserved regulatory mechanism in multiple species possibly involved in UPR. As both Fic and BiP are highly conserved in many organisms, it will be worth investigating the role of AMPylation in other species (Ham, 2014).
Understanding the regulatory mechanism of BiP is of utmost importance, as misregulation of BiP is associated with numerous diseases including neurological disorders and various cancers. An increased level of BiP is a critical factor for tumor progression and has been shown to confer chemoresistance to a variety of cancer cell lines. Accordingly, inhibition of BiP activity is emerging as an important cancer target. The discovery of BiP AMPylation and deAMPylation presents a new targetable avenue for drug development (Ham, 2014).
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
It was 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, any ATF4 consensus binding sequence (5'-RTTRCRTCA-3') was not found 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 chronic ER stress model. In a mammalian model
of chronic ER stress, the IRE1 branch of the UPR activates the JNK
pathway to trigger apoptosis thanks to TRAF2/ASK1. In the chronic
ER stress, depletion of traf2
has no effect. Instead, it was shown for the first time 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
The study characterized the components of the JNK signaling that is activated in response to chronic ER stress in Drosophila wing imaginal discs. 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. 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 shows 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).
Nagy, P., Varga, A., Pircs, K., Hegedűs, K. and Juhász, G. (2013). Myc-driven overgrowth requires unfolded protein response-mediated induction of autophagy and antioxidant responses in Drosophila melanogaster. PLoS Genet 9: e1003664. PubMed ID: 23950728
Autophagy and antioxidant responses have been considered to act as tumor suppressor pathways in normal cells and during early stages of tumorigenesis, while activation of these processes may also confer advantages for cancer cells. Lack of proper vasculature in solid tumors causes hypoxia and nutrient limitation. These stresses in the tumor microenvironment have been suggested to elevate UPR and autophagy to promote survival of cancer cells. This study demonstrates that genetic alterations similar to those observed in cancer cells (that is, deregulated expression of Myc) can also activate the UPR, autophagy and antioxidant pathways in a cell-autonomous manner in Drosophila. These processes are likely also activated as a consequence of deregulated Myc expression in human cancer cells based on a number of recent reports, similar to the findings in Drosophila presented in this study. First, chloroquine treatment that impairs all lysosomal degradation pathways is sufficient to reduce tumor volume in Myc-dependent lymphoma models. Second, ER stress and autophagy induced by transient Myc expression increase survival of cultured cells, and PERK-dependent autophagy is necessary for tumor formation in a mouse model. Data suggest that UPR-mediated autophagy and antioxidant responses may also be necessary to sustain the increased cellular growth rate driven by deregulated expression of Myc (Nagy, 2013).
Myc has proven difficult to target by drugs. Myc-driven cancer
cell growth could also be selectively prevented by blocking
cellular processes that are required in cancer cells but
dispensable in normal cells, known as the largely unexplored
non-oncogene addiction pathways. Previous genetic studies
establish that autophagy is dispensable for the growth and
development of mice, although knockout animals die soon after
birth due to neonatal starvation after cessation of placental
nutrition. Tissue-specific Atg knockout mice survive and
the animals are viable, with potential adverse effects only
observed in aging animals. Genetic deficiencies linked to p62
are also implicated in certain diseases, but knockout mice grow
and develop normally and are viable. Similarly, Nrf2
knockout mice are viable and adults exhibit no gross
abnormalities, while these animals are hypersensitive to oxidants.
Mice lacking PERK also develop normally and are viable. All these
knockout studies demonstrate that these genes are largely
dispensable for normal growth and development of mice, and that
progressive development of certain diseases is only observed later
during the life of these mutant animals. There are currently no
data regarding the effects of transient inhibition of these
processes, with the exception of the non-specific lysosomal
degradation inhibitor chloroquine, originally approved for the
treatment of malaria, which is already used in the clinic for
certain types of cancer (Nagy, 2013).
Elucidation of the genetic alterations behind increased UPR, autophagy and antioxidant responses observed in many established human cancer cells may allow specific targeting of these pathways, and potentially have a tremendous benefit for personalized therapies. In addition to non-specific autophagy inhibitors such as chloroquine, new and more specific inhibitors of selected Atg proteins are being developed. Given the dual roles of autophagy during cancer initiation and progression, a major question is how to identify patients who would likely benefit from taking these drugs. For example, no single test can reliably estimate autophagy levels in clinical samples, as increases in autophagosome generation or decreases in autophagosome maturation and autolysosome breakdown both result in accumulation of autophagic structures. Based on this study's data and recent mammalian reports, elevated Myc levels may even turn out to be useful as a biomarker before therapeutic application of inhibitors for key autophagy, UPR or antioxidant proteins in cancer patients (Nagy, 2013).
Roussel, B.D., Newton, T.M., Malzer, E., Simecek, N., Haq, I., Thomas, S.E., Burr, M.L., Lehner, P.J., Crowther, D.C., Marciniak, S.J. and Lomas, D.A. (2013). Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB. Hum Mol Genet 22: 4616-4626. PubMed ID: 23814041
After creating a cellular model to investigate FENIB, this study identified the E3 and E2 ligases involved in neuroserpin degradation. This was performed with the use of a Drosophila model of FENIB that shows accumulation of G392E neuroserpin when E3 and E2 orthologues are knocked down. It is interesting to note that the CG5623 depletion (UBE2j1 and j2) did not show any accumulation while in HeLa cells UBE2j1 shows a clear effect. These discrepancies between insects and mammals are well characterized in the literature, for example p53 is only implicated in cell death in flies while in mammals it has a role in the control of both cell death and cell cycle regulation. While a previous study has found the E3 ligases gp78 and hrd1 to be involved in neuroserpin degradation, this study identified differences in the specificity of these proteins. Gp78, and its partner E2 ligase UBE2g2, target unfolded, truncated forms of neuroserpin, while hrd1 and UBE2j1 target ordered polymers of neuroserpin for degradation by ERAD. Hrd1 and gp78 have also been well-characterized in the degradation process of mutant alpha-1-antitrypsin and many other misfolded proteins (Roussel, 2013).
Microarray analysis, and subsequent quantitative PCR, revealed
that the expression of ordered polymers is associated with an
activation of the sterol biosynthesis pathway. Previous studies
have shown an interaction between ERAD and cholesterol
biosynthetic pathway. HMGCoA reductase, the rate limiting enzyme
in cholesterol biosynthesis, can interact with the E3 ligase gp78
to mediate its sterol-accelerated degradation. Moreover, in vitro
work has demonstrated that Hrd1 is involved in the dislocation and
the degradation of HMGCoA reductase. This study speculates that
accumulation of mutant neuroserpin leads to the activation of the
cholesterol biosynthetic pathway. However, only inhibition of
HMGCoA reductase and not other enzymes on this pathway increases
the retention of mutant neuroserpin; HMGCoA reductase is the rate
limiting enzyme of the pathway and so it is likely that
accumulation of mutant neuroserpin increases HMGCoA reductase, and
hence upregulates the whole pathway (Roussel, 2013).
Taken together, these experiments reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. The accumulation of intracellular polymers of neuroserpin leads to the induction of genes involved in cholesterol synthesis, while inhibition of sterol biosynthesis impairs the ubiquitination of mutant neuroserpin and thus causes its accumulation (Roussel, 2013).
Brown, M.K., Chan, M.T., Zimmerman, J.E., Pack, A.I., Jackson, N.E. and Naidoo, N. (2014). Aging induced endoplasmic reticulum stress alters sleep and sleep homeostasis. Neurobiol Aging 35: 1431-1441. PubMed ID: 24444805
Aging also impairs quality control systems that are necessary for protein homeostasis. Earlier work has demonstrated that aging decreases BiP levels and increases pro-apoptotic factors such as CCAAT/enhancer-binding protein-homologous protein (CHOP) in the cerebral cortex of mice. Young flies experience less ER stress and their adaptive UPR is fully functional. Aged flies, on the other hand, are under chronic ER stress conditions with a diminished adaptive UPR. Therefore, basal ER stresses for the two groups are vastly different and likely contributes to the disparate responses to sleep deprivation. The young flies are readily able to handle acute sleep deprivation, whereas aged flies, already under stress, are overwhelmed by the secondary stressor. Addition of PBA has little effect on recovery sleep in young flies; however, PBA treatment ameliorates existing ER stress and suppresses further UPR induction in aged flies. As a result, treated aged flies display more efficient recovery sleep or are able to discharge sleep debt more efficiently than untreated aged flies (Brown, 2014).
Although no significant differences were found in BiP expression in flies treated with PBA, XBP1 was significantly affected by PBA treatment. PBA not only reduces the amount of spliced XBP1, it also reduces the expression of the unspliced variant as well. PBA likely suppresses UPR induction by chaperoning unfolded proteins, so that BiP stays bound to IRE1 and PERK and delays or prevents its activation, even under the condition of sleep deprivation. Phospho-IF2α levels are significantly reduced in the PBA treated aged flies subjected to sleep deprivation. Changes in p-eIF2α can be directly linked to the behavioral changes in the aged flies given that increased p-eIF2α facilitates baseline and recovery sleep. The reduced eIF2α phosphorylation in PBA treated animals may account for the improved homeostatic response in the aged SD flies (Brown, 2014).
This study also chemically induced ER stress in young flies and then subjected them to sleep deprivation. This acute tunicamycin treatment was found to decrease and fragment baseline sleep and phenocopy the recovery sleep behavior that is seen in the aged flies. There are two possible explanations for the tunicamycin results. First, tunicamycin activity leads to inhibition of glycosylation and general misfolding of proteins, which will ultimately result in ER stress. Secondly, tunicamycin could potentially prevent maturation of glycoproteins required for sleep maintenance. This data demonstrates that ER stress induces sleep fragmentation. Together with previous findings that sleep deprivation caused ER stress and activated the UPR, these results suggests that both processes are intimately linked and feedback on one another (Brown, 2014).
PBA has been shown to significantly increase life span in Drosophila, purportedly through inhibition of HDAC activity. The moderate effects of PBA on sleep consolidation in young sssp1 mutant flies suggest that protein stabilization by PBA is only partially ameliorating defects in these animals. The decrease in sleep in the ctrl sss background strain treated with PBA supports the increase and consolidation of wake in the wild-type lines. It has been previously shown that induction of ER stress impairs waking in mice. The study speculates that application of PBA also promotes wake-active neuronal function and consolidates wake in the flies by reducing ER stress. The issue is not the accumulation of sleep debt, but the discharge of sleep debt that is impaired in the aged flies. By ameliorating ER stress and suppressing activation of the UPR, PBA allows for more efficient recovery sleep in the aged flies. Since PBA ameliorates ER stress by directly reducing aberrant protein load, treating Drosophila with PBA supplements chaperone activity and decreases the burden of misfolded proteins that occurs as a consequence of an external stressor, sleep deprivation, as well as normal aging (Brown, 2014).
Baqri, R.M., Pietron, A.V., Gokhale, R.H., Turner, B.A., Kaguni, L.S., Shingleton, A.W., Kunes, S. and Miller, K.E. (2014). Mitochondrial chaperone TRAP1 activates the mitochondrial UPR and extends healthspan in Drosophila. Mech Ageing Dev 141-142: 35-45. PubMed ID: 25265088
Kim, A.Y., Seo, J.B., Kim, W.T., Choi, H.J., Kim, S.Y., Morrow, G., Tanguay, R.M., Steller, H. and Koh, Y.H. (2015). The pathogenic human Torsin A in Drosophila activates the unfolded protein response and increases susceptibility to oxidative stress. BMC Genomics 16: 338. PubMed ID: 25903460
Chow, C.Y., Wolfner, M.F. and Clark, A.G. (2013). Using natural variation in Drosophila to discover previously unknown endoplasmic reticulum stress genes. Proc Natl Acad Sci U S A 110: 9013-9018. PubMed ID: 23667151
Functional testing demonstrated that many of the candidates identified in the association study have potential roles in TM-induced ER stress response. It was found that 76% of the tested candidates with an effect on survival have no known role in ER stress. For example, CG15611 encodes a putative Rho guanyl-nucleotide exchange factor (RhoGEF) that is thought to regulate vesicle budding from the Golgi. CG15611 may regulate protein trafficking between the ER and Golgi and function in eliminating misfolded proteins. Megator encodes a nuclear pore protein. Megator has no known function in ER stress, but Ire1 has been shown to interact with some components of the nuclear pore complex in yeast. Although extensive functional studies are needed to identify specific functions in ER stress response, these verified candidates provide a promising pool of unstudied ER stress genes (Chow, 2013).
Nominating genes for future study can be challenging. Genes identified by both approaches in this study might be especially important. There are four genes in common between the candidate genes in the association study and genes whose expression is induced during the early time point (CG10962, CG11594, KrT95D, and tkv). There are two genes in common between the association study and the late time point (CG10962 and CG34228). Strikingly, CG10962 was found in the association study and both time points. CG10962 is orthologous to the human gene DHRS11 and is a member of the short-chain dehydrogenases/reductases (SDR) family. Proteins in the SDR family may have a previously unappreciated role in ER stress (Chow, 2013).
The ultimate goal of this study was to use Drosophila natural variation as a model to identify ER stress genes that may contribute to variation in the human ER stress response. Because the basic UPR pathways are well conserved between Drosophila and human, Drosophila is a good model for studying ER stress. The potential relevance of these results to humans was quantified by identifying the genes with human orthologs. It was found that 52%, 68%, and 63% of ER stress genes in this study have human orthologs (early response, late response, and association study, respectively). The majority of genes that contribute to variation in ER stress response in Drosophila are conserved and are very likely to have similar roles in the human ER stress response. To test the parallels between the current study's in vivo Drosophila results and those in mammals, results were compared to in vivo TM-induced responses in the mouse. Of the genes in the study with clear mouse orthologs, 47%, 48%, and 21% (early response, late response, and association study, respectively) overlap with the in vivo TM-induced liver transcriptional response in mouse (Chow, 2013).
Because ER stress is a major contributor to disease, genes having human orthologs that have been implicated in human disease were also identified. Genes that cause Mendelian-inherited disease, or are associated with disease, have entries in the Online Mendelian Inheritance in Man database (OMIM). It was found that 32%, 23%, and 28% of conserved ER stress genes in the study (early response, late response, and association, respectively) have orthologs with entries in OMIM and contribute to human disease. Many diseases are affected by these ER stress genes. For example, orthologs of genes associated with neurological disease were identified in the microarray and association study, including RhoGEF3 (human gene: ARHGEF9; disease: epileptic encephalopathy), CG7804 (TARDBP; ALS), CG10420 (SIL1; Marinesco–Sjogren syndrome), Tsp29Fb (TSPAN7; X-linked mental retardation), CG13423 (BLMH; Alzheimer’s disease), and Cbs (CBS; homocystinuria). Orthologs of genes associated with cancer were also identified, including l(2)37Cc (PHB; breast cancer), and RunxA (RUNX1; leukemia). Further, orthologs involved in diabetes, metabolic disorders, and developmental disorders were identified. Knowledge of the role of these genes in ER stress may lead to a better understanding of how ER stress contributes to the pathophysiology of their associated diseases (Chow, 2013).
Moustaqim-Barrette, A., Lin, Y.Q., Pradhan, S., Neely, G.G., Bellen, H.J. and Tsuda, H. (2014). The amyotrophic lateral sclerosis 8 protein, VAP, is required for ER protein quality control. Hum Mol Genet 23: 1975-1989. PubMed ID: 24271015
Defects in ER proteostasis have been implicated in the pathology of ALS. The ER constantly requires maintenance of protein homeostasis, or proteostasis. To this end, the ER carries the burden of continuously modulating protein folding and degradation to avoid accumulation of misfolded proteins. An overloaded ERQC induces ER stress, and severe or prolonged ER stress leads to cell dysfunction and eventually cell death. Various proteins involved in ERQC have been identified, including VCP, Derlin-1 and Calreticulin. Moreover, a familial form of ALS is associated with mutations in VCP and mutations in SOD1 have been shown to inhibit the function of Derlin-1 and reduce the levels of Calreticulin and trigger ER stress. In SOD1 mice, the ER stress seems to contribute to selective motor neuron degeneration and ER stress has also been implicated in numerous patients with sporadic ALS. Together, these observations suggest that defects in ER proteostasis might be a common pathological feature of ALS. In many neurodegenerative diseases, only a subpopulation of neurons is initially targeted. However, in most cases the disease-causing proteins are ubiquitously expressed. Why some regions of the brain are more susceptible to defects remains unknown. Specific defects in some subtypes of neurons in the vap null mutants were found. The defects are not limited to motor neurons. Interestingly, VapB levels decrease concomitantly with the disease's progression in the SOD1 mouse model, and sporadic ALS patients have been reported to have decreased levels of the VapB protein, suggesting that impaired VapB function may contribute to the pathogenesis of familial and sporadic forms of ALS. It is therefore likely that the molecular mechanism by which loss of Vap affects the cells is not restricted to ALS8 (Moustaqim-Barrette, 2014).
An important question raised by results of this study is how VapB functions in protein homeostasis in the ER. The ERQC is involved in identifying aberrantly misfolded proteins, retrotranslocating the misfolded proteins and processing the degraded retrotranslocated proteins. These processes seem to be tightly linked. Indeed, many proteins function in multiple different steps in the ERQC. However, Vap is unlikely to function in chaperone-dependent refolding, since a molecular chaperone, Bip, is upregulated in vap null mutants. Moreover, Bip overexpression fails to rescue the ER stress in the vap null mutants (Moustaqim-Barrette, 2014).
Restoring the levels of an Osbp that is not dependent on Vap binding suppresses the ER defects caused by loss of Vap, suggesting that this pathway is required for the execution of the ERQC. Mammalian Osbp and Osbp-related protein (Orp) constitute a large eukaryotic gene family characterized by a conserved C-terminal sterol binding domain. This domain organization suggests that a primary function of Osbp/Orp is to sense cholesterol or oxysterols to control transport between target membranes. Differential localization of Osbp between organelles in response to exogenous and endogenous sterol ligands indicates that Osbps transfer cholesterol and/or oxysterols between these organelles. Although the ER membrane is cholesterol poor (3–6% of total lipids), acute cholesterol depletion in culture medium impairs the mobility of membrane proteins and protein secretion from the ER in cultured cells. Hence, the defects in ER proteostasis might be due to decreased levels of cholesterol in the ER caused by loss of Osbp/Vap function. In addition, Osbps are also coupled to the activation of Cert and SM synthesis through increased activity of PI4KIIα, a cholesterol sensitive PI 4-kinase. This suggests that loss of Vap may also cause defects in ceramide transport from the ER, which may result in accumulation of ceramide and defects in ER proteostasis, consistent with the observation that ceramide accumulates in ER stress (Moustaqim-Barrette, 2014).
This study documents the defects caused by mutant VapALS8 protein expressed under the control of its endogenous regulatory elements in a genomic rescue construct. Previous studies suggest that the VapALS8 protein causes dominant negative defects when the mutant protein is overexpressed in flies or cultured cells. However, overexpression of VapALS8 rescues the lethality associated with the vap null mutant and the data presented in this study based on the vapALS8 genomic transgene strongly suggests that the defects caused by the ALS8 mutant protein are hypomorphic rather than dominant negative. Consistent with these findings, another study reports two mutations (A145 V and S160Δ) in the vapB gene in ALS patients; these mutations (A145 V and S160Δ) partially fail to rescue the defects caused by loss of vapB in zebrafish. This study therefore proposes that the ALS8 mutation in patients is a partial loss of function mutation and that vapB is haploinsufficient in humans (Moustaqim-Barrette, 2014).
Appocher, C., Klima, R. and Feiguin, F. (2014). Functional screening in Drosophila reveals the conserved role of REEP1 in promoting stress resistance and preventing the formation of Tau aggregates. Hum Mol Genet 23: 6762-6772. PubMed ID: 25096240
In agreement with this view, the human homolog protein REEP1 in flies was identified and demonstrated to be a conserved protein that localizes in the tubule-vesicular membranes of the ER where it promotes the intracellular resistance against stressful situations like heat shock or the abnormal accumulation of misfolded proteins in the cytoplasm. In agreement with these results, it has been previously reported that HVA22, the plant homolog gene of D-reep1, is required to tolerate stressful situations by inhibiting the activation of programmed cell death in plants growing under adverse conditions. In Drosophila, it was found that D-reep1 null alleles do not present evident problems during development or neurological defects associated with aging or alterations in the life span. Similar results have been observed in yeast, where null mutations in the D-reep1 homolog gene of YOP1p did not affect cells viability. On the contrary, the suppression of REEP1 in mice interferes with the ER structure and reduces the long-term neuronal survival, indicating that species-specific differences in the gene function may exist. Regarding to that, it was observed that D-reep1 and human REEP1 are identically localized in the ER of Drosophila and required to confer stress resistance against the accumulation of unfolded proteins induced by tunicamycin. In correlation with these data, it was found that D-reep1 function is necessary to prevent Tau-mediated neurodegeneration and aggregates formation, suggesting that these results might be connected. Concerning this hypothesis, it was found that increased ER stress enhances the neurodegenerative effect produced by Tau expression in vivo. Moreover, it was detected that modifications in Tau levels increase the expression amounts of D-reep1 mRNA, indicating that D-reep1 may form part of the cellular machinery in charged to respond, signal or protect against ER stress in vivo (Appocher, 2014).
Regarding the potential mechanisms behind neuronal protection, it was found that alterations in D-reep1 or REEP1 function do not affect the total levels of Tau present in Drosophila heads suggesting that these proteins might be required to prevent the formation or accumulation of Thioflavin-S-positive Tau clusters rather than promoting the degradation of the aggregates. Nevertheless, a potential role of D-reep1 in regulating the removal of the Tau insoluble clusters through ER-dependent autophagy could not be discarded; further experiments will be necessary to clarify these issues. In summary, this study uncoveres a novel and conserved function of the D-reep1 gene in promoting ER stress resistance and preventing Tau-mediated degeneration in vivo, most probably by inhibiting the pathological accumulation of Thioflavin-S-positive Tau aggregates. Finally, the study also predicts that defects in REEP1 function, in patients with HSP, may lead to alterations in the neuronal responses to ER stress with defects in the metabolism of Tau and general problems in the maintenance of the structural organization and intracellular transport in long axons (Appocher, 2014).
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
The consequences of perturbing ER homeostasis in the intestinal epithelium are reminiscent of similar effects in Xbp1-deficient mice, where loss of Xbp1 promotes ISC proliferation and intestinal tumorigenesis. At the same time, a recent study suggests that UPRER components are primarily expressed in transit amplifying cells of the intestinal epithelium, and that activation of the UPRER (specifically the PERK branch) promotes differentiation of intestinal epithelial stem cells. The Drosophila midgut epithelium does not contain a transit amplifying cell population, yet data suggest that a role for the UPRER in the control of ISC activity is conserved (Wang, 2014).
The requirement for CncC inhibition in ER stress-mediated activation of ISC proliferation highlights the integrated control of ISC activity by oxidative and ER stress signals. The study proposes that Xbp1, by promoting ER homeostasis, limits ROS accumulation in ISCs and thus maintains ISC quiescence. Excessive ROS results in JNK activation, which in turn activates Fos and inhibits CncC in ISCs, triggering proliferation (Wang, 2014).
This coordination of ER and oxidative stress responses by CncC and the UPRER is likely to be complex. In C. elegans the UPRER coordinates transcriptional regulation of anti-oxidant genes with the CncC homologue SKN-1. Interestingly, SKN-1 can also directly control the expression of UPRER components (including Xbp1, ATF-6 and Bip) by binding to their promoter regions, independent of oxidative stress. Studies in worms have further established the UPRER as a critical determinant of longevity, and Xbp1 extends lifespan by improving ER stress resistance. Strikingly, local activation of the UPRER can trigger UPRER responses in distant tissues, indicating that endocrine processes exist that coordinate such stress responses across cells and tissues. Results support the notion that improving proteostasis by boosting ER folding capacity improves long-term tissue homeostasis. These effects seem to be largely mediated by cell-autonomous integration of the UPRER and redox response by JNK and CncC, but non-autonomous effects of ER stress on ISC proliferation are also observed when knocking down Xbp1 in EBs or ECs selectively. Furthermore, JNK is activated broadly in the intestinal epithelium when Xbp1 or Hrd1 are knocked down in ISCs and EBs, suggesting that non-autonomous interactions between cells experiencing ER stress also influence the regenerative response of this tissue. The molecular events regulating the coordination between cell-autonomous and non-autonomous events in the ER stress response of ISCs are subject of current investigation (Wang, 2014).
In the small intestine of mice, the UPRER influences regenerative activity not only by influencing ISCs and transit amplifying cells directly, but also by influencing intestinal immune homeostasis. Loss of Xbp1 in intestinal epithelial cells (IECs) leads to apoptosis of secretory Paneth cells and goblet cells, and this pathology is associated with inflammation and higher risk of IBD. Deregulation of innate immune responses by the UPRER is also found in human patients, as well as in C. elegans. It can therefore be anticipated that the age-related increase in ER stress in the fly intestine also influences innate immune homeostasis and may contribute to commensal dysbiosis, which has been recently shown to be a driving factor in the age-related loss of proliferative homeostasis of the fly intestine. It will be intriguing to dissect the interaction between the UPRER machinery, innate immune signaling in ECs, commensal homeostasis and stem cell function in detail, and it can be anticipated that these interactions have a significant effect on overall lifespan of the organism (Wang, 2014).
Liang, C.J., Chang, Y.C., Chang, H.C., Wang, C.K., Hung, Y.C., Lin, Y.E., Chan, C.C., Chen, C.H., Chang, H.Y. and Sang, T.K. (2014). Derlin-1 regulates mutant VCP-linked pathogenesis and endoplasmic reticulum stress-induced apoptosis. PLoS Genet 10: e1004675. PubMed ID: 25255315
Zacharogianni, M., Gomez, A.A., Veenendaal, T., Smout, J. and Rabouille, C. (2014). A stress assembly that confers cell viability by preserving ERES components during amino-acid starvation. Elife 3. PubMed ID: 25386913
Ryoo, H.D., Li, J. and Kang, M.J. (2013). Drosophila XBP1 expression reporter marks cells under endoplasmic reticulum stress and with high protein secretory load. PLoS One 8: e75774. PubMed ID: 24098723
Plongthongkum, N., Kullawong, N., Panyim, S. and Tirasophon, W. (2007). Ire1 regulated XBP1 mRNA splicing is essential for the unfolded protein response (UPR) in Drosophila melanogaster. Biochem Biophys Res Commun 354: 789-794. PubMed ID: 17266933
Ryoo, H.D. (2015). Drosophila as a model for unfolded protein response research. BMB Rep [Epub ahead of print]. PubMed ID: 25999177
Anholt, R.R. and Carbone, M.A. (2013). A molecular mechanism for glaucoma: endoplasmic reticulum stress and the unfolded protein response. Trends Mol Med 19: 586-93. PubMed ID: 23876925
PERK limits Drosophila lifespan by promoting intestinal stem cell proliferation in response to ER stress
An orchestrated program regulating secretory pathway genes and cargos by the transmembrane transcription factor CREB-H
Ubiquitin conjugating enzymes and the degradation of ER membrane proteins
Date revised: 13 August 2015
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