Protein Kinase R-like Endoplasmic Reticulum Kinase Activity Research Articles

Astaxanthin inhibits apoptosis in a cell model of tauopathy by attenuating endoplasmic reticulum stress and unfolded protein response

The accumulation of misfolded proteins is a common pathological characteristic shared by many neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. The disruption of proteostasis triggers endoplasmic reticulum (ER) stress, during which the unfolded protein response (UPR) is initiated by the activation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). These three branches of UPR signals act in concert to reduce the levels of abnormal proteins and restore ER homeostasis. However, the overactivation of UPR impairs cell function and induces apoptosis, which has been implicated in neurodegeneration. Astaxanthin is a xanthophyll carotenoid which has been shown to have neuroprotective effects in both cell and animal models; however, its effects on ER stress and UPR induced by disrupted proteostasis remain unclear. In this study, the effects of astaxanthin on ER stress and cytotoxicity were investigated in N2a cells stably expressing the pro-aggregant tau repeat domain carrying FTDP-17 mutation ΔK280 (Tau4RDΔK280). The results demonstrated that astaxanthin significantly inhibited Tau4RDΔK280-induced loss of cell viability and apoptosis, attenuating Tau4RDΔK280-induced caspase-3 activation and decrease of Bcl-2. Further studies revealed that astaxanthin treatment alleviated Tau4RDΔK280-induced ER stress and suppressed the activation of PERK, IRE1 and ATF6 signaling pathways. These findings suggested that astaxanthin might inhibit Tau4RDΔK280-induced cytotoxicity by attenuating UPR and ER stress. In addition, astaxanthin treatment resulted in a great reduction in the production of intracellular reactive oxygen species and a significant decrease in calcium influx induced by Tau4RDΔK280, which also contributed to the protective effects of astaxanthin against Tau4RDΔK280-induced cytotoxicity.

VPS34-IN1 induces apoptosis of ER+ breast cancer cells via activating PERK/ATF4/CHOP pathway

VPS34-IN1 is a specific selective inhibitor of Class III Phosphatidylinositol 3-kinase (PI3K) and has been shown to exhibit a significant antitumor effect in leukemia and liver cancer. In current study, we focused on the anticancer effect and potential mechanism of VPS34-IN1 in estrogen receptor positive (ER+ ) breast cancer. Our results revealed that VPS34-IN1 inhibited the viability of ER+ breast cancer cells in vitro and in vivo. Flow cytometry and western blot analyses showed that VPS34-IN1 treatment induced breast cancer cell apopotosis. Interestingly, VPS34-IN1 treatment activated protein kinase R (PKR)-like ER kinase (PERK) branch of endoplasmic reticulum (ER) stress. Furthermore, knockdown of PERK by siRNA or inhibition of PERK activity by chemical inhibitor GSK2656157 could attenuate VPS34-IN1-mediated apoptosis in ER+ breast cancer cells. Collectively, VPS34-IN1 has an antitumor effect in breast cancer, and it may result from activating PERK/ATF4/CHOP pathway of ER stress to induce cell apoptosis. These findings broaden our understanding of the anti-breast cancer effects and mechanisms of VPS34-IN1 and provide new ideas and reference directions for the treatment of ER+ breast cancer.

Abstract 267: Vascular Smooth Muscle Cell-specific Deletion Of Protein Kinase R-like Endoplasmic Reticulum Kinase Attenuates Microbiome-enhanced Abdominal Aortic Aneurysm

Background: We have previously linked the microbiome-derived metabolite trimethylamine N-oxide (TMAO) to abdominal aortic aneurysm (AAA) in both associative human and mechanistic mouse studies. TMAO is reported to bind protein kinase R-like endoplasmic reticulum kinase (PERK), resulting in selective activation of the unfolded protein response (UPR). Concordantly, our previous RNA sequencing results indicate that TMAO augments PERK-mediated UPR pathways in the aneurysm wall. Thus, the current study will determine the functional role of TMAO-mediated PERK activation in aortic VSMCs to AAA pathogenesis. Methods & Results: Ldlr -/- mice with VSMC-specific deletion of Perk were produced by breeding hemizygous male mice expressing Cre under the control of the Transgelin promoter ( B6.Cg-TG(Tagln-cre)1Her/J , Jackson Labs) to female Perk floxed mice ( Eif2ak3 tm1.2Drc /J , Jackson Labs). Specific deletion of PERK in VSMCs was validated by western blot. Male mice were subjected to angiotensin II (AngII) infusion via osmotic mini pump (1,000 ng/kg/min; 28 days), while female mice underwent laparotomy and application of topical elastase (10 mg/mL porcine pancreatic elastase 5 minutes) to induce AAA. To augment circulating TMAO levels, mice were fed a high choline diet (0.2% total cholesterol + 1.2% choline supplementation) for one week prior to and throughout the study. Male mice with VSMC specific PERK knockout demonstrated significantly reduced AngII-induced aortic dilation (Cre+: 0.90 ± 0.048 mm; Cre-: 1.57 ± 0.156 mm; P = 0.001) and rupture induced death relative to control. Similarly, female mice with VSMC specific PERK knockout displayed significantly reduced aortic diameter (Cre+: 1.17 ± 0.069 mm; Cre-: 2.65 ± 0.432 mm; P = 0.002) and rupture induced mortality as compared to control. Additionally, male and female mice with VSMC specific PERK knockout had significantly greater Type I collagen and reduced macrophage accumulation compared to control. Conclusions: These results indicate that VSMC specific knockout of PERK blunts AAA formation in two independent murine models. Further studies are needed to elucidate the mechanism of PERK mediated ER stress in AAA, which may reveal novel therapeutic targets for AAA treatment.

Acute Pharmacological Inhibition of Protein Kinase R-Like Endoplasmic Reticulum Kinase Signaling After Spinal Cord Injury Spares Oligodendrocytes and Improves Locomotor Recovery.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is a major signal transducer of the endoplasmic reticulum stress response (ERSR) pathway. Outcomes of PERK activation range from abrogating ER stress to induction of cell death, dependent on its level, duration, and cellular context. Current data demonstrate that after mouse spinal cord injury (SCI), acute inhibition of PERK (0-72 h) with the small molecule inhibitor GSK2656157 reduced ERSR while improving white matter sparing and hindlimb locomotion recovery. GSK2656157-treated mice showed increased numbers of oligodendrocytes at the injury epicenter. Moreover, GSK2656157 protected cultured primary mouse oligodendrocyte precursor cells from ER stress-induced cytotoxicity. These findings suggest that in the context of SCI, excessive acute activation of PERK contributes to functionally relevant white matter damage. Pharmacological inhibition of PERK is a potential strategy to protect central nervous system (CNS) white matter following acute injuries, including SCI.

Neurodegeneration risk factor, EIF2AK3 (PERK), influences tau protein aggregation

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms-unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling invitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.

Promotion of osteogenesis in BMSC under hypoxia by ATF4 via the PERK-eIF2α signaling pathway.

Mandibular distraction osteogenesis (MDO) is an endogenous tissue engineering technology in which bone marrow mesenchymal stem cells (BMSC) play a key role in MDO-related osteogenesis. Activating transcription factor 4 (ATF4) is involved in osteogenesis through activation of PERK (Protein kinase R-like endoplasmic reticulum kinase) in endoplasmic reticulum stress (ERS) condition under hypoxia. However, the specific role of ATF4 in MDO with BMSC remains unknown. The aim of this study was to explore the effects of ATF4 in MDO with BMSC under hypoxia. Briefly, canine BMSCs were cultured in a hypoxic chamber, and effects of hypoxia were evaluated using cell migration assay and Alizarin Red S staining. Expression levels of protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2α, ATF4, osteocalcin, and bone sialoprotein were evaluated using quantitative polymerase chain reaction and western blotting. BMSCs were transduced with the ATF4-small interfering RNA lentivirus. The effects were evaluated using all the aforementioned experiments. The results showed that hypoxia promoted migration, osteoblast differentiation, and ATF4 expression in BMSC. ATF4 knockdown in BMSC significantly inhibited migration and osteoblast differentiation abilities, while hypoxia reversed these effects to some extent. In addition, the molecular mechanism partly depended on the ERS signaling pathway, with ATF4 as the key factor. In summary, we presented a novel mechanism of ATF4-mediated regulation of BMSC under hypoxia.

Role of the Interaction Between NCK1 and PERK in Pancreatic Beta-cell Function and Survival

Preventing pancreatic β-cell failure and death, features of diabetes pathogenesis, could help treat diabetes. PERK (Protein Kinase R–Like Endoplasmic Reticulum Kinase), a player in the unfolded protein response, is essential for β-cell function and survival. PERK activity is modulated by binding of the adaptor protein NCK1 (Non-Catalytic region of tyrosine Kinase). Silencing of Nck1 in β cells in vitro enhances basal PERK activity and prevents its pathological hyperactivation under diabetogenic stresses, improving β-cell function and survival.

Abstract LB014: Carbon monoxide induced PERK regulated autophagy reprograms anti-tumor T cells with robust immunometabolic phenotype

Abstract Mitochondria and Endoplasmic reticulum (ER) share structural and functional networks to maintain cellular homeostasis and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS condition drives mitochondrial biogenesis and function. So far, various strategies have been tested to enhance the mitochondrial function in T cells to improve their anti-tumor potential. However, strategies targeting ER-Mitochondria crosstalk (by employing moderate ERS condition) to boost the anti-tumor T cell function have not been exploited yet. Thus, in the present study, we used carbon monoxide (CO), a short-lived gaseous molecule, to test if engaging moderate ERS conditions can improve T cells mitochondrial biogenesis, function, and anti-tumor therapeutic efficacy. Using melanoma antigen gp100 reactive T cells, we identify that CO induced transient activation of ERS sensor ‘protein kinase R-like endoplasmic reticulum kinase (PERK)’ dramatically increase anti-tumor T cell function. Furthermore, mechanistically we found that CO-induced PERK activation temporarily halts protein translation and induces protective autophagy (that includes mitophagy). To get further insight into how CO treatment kinetically regulates autophagic activity in T cells, we used LC3-GFP to differentiate between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter into the process (LC3-GFPneg). Using Ag-specific LC3-GFP expressing T cells, we demonstrate that LC3-GFPpos T cells show robust anti-tumor potential. Moreover, LC3-GFPneg shows a T regulatory-like phenotype, harbors dysfunctional mitochondria, and accumulates abnormal metabolite content. These anomalous ratios of metabolites render the cells with a hypermethylated state and distinct epigenetic profile limiting their anti-tumor activity. Overall, this preclinical data highlights that ERS-mediated activation of autophagy pathways modifies the mitochondrial function and epigenetically reprogram the T cells towards a superior anti-tumor phenotype and can be therapeutically translated to achieve robust tumor control in adoptive transfer therapies. Citation Format: Paramita Chakraborty, Rasesh Y. Parikh, Seungho Choi, Meenal Mehrotra, Eduardo N. Maldonado, Hongjun Wang, J. Alan Diehl, Vamsi K. Gangaraju, Shikhar Mehrotra. Carbon monoxide induced PERK regulated autophagy reprograms anti-tumor T cells with robust immunometabolic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB014.

Targeting Autophagy with Carbon-monoxide Reprograms Anti-Tumor T Cells with Robust Immunometabolic Phenotype

Abstract Mitochondria and Endoplasmic reticulum (ER) share structural and functional networks and activates well-orchestrated signaling processes to shape a cell’s fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS condition promotes mitochondrial function. Strategies to boost anti-tumor T cell function by targeting ER-Mitochondria cross talk have not been exploited yet. Thus, we used carbon monoxide (CO), a short-lived gaseous molecule, to test if engaging moderate ERS conditions can improve T cells mitochondrial function and anti-tumor function. Using melanoma antigen specific T cells, we identify that CO induced transient activation of ERS sensor ‘protein kinase R-like endoplasmic reticulum kinase (PERK)’ dramatically increase anti-tumor T cell function. Furthermore, CO-induced PERK activation temporarily halts protein translation and induces protective autophagy (that includes mitophagy). To get further insight, we used LC3-GFP to differentiate between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter into the process (LC3-GFPneg). We found that LC3-GFPpos T cells show robust anti-tumor potential whereas, LC3-GFPneg cells show T regulatory-like phenotype, harbor dysfunctional mitochondria, and accumulate abnormal metabolite content. These anomalous ratios of metabolites render the cells with a hypermethylated state and distinct epigenetic profile limiting their anti-tumor activity. Overall, the study highlights that ERS activated autophagy pathways modify mitochondrial function and epigenetically reprogram the T cells towards a superior anti-tumor phenotype to achieve robust tumor control.

Carbon Monoxide Activates PERK-Regulated Autophagy to Induce Immunometabolic Reprogramming and Boost Antitumor T-cell Function.

Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

Activation of the Protein Kinase R-Like Endoplasmic Reticulum Kinase (PERK) Pathway of the Unfolded Protein Response after Experimental Traumatic Brain Injury and Treatment with a PERK Inhibitor.

Neurodegeneration after traumatic brain injury (TBI) is increasingly recognized as a key factor contributing to poor chronic outcomes. Activation (i.e., phosphorylation) of the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway has been implicated in neurodegenerative conditions with pathological similarities to TBI and may be a potential target to improve TBI outcomes. Here, we aimed to determine whether a moderate TBI would induce activation of the PERK pathway and whether treatment with the PERK inhibitor, GSK2606414, would improve TBI recovery. Male mice were administered a lateral fluid percussion injury (FPI) or sham injury and were euthanized at either 2 h, 24 h, or 1 week post-injury (n = 5 per injury group and time point) to assess changes in the PERK pathway. In the injured cortex, there was increased phosphorylated-PERK at 2 h post-FPI and increased phosphorylation of eukaryotic translation initiation factor α at 24 h post-FPI. We next examined the effect of acute treatment with GSK2606414 on pathological and behavioral outcomes at 4 weeks post-injury. Thus, there were a total of four groups: sham + VEH (n = 9); sham + GSK4606414 (n = 10); FPI + VEH (n = 9); and FPI + GSK2606414 (n = 9). GSK2606414 (50 mg/kg) or vehicle treatment was delivered by oral gavage beginning at 30 min post-injury, followed by two further treatments at 12-h increments. There were no significant effects of GSK2606414 on any of the outcomes assessed, which could be attributable to several reasons. For example, activation of PERK may not be a significant contributor to the neurological consequences 4 weeks post-FPI in mice. Further research is required to elucidate the role of the PERK pathway in TBI and whether interventions that target this pathway are beneficial.

Elucidation of the mechanism of NEFA-induced PERK-eIF2α signaling pathway regulation of lipid metabolism in bovine hepatocytes

During the periparturient transition period, negative energy balance (NEB) characterized by high concentrations of non-esterified fatty acids (NEFA) may cause fatty liver and ketosis in dairy cows. Previous studies have shown that the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the endoplasmic reticulum stress (ERS) response plays an important role in lipid metabolism in hepatocytes. This study, therefore, investigated the role of the PERK-branch in NEFA-induced fatty liver. Different concentrations of NEFA or GSK2656157 (a novel catalytic inhibitor of PERK) were used to treat hepatocytes isolated from calves. The NEFA treatment significantly increased the triacylglycerol (TG) content, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the abundance of glucose-regulated protein 78 (Grp78), C/EBP homologous protein (CHOP), sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FASN), peroxisome proliferator-activated receptor-α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), apolipoprotein B (APOB), and the low-density lipoprotein receptor (LDLR). Compared with the 1.2 mM NEFA group, inhibition of PERK activity further increased the TG content in hepatocytes, the very-low-density lipoprotein (VLDL) content in the supernatant and the protein abundance of APOB while reducing the expression and nuclear levels of SREBP-1c and PPARα, as well as the expression of CPT1A and CPT2. In conclusion, the results showed that the NEFA-induced PERK-eIF2α signaling pathway promotes lipid synthesis, lipid oxidation, but inhibits the assembly and secretion of VLDL. Therefore, during the transition period, the activation of the PERK-eIF2α signaling pathway in the liver of dairy cows could defeat the acid-induced lipotoxicity and provide energy to alleviate NEB.

Helicobacter Pylori Infection Triggers PERK-Associated Survivin Loss in Gastric Tissue Samples and Cell Lines

Infection by Helicobacter pylori (Hp) is the main risk factor associated with the development and progression of precancerous lesions to gastric cancer. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated by endoplasmic reticulum (ER) stress, and ER stress-induced apoptotic cell death has been associated with Hp infection. Survivin, an inhibitor of apoptosis, is downregulated in the mucosa of Hp infected subjects and loss of survivin in gastric cells correlates with reduced viability. Here, we determined whether Hp-induced changes in PERK contribute to the loss of survivin, previously associated with the genesis of gastric cancer precancerous lesions. Our results show that PERK is activated in the early stages of Hp infection in the human gastric mucosa affected by gastritis and PERK activation coincided with reduced survivin levels compared with Hp-negative gastritis. Upon Hp infection in vitro, PERK silencing restored survivin abundance and its activity increased survivin loss, in parallel with a partial downstream activation of the α- subunit of eukaryotic initiation factor-2 (eIF2α). Our results suggest a novel mechanism by which Hp-stimulated PERK reduces gastric cell viability/proliferation by downregulating survivin and is likely to favour the genesis of gastric cancer precancerous lesions. Supplementary-file

Chromosome 22q11.2 deletion causes PERK-dependent vulnerability in dopaminergic neurons.

BackgroundThe chromosome 22q11.2 deletion is an extremely high risk genetic factor for various neuropsychiatric disorders; however, the 22q11.2 deletion-related brain pathology in humans at the cellular and molecular levels remains unclear.MethodsWe generated iPS cells from healthy controls (control group) and patients with 22q11.2 deletion (22DS group), and differentiated them into dopaminergic neurons. Semiquantitative proteomic analysis was performed to compare the two groups. Next, we conducted molecular, cell biological and pharmacological assays.FindingsSemiquantitative proteomic analysis identified ‘protein processing in the endoplasmic reticulum (ER)’ as the most altered pathway in the 22DS group. In particular, we found a severe defect in protein kinase R-like endoplasmic reticulum kinase (PERK) expression and its activity in the 22DS group. The decreased PERK expression was also shown in the midbrain of a 22q11.2 deletion mouse model. The 22DS group showed characteristic phenotypes, including poor tolerance to ER stress, abnormal F-actin dynamics, and decrease in protein synthesis. Some of phenotypes were rescued by the pharmacological manipulation of PERK activity and phenocopied in PERK-deficient dopaminergic neurons. We lastly showed that DGCR14 was associated with reduction in PERK expression.InterpretationOur findings led us to conclude that the 22q11.2 deletion causes various vulnerabilities in dopaminergic neurons, dependent on PERK dysfunction.FundingThis study was supported by the 10.13039/100010463AMED under grant nos JP20dm0107087, JP20dm0207075, JP20ak0101113, JP20dk0307081, and JP18dm0207004h0005; the MEXT KAKENHI under grant nos. 16K19760, 19K08015, 18H04040, and 18K19511; the 10.13039/100008732Uehara Memorial Foundation under grant no. 201810122; and 2019 iPS Academia Japan Grant.

Expression of cancer cell-associated Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) regulates protective anti-tumor immunity in melanoma

Abstract Activation of moderated endoplasmic reticulum (ER) stress in cancer cells promotes cellular adaptation and survival in response to the adverse conditions of the tumor microenvironment (TME). However, how modulation of ER stress in cancer cells culminates in the development of an immunosuppressive TME remains poorly understood and therapeutically unexploited. Upon disruption of ER homeostasis, mediator proteins IRE1a, ATF6 and PERK induce activation of the unfolded protein response (UPR). Although maladaptive priming of UPR mediators in immune cells deactivates their capacity for anti-tumor immunity, the immunosuppressive potential of UPR mediators in cancer cells, in particular PERK, remains unknown. Thus, in this study, we hypothesized that activation of PERK in tumor cells shapes the formation of the immunosuppressive TME and impairs the development of anti-tumor immunity. Elimination of PERK in cancer cells impaired survival upon chemically-induced ER stress and promoted caspase-independent cell death. Interestingly, in vivo ablation of PERK in melanoma impaired tumor growth, which surprisingly occurred in a T cell-dependent manner. Additionally we observed that ablation of PERK shifted the TME from an immunosuppressive to immunostimulatory environment, enhanced accumulation of effector T cells and directed reprogramming of myeloid derived suppressor cells (MDSCs) into immunostimulatory cells. Additional experiments indicated that ablation of PERK induced expression of mediators associated with immunogenic cell death. Collectively, our findings demonstrate the immunoregulatory role of PERK in tumor cells and highlight the therapeutic potential of targeting UPR mediators and tumor adaptive processes.

Autophagy Induced due to Moderate Endoplasmic Reticulum Stress in T Cells Enhances Immunotherapeutic Tumor Control

Abstract A number of external or internal stimuli cause endoplasmic reticulum (ER) stress conditions. In response to ER stress, stressed cells activate well-orchestrated cellular signaling processes to resolve the stress or remove itself by triggering apoptosis. In the tumor microenvironment, induction of sustained ER stress is known to dampen the antitumor activity of T cells. However, how T cells respond to suboptimal ER stimuli is unknown. Using melanoma antigen gp100 reactive T cells, we found that T cells transiently activate protein kinase R-like endoplasmic reticulum kinase (PERK) dependent signaling network to ameliorate low level of ER stress. While transient PERK activation in T cell enhances stemness, promotes mitochondrial biogenesis, increases T cells anti-tumor immunity in B16 melanoma bearing mouse, overexpression of PERK triggers T cell dysfunctionality and death. Mechanistically, we discovered that transient activation of PERK decreases overall protein translation and induces protective autophagy to prevent cell damage. Conversely, knockdown of PERK abrogates autophagy activation, hampers mitochondrial biogenesis in response to suboptimal ER stress, which in turn compromises antitumor function. Furthermore, blocking autophagy in T cells hampers T cell anti-tumor activity. Lastly, using gp100 reactive LC3-GFP expressing T cells we demonstrated that, T cells that initiate autophagy due to suboptimal ER stress show better anti-tumor potential compared to those, that fail to enter into the process. Overall, these preclinical data highlight that, both transient ER stress and autophagy pathways are important for healthy T cell function and therapeutically, can be manipulated to regulate their antitumor potential.

The kinase PERK represses translation of the G-protein–coupled receptor LGR5 and receptor tyrosine kinase ERBB3 during ER stress in cancer cells

As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.

Simultaneous Measurement of Metabolite Concentration and Isotope Incorporation by Mass Spectrometry.

Studies of the topology, functioning, and regulation of metabolic systems are based on two main types of information that can be measured by mass spectrometry: the (absolute or relative) concentration of metabolites and their isotope incorporation in 13C-labeling experiments. These data are currently obtained from two independent experiments because the 13C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the 13C-mass fractions from which its 13C-isotopologue distribution (CID) is quantified. Here, we developed a generic method with a dedicated processing workflow to obtain these two sets of information simultaneously in a unique sample collected from a single cultivation, thereby reducing by a factor of 2 both the number of cultivations to perform and the number of samples to collect, prepare, and analyze. The proposed approach is based on an IS labeled with other isotope(s) that can be resolved from the 13C-mass fractions of interest. As proof-of-principle, we analyzed amino acids using a doubly labeled 15N13C-cell extract as IS. Extensive evaluation of the proposed approach shows a similar accuracy and precision compared to state-of-the-art approaches. We demonstrate the value of this approach by investigating the dynamic response of amino acids metabolism in mammalian cells upon activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a key component of the unfolded protein response. Integration of metabolite concentrations and isotopic profiles reveals a reduced de novo biosynthesis of amino acids upon PERK activation. The proposed approach is generic and can be applied to other (micro)organisms, analytical platforms, isotopic tracers, or classes of metabolites.

Evolutionarily Conserved Regulation of Sleep by the Protein Translational Regulator PERK.

Sleep is a cross-species phenomenon whose evolutionary and biological function remain poorly understood. Clinical and animal studies suggest that sleep disturbance is significantly associated with disruptions in protein homeostasis-or proteostasis-in the brain, but the mechanism of this link has not been explored. In the cell, the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway modulates proteostasis by transiently inhibiting protein synthesis in response to proteostatic stress. In this study, we examined the role of the PERK pathway in sleep regulation and provide the first evidence that PERK signaling is required to regulate normal sleep in both vertebrates and invertebrates. We show that pharmacological inhibition of PERK reduces sleep in both Drosophila and zebrafish, indicating an evolutionarily conserved requirement for PERK in sleep. Genetic knockdown of PERK activity also reduces sleep in Drosophila, whereas PERK overexpression induces sleep. Finally, we demonstrate that changes in PERK signaling directly impact wake-promoting neuropeptide expression, revealing a mechanism through which proteostatic pathways can affect sleep and wake behavior. Taken together, these results demonstrate that protein synthesis pathways like PERK could represent a general mechanism of sleep and wake regulation and provide greater insight into the relationship between sleep and proteostasis.

Asparagine Synthetase Is Highly Expressed at Baseline in the Pancreas Through Heightened PERK Signaling.

Asparaginase (ASNase) causes pancreatitis in approximately 10% of leukemia patients, and the mechanisms underlying this painful complication are not known. ASNase primarily depletes circulating asparagine, and the endogenously expressed enzyme, asparagine synthetase (ASNS), replenishes asparagine. ASNS was suggested previously to be highly expressed in the pancreas. In this study, we determined the expression pattern of ASNS in the pancreas and the mechanism for increased pancreatic ASNS abundance. Compared with other organs, ASNS was highly expressed in both the human and mouse pancreas, and, within the pancreas, ASNS was present primarily in the acinar cells. The high baseline pancreatic ASNS was associated with higher baseline activation of protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in the pancreas, and inhibition of PERK in acinar cells lessened ASNS expression. ASNase exposure, but not the common pancreatitis triggers, uniquely up-regulated ASNS expression, indicating that the increase is mediated by nutrient stress. The up-regulation of acinar ASNS with ASNase exposure was owing to increased transcriptional rather than delayed degradation. Knockdown of ASNS in the 266-6 acinar cells provoked acinar cell injury and worsened ASNase-induced injury, whereas ASNS overexpression protected against ASNase-induced injury. In summary, ASNS is highly expressed in the pancreatic acinar cells through heightened basal activation of PERK, and ASNS appears to be crucial to maintaining acinar cell integrity. The implications are that ASNS is especially hardwired in the pancreas to protect against both baseline perturbations and nutrient deprivation stressors, such as during ASNase exposure.