Gadd45 Protein Research Articles

Alternative splicing and characterisation of the tauopathy‐induced gene Growth Arrest DNA Damage Induced 45 alpha

AbstractBackgroundThe Gadd45 family are a set of 3 genes classically induced by DNA damage. They are also induced by wider stresses such as axonal injury, ischemia and degenerative diseases like ALS. Increased Gadd45 protein staining is observed in AD brains, however their association with pathogenic tau is unknown. The Gadd45 genes undergo alternative splicing to generate 8 functionally distinct proteins, whose significance in neuronal systems are poorly understood. We aim to investigate if the Gadd45 family are upregulated in tauopathies and whether this is accompanied by changes to mRNA splicing of these genes, with a view to understand whether these differentially affect cell health.MethodTransgenic mice (rTg4510) over‐expressing and FTD‐associated mutant tau (TauP301L) were used to investigate tau‐associated upregulation of the Gadd45 genes. Subsequently, we analyse which splice variants are present in primary cortical neurons and express plasmid constructs of these variants to investigate their sub‐cellular localisation and function, using apoptotic cell death as a read‐out.ResultGadd45α, but not β or γ members of the same family, is upregulated in response to pathological tau. Untreated cortical neurones express a single isoform of the Gadd45α gene, however upon cellular stress 2 additional isoforms are induced. The identified variants show distinct sub‐cellular distribution, including changes to nuclear localisation and the development of punctate staining. Furthermore, these differentially affect apoptosis.ConclusionGadd45α is selectively upregulated by tau pathology. The gene undergoes alternative splicing in murine neurones in response to cellular stress, and these variants show evidence for distinct functions. We will next investigate the association of these variants with pathological tau.We thank the Alzheimer’s Society for funding this work.

A highly efficient human cell-free translation system.

Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer’s disease mouse model: a role for ER-mitochondria interaction

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer’s disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

Inhibition of the integrated stress response reverses oxidative stress damage-induced postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD) is a common complication following anesthesia and surgery that might lead to a decline in learning and memory. Oxidative stress damage is one of the pathogenic mechanisms underlying POCD. Recent studies had shown that the integrated stress response (ISR) is closely related to oxidative stress. The core response of the ISR is phosphorylation of eIF2α. Various cellular stress stimuli trigger activation of eIF2α kinases, thus causing phosphorylation of eIF2α. ISR is associated with many neurodegenerative diseases; however, the relationship between POCD and ISR has not been defined. In the present study, the tibias in 4-month-old male C57BL/6 mice were fractured under isoflurane anesthesia to establish the POCD animal model. Cognitive function was assessed by fear conditioning tests and the Y-maze from 3 to 14 days post-surgery. Western blot was used to determine the levels of PeIF2α, eIF2α, ATF4, GADD34, CHOP, BDNF, proBDNF, and p-NR2B expression. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured to determine oxidative stress in hippocampal tissues. After tibial fracture surgery in mice, the hippocampus had increased levels of PeIF2α, ATF4, GADD34, and CHOP protein, ROS-positive cells, and average fluorescence intensity, SOD activity was decreased, and the MDA level was increased. The ISR inhibitor, ISRIB, reduced the levels of PeIF2α, ATF4, GADD34, and CHOP protein, and alleviated oxidative stress in the hippocampus of POCD mice. Moreover, ISRIB ameliorated cognitive dysfunction in POCD mice. Our findings suggested that targeting ISR may represent an effective approach to combat POCD.

Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform.

In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS.

Effects of <i>Buyang Huanwu</i> Decoction on Endoplasmic Reticulum Stress-Autophagy Response and GADD34 Expression in Mice with CIRI

<i>Objective</i>: To investigate the effects of <i>Buyang Huanwu Decoction</i> on endoplasmic reticulum stress-autophagic response and the expression of GADD34 in ischemic penumbra of mice with CIRI. <i>Methods</i>: The mice were randomly divided into sham operation group, model group and <i>Buyang Huanwu Decoction</i> low-, medium-, and high-dosage groups. The mice were treated by gavage for 7 days before modeling and 24 hours after modeling. CIRI model was established by the thread embolization method. The neurological function was evaluated on the 48 hours after modeling; TTC staining was used to observe the cerebral infarct volume; HE staining was used to observe the pathological changes of ischemic penumbra; TUNEL was performed to assess cell apoptosis; Western blot was performed to assess the expression of endoplasmic reticulum stress marker protein GRP78 and autophagy marker proteins LC3 and p62; Immunohistochemistry and real-time PCR were applied to examine the protein and mRNA expressions of GADD34. <i>Results</i>: Compared with the model group, neurological function score and volume of cerebral infarction were decreased, reperfusion injury of ischemic penumbra was improved with varying degrees, number of apoptotic cells was decreased, expression of GRP78 and p62 protein were down-regulated while ratio of LC3-II/ LC3 –I was up-regulated, and expression of GADD34 protein and mRNA were up-regulated in each <i>Buyang Huanwu Decoction</i> group, with statistical significance (<i>P</i><0.01). <i>Conclusion Buyang Huanwu Decoction</i> can significantly improve the damaged neurological function, cerebral infarction volume and histopathological damage of ischemic penumbra, as well as reduce the apoptosis cells, which plays a neuroprotective role in mice with CIRI. Its mechanism may be related to up regulating the expression of GADD34, regulating the steady-state balance of endoplasmic reticulum stress autophagy response in ischemic penumbra.

Integrated stress response activation after recovery of cardiopulmonary bypass in rats

The systemic inflammation response syndrome (SIRS) is a consequence of the cardiopulmonary bypass (CPB) in cardiac surgery that can trigger multiple organ dysfunction (MOD). Pulmonary, renal and cerebral dysfunctions are the most common post-operative complications of CPB. The sustained of activation of integrated stress response (ISR) pathway could impact patient outcomes. This project aims to decipher pathophysiological process in SIRS and organ dysfunction after recovery of CPB in a rat model. After isoflurane induction (2%, O 2 : 0.5 L/min) surgical procedure for CPB or Sham was performed under propofol-fentanyl infusion (30–4 μg/kg/h) on mechanically ventilated 15-week old rats. The CPB circuit allows blood to be drawn by the vena cava and reperfused in the aorta after oxygenation. Hemodynamic and biochemical parameters were monitored during the procedure. Mean Arterial Pressure (MAP) was maintained to a minimum of 60 mmHg with a FiO 2 1.0 for 1 h. Five hours post-recovery, behavior, respiratory rate and hemodynamic were measured. Blood and organs were collected to evaluate the integrated stress response pathway. Compared to Sham, CPB leads to a decrease of MAP (88.3 ± 2.6 vs 60.0 ± 9.4 mmHg, P < 0.05, N = 4), an increase of Lactate (1.4 ± 0.4 vs 4.1 ± 0.9 mM, P < 0.05), Creatinine (34.7 ± 1.0 vs 152.7 ± 7.7 μM, P < 0.05), and pro-inflammatory cytokines IL-6 (Sham: 48 ± 24 pg/mL; CPB: 8468 ± 1 pg/mL, P < 0.05). Rat Behavior and reflex, and lung inflammation were significantly altered 5 h after recovery when compared to Sham. ISR pathway activation was shown by western blot on lung, kidney and brain tissues from CPB rats with respectively a 3-, 2- and 3-fold increase in GADD34 protein levels. On our CPB rat model, we highlight organs dysfunctions associated with an activation of the ISR pathway. We will now evaluate the potential impact of ISR activation on MOD.

Induction of reporter genes expression by ionizing radiation and toxins influence on the Drosophila melanogaster genome

In the human environment, there are many factors that can cause genotoxic and mutagenic effects and one of them is ionizing radiation. Radioactive substances like radon, thorium, radium and their decay products during decomposition are able to emit not only highly penetrating gamma radiation, but also form large heavy alpha-particles. Alpha-particles have a low penetrating power, but high ionization energy, about 10 times greater than gamma-rays. Consequently, we researched genotoxic effects of alpha-radiation using a test-system with reporter genes on the Drosophila melanogaster. In nature, the main source of alpha-particles is colorless radioactive gas radon, its isotopes and daughter decay products. It is technically difficult to create sufficiently intense and directed beams of α-radiation on the long-lived radon isotope – 222Rn. Therefore, for experiments was chosen the 238Pu (Plutonium) isotope, which has the necessary α-line at decay equal to 5.5 MeV. The genotoxic activity of alpha-particles was tested on Drosophila melanogaster with genotype containing genes of luminous proteins. Working of the genetic construction based on the GAL4-UAS system, often used in research to determine gene expression. Genotype also included GADD45 protein gene and GFP gene (green fluorescent protein). This genes combination allows to observe glow of Drosophila organs and tissues after stress factor impact. The sensitivity of construction was checked by chemical mutagen cisplatin, which led to the larvae death at high concentrations, and caused glow of organs at low concentrations. The larvae were exposed to alpha-radiation for 20 hours. Preparations were analyzed on a light microscope with a blue filter and glow of the larval organs was observed. The glow intensity was depending from radiation source. In other words, higher radiation dose caused brighter glow, which correlates with synthesis of luminous protein level. This proves that alpha-radiation from radon and its decay daughter products has a genetic effect. Keywords: alpha radiation, radon, Drosophila, GFP, genotoxicity.

Индукция экспрессии репортерных генов при воздействии ионизирующей радиации и токсинов на геном Drosophila melanogaster

In the human environment, there are many factors that can cause genotoxic and mutagenic effects and one of them is ionizing radiation. Radioactive substances like radon, thorium, radium and their decay products during decomposition are able to emit not only highly penetrating gamma radiation, but also form large heavy alpha-particles. Alpha-particles have a low penetrating power, but high ionization energy, about 10 times greater than gamma-rays. Consequently, we researched genotoxic effects of alpha-radiation using a test-system with reporter genes on the Drosophila melanogaster. In nature, the main source of alpha-particles is colorless radioactive gas radon, its isotopes and daughter decay products. It is technically difficult to create sufficiently intense and directed beams of α-radiation on the long-lived radon isotope – 222Rn.&nbsp; Therefore, for experiments was chosen the 238Pu (Plutonium) isotope, which has the necessary α-line at decay equal to 5.5 MeV. The genotoxic activity of alpha-particles was tested on Drosophila melanogaster with genotype containing genes of luminous proteins. Working of the genetic construction based on the GAL4-UAS system, often used in research to determine gene expression. Genotype also included GADD45 protein gene and GFP gene (green fluorescent protein). This genes combination allows to observe glow of Drosophila organs and tissues after stress factor impact. The sensitivity of construction was checked by chemical mutagen cisplatin, which led to the larvae death at high concentrations, and caused glow of organs at low concentrations. The larvae were exposed to alpha-radiation for 20 hours. Preparations were analyzed on a light microscope with a blue filter and glow of the larval organs was observed. The glow intensity was depending from radiation source. In other words, higher radiation dose caused brighter glow, which correlates with synthesis of luminous protein level. This proves that alpha-radiation from radon and its decay daughter products has a genetic effect. Keywords: alpha radiation, radon, Drosophila, GFP, genotoxicity.

Halofuginone protects against advanced glycation end products‑induced injury of H9C2 cells via alleviating endoplasmic reticulum stress‑associated apoptosis and inducing autophagy.

Advanced glycation end products (AGEs) have been reported to serve an important role in the stiffening of cardiac tissues and myocardial cell injury. Serious myocardial cell injury can result in various heart diseases with high mortality. Halofuginone (HF), which possesses marked anti-inflammatory and antifibrotic effects, has recently been applied to inhibit the effects of cardiac stress. The present study aimed to investigate the potential effects of HF and its underlying mechanism in the treatment of AGEs-induced H9C2 cardiomyocyte damage. The western blot results of the present study demonstrated that HF may reduce the expression levels of myocardial injury markers, including myoglobin, creatine kinase MB and cardiac troponin I. In addition, flow cytometric analysis indicated that the production of reactive oxygen species (ROS) was significantly decreased by HF. Additionally, endoplasmic reticulum (ER) stress was suppressed in response to treatment with HF, as observed by low expression levels of ER stress-associated proapoptotic proteins (CCAAT/enhancer-binding protein homologous protein and cleaved caspase-12); overexpression of prosurvival proteins (growth arrest and DNA damage-inducible protein GADD34 and binding immunoglobulin protein) was also reported. Furthermore, the expression levels of microtubule-associated proteins 1A/1B light chain 3B (LC3)II/LC3I and Beclin 1 were elevated, whereas P62 expression levels were reduced following treatment with HF. These findings, together with immunofluorescence staining of LC3, indicated that HF may induce autophagy. Finally, the protective effects of HF on AGEs-treated H9C2 cells were reversed following treatment with the inhibitor 3-methyladenine, as indicated by inhibition of autophagy, and increases in apoptosis, ROS production and the ER stress response. Collectively, the findings of the present study suggested that the protective effects of HF against AGEs-induced myocardial cell injury may be associated with the induction of autophagy and amelioration of ROS-mediated ER stress and apoptosis. These findings may contribute to the development of a novel therapeutic method to inhibit the progression of myocardial cell injury.

Effect of cardiopulmonary bypass on a stress response pathway in a recovery rat's model

The systemic inflammation response syndrome (SIRS) is a common side-effect of the cardiopulmonary bypass (CPB) in cardiac surgery. SIRS can trigger multiple organ dysfunction which can lead to patient death. Pulmonary dysfunction is the most common post-operative complication of CPB. However, the lack of animal models recapitulating the disease prevents the investigation of pathophysiological mechanisms of CPB's complications. This project aims to develop a robust rat model recapitulating the SIRS during an extracorporeal circulation. This model mimics the clinical procedure and will be used to investigate the activation of stress response pathway (ISR). Surgery was made under isoflurane (3%, 02: 0.4L/min) and CPB was performed under propofol infusion (5μg/kg/hr) on mechanically ventilated 12 weeks old rats. Blood was drawn from cava vein then reinjected in the aorta. Hemodynamic parameters were measured in femoral artery. The circuit was primed with Ringer Lactate (< 1/3 of rats’ volemia). To oxygenate the blood, an oxygenator was in line with the peristaltic pump. Hemodynamic and blood gases were monitored during the procedure. After 30 mins of CPB, rats were culled. Blood and organs were collected for ISR pathway analyses. Mean blood pressure was maintained at 50mmHg with a PaO2 > 380mmHg. Lactate and kaliemia were increased after CPB (pre: 1.31 ± 0.49 vs post: 5.32 ± 3.67 and pre: 4.73 ± 0.15 vs post: 6.83 ± 2.22, respectively, N = 3). An activation of the ISR pathway was demonstrated by western blot on lung from CPB rats compared to Sham rats, with a 10-fold increase in p(ser51)-eIF2α/eIF2α ratio, a 2-fold increase in CHOP and GADD34 protein levels. We have developed a pre-clinical rat model of CPB in which preliminary data highlight an early activation of ISR pathway in lung. This model will be used to decipher the pathophysiological mechanisms of CPB's complications.

The DNA Repair-Associated Protein Gadd45γ Regulates the Temporal Coding of Immediate Early Gene Expression within the Prelimbic Prefrontal Cortex and Is Required for the Consolidation of Associative Fear Memory.

We have identified a member of the growth arrest and DNA damage (Gadd45) protein family, Gadd45γ, which is known to be critically involved in DNA repair, as a key player in the regulation of immediate early gene (IEG) expression underlying the consolidation of associative fear memory in adult male C57BL/6 mice. Gadd45γ temporally influences learning-induced IEG expression in the prelimbic prefrontal cortex (PLPFC) through its interaction with DNA double-strand break (DSB)-mediated changes in DNA methylation. Our findings suggest a two-hit model of experience-dependent IEG activity and learning that comprises (1) a first wave of IEG expression governed by DSBs and followed by a rapid increase in DNA methylation, and (2) a second wave of IEG expression associated with the recruitment of Gadd45γ and active DNA demethylation at the same site, which is necessary for memory consolidation.SIGNIFICANCE STATEMENT How does the pattern of immediate early gene transcription in the brain relate to the storage and accession of information, and what controls these patterns? This paper explores how Gadd45γ, a gene that is known to be involved with DNA modification and repair, regulates the temporal coding of IEGs underlying associative learning and memory. We reveal that, during fear learning, Gadd45γ serves to act as a coordinator of IEG expression and subsequent memory consolidation by directing temporally specific changes in active DNA demethylation at the promoter of plasticity-related IEGs.

Effect of neoadjuvant chemotherapy and its correlation with HPV status, EGFR, Her-2-neu, and GADD45 expression in oral squamous cell carcinoma

BackgroundHead and neck cancers are the commonest cancer in Southeast Asia. Despite being a surface cancer, it is associated with significant morbidity as despite early detection by the patients they often report for treatment late and hence are associated with poor prognosis. The role of neoadjuvant chemotherapy in head and neck cancer is still under evaluation; there is a large subgroup of population that does not respond to chemotherapy, and hence, most studies have failed to show any survival benefit. This study evaluated the role of neoadjuvant therapy with docetaxel and carboplatin in patients with oral cancer and correlated the response to human papilloma virus, EGFR1, EGFR2, and GADD45 expression.MethodsA total of 24 locally advanced, non-metastatic oral cancer patients were included in the study. Tumor biopsies were taken prior to the start of neoadjuvant therapy for expression of EGFR, Her-2-Neu, and GADD45 by immunohistochemistry and for HPV by PCR. The response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) criteria after three cycles of chemotherapy. Statistical analysis was performed using correlation and Kaplan–Meier analysis; the difference in survival was calculated with log rank test.ResultsA total of 21 male and 3 female with a mean age of 53.12 years were enrolled. Sixty-five percent of these received three cycles of chemotherapy. Five patients were positive for HPV 16 and none for HPV 18. Twenty-two of 24 patients showed GADD45 expression, 3 showed expression of Her-2-Neu while all 24 showed expression for EGFR1 protein. Two-year overall survival was 81%; GADD45 expressions were found to significantly affect the overall and disease-free survival, while any of the other protein expression studied and HPV status was not significant.ConclusionThe result of the present study shows significant downgrading of the oral cancers with neoadjuvant chemotherapy suggesting its utility in borderline operable cases. However, the response of chemotherapy does not appear to be related to the expression of EGFR, Her-2-Neu, and GADD45 protein or presence of HPV. Bone involvement, perineural invasion, and GADD45 expression significantly predict OS and DFS. All patients who did not express Gadd45 died before 2 years. Study with more subjects and longer follow-up should be carried out to elucidate this relation further.

Chronic oxidative stress promotes GADD34-mediated phosphorylation of the TAR DNA-binding protein TDP-43, a modification linked to neurodegeneration

Oxidative and endoplasmic reticulum (ER) stresses are hallmarks of the pathophysiology of ALS and other neurodegenerative diseases. In these stresses, different kinases phosphorylate eukaryotic initiation factor eIF2α, enabling the translation of stress response genes; among these is GADD34, the protein product of which recruits the α-isoform of protein phosphatase 1 catalytic subunit (PP1α) and eIF2α to assemble a phosphatase complex catalyzing eIF2α dephosphorylation and resumption of protein synthesis. Aberrations in this pathway underlie the aforementioned disorders. Previous observations indicating that GADD34 is induced by arsenite, a thiol-directed oxidative stressor, in the absence of eIF2α phosphorylation suggest other roles for GADD34. Here, we report that arsenite-induced oxidative stress differs from thapsigargin- or tunicamycin-induced ER stress in promoting GADD34 transcription and the preferential translation of its mRNA in the absence of eIF2α phosphorylation. Arsenite also stabilized GADD34 protein, slowing its degradation. In response to oxidative stress, but not ER stress, GADD34 recruited TDP-43, and enhanced cytoplasmic distribution and cysteine modifications of TDP-43 promoted its binding to GADD34. Arsenite also recruited a TDP-43 kinase, casein kinase-1ϵ (CK1ϵ), to GADD34. Concomitant with TDP-43 aggregation and proteolysis after prolonged arsenite exposure, GADD34-bound CK1ϵ catalyzed TDP-43 phosphorylations at serines 409/410, which were diminished or absent in GADD34-/- cells. Our findings highlight that the phosphatase regulator, GADD34, also functions as a kinase scaffold in response to chronic oxidative stress and recruits CK1ϵ and oxidized TDP-43 to facilitate its phosphorylation, as seen in TDP-43 proteinopathies.

Abstract A28: Oxygen-dependent translatome remodeling is controlled by eIF2α dephosphorylation

Abstract Introduction: Eukaryotic translation initiation factor 2α (eIF2α) is a core component of the cellular translation machinery. Alterations in eIF2α phosphorylation constitute a key adaptation during a variety of physiological stresses, including oxygen deprivation (hypoxia). We recently found that cancer cells respond to hypoxia through the systemic reprogramming of cap-dependent mRNA translation efficiencies by switching to the hypoxic eIF4F, eIF4FH. In the present study, we sought to elucidate the role(s) of eIF2α phosphorylation and dephosphorylation in this translatome remodeling process. In particular, the growth arrest and DNA damage-inducible protein GADD34 has been reported as an adaptive, stress-activated mediator of eIF2α dephosphorylation. We aimed to determine the role of GADD34 in the cellular hypoxic response. Findings: Our results indicate that hypoxia-induced eIF2α phosphorylation is a transient response with corresponding effects on translation efficiencies. Using next-generation mRNA sequencing (mRNA-seq), we reveal the dynamic profile of the translatome remodeling process during acute and chronic hypoxia. Our findings demonstrate that GADD34-mediated eIF2α dephosphorylation is required for hypoxic protein synthesis. GADD34 silencing results in high levels of eIF2α phosphorylation and severe global translational inhibition. Notably, GADD34 regulates the translation of hypoxia-inducible proteins in a hypoxia-inducible factor 2α (HIF-2α)-dependent manner. Finally, high throughput mass spectrometry (MS) analysis reveals several potential hypoxia-regulated GADD34 post-translational modifications (PTMs). Efforts are underway to validate and determine the effect(s) of these PTMs on GADD34 protein stability and/or activity. Conclusion: We demonstrate that GADD34-mediated eIF2α dephosphorylation regulates oxygen-dependent translatome reprogramming in cancer cells. This pathway represents an exciting potential target for cancer therapeutics. Citation Format: J.J. David Ho, Nathan C. Balukoff, Grissel Cervantes, Ayalivis De La Rosa, Stephen Lee. Oxygen-dependent translatome remodeling is controlled by eIF2α dephosphorylation. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A28.

Protective effects of Rg2 against UVB-induced DNA damage in HaCaT cells is dependent on p53 expression

ABSTRACTOur previous studies have shown that ginsenoside Rg2 protects the genotoxicity of UVB via earlier upregulation of p53 and GADD45 proteins. In the present study, we investigated the effects of Rg2 on the genotoxicity of UVB in cells transfected with p53 siRNA. The cells transfected with control siRNA, exposed to UVB and then post-incubated with 100 μM Rg2 showed increase in cell viability to about 20%, as compared to no significant increase in cell viability in cells transfected with p53 siRNA. The UVB-induced apoptosis was also decreased by 100 μM Rg2 to about 30% in control siRNA-transfected cells, as compared to only 10% decrease in the apoptosis in p53 siRNA-transfected cells. The p53 and GADD45 protein levels in control siRNA-transfected cells after UVB exposure increased to about 3.5 and 2.7 fold, respectively, as compared to the non-treated control. The increased p53 and GADD45 protein levels were downregulated by Rg2 in an Rg2 concentration-dependent manner. However, the protective effects of Rg2 were not shown in p53 siRNA-transfected cells. All these results suggest that Rg2 protects cells against UVB-induced genotoxicity by increasing DNA repair, in possible association with modulation of protein levels involved in p53 signaling pathway.

The Gadd45 Family As Mediators of Hematopoietic Stem Cell Differentiation - from Genotoxic Stress to Cytokine-Induced Differentiation

The growth arrest and DNA-damage-inducible 45 (Gadd45) protein family is encoded by three genes, Gadd45a, b and g. All members of the family are early responders of cellular stress with tumor-suppressive function. In leukemia, the Gadd45 genes are often epigenetically silenced. Lately, we identified the GADD45 Gamma as the molecular link of differentiation-promoting cytokines to induce differentiation in HSCs (1). Here, we unraveled the function of the genotoxic stress-induced family member GADD45 Alpha (GADD45A) in hematopoiesis. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains highly controversial.We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution using time-lapse microscopy-based HSC tracking. In contrast to other cellular systems, GADD45A expression neither caused a cell cycle arrest nor an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 h. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multi-lineage differentiation in vivo. The differentiation induction by GADD45A was transmitted by activating p38 MAPK signaling, and allowed the generation of megakaryocytic-erythroid, myeloid and lymphoid lineages.These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system. As the differentiation induction is conserved throughout the GADD45 family our study establishes this cell fate as an HSC-specific DNA-damage escape mechanism. Comparative analyses of the three proteins will further dissect the induced mechanisms at the molecular level.(1) Thalheimer, F.B., Wingert, S., De Giacomo, P., Haetscher, N., Rehage, M., Brill, B., Theis, F.J., Hennighausen, L., Schroeder, T., Rieger, M.A. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells. Stem Cell Reports 3(1):34-43. (2014) DisclosuresNo relevant conflicts of interest to declare.

C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells.

GADD34 is a member of a growth arrest and DNA damage (GADD)-inducible gene family. Here, we established a novel Chinese hamster ovary (CHO)-K1-derived cell line, CHO-K1-G34M, which carries a nonsense mutation (termed the Q525X mutation) in the GADD34 gene. The Q525X mutant protein lacks the C-terminal 66 amino acids required for GADD34 to bind to and activate protein phosphatase 1 (PP1). We investigated the effects of GADD34 with or without the Q525X mutation on the phosphorylation status of PP1 target proteins, including the α subunit of eukaryotic initiation factor 2 (eIF2α) and glycogen synthase kinase 3β (GSK3β). CHO-K1-G34M cells had higher levels of eIF2α phosphorylation compared to the control CHO-K1-normal cells both in the presence and absence of endoplasmic reticulum stress. Overexpression of the wild-type GADD34 protein in CHO-K1-normal cells largely reduced eIF2α phosphorylation, while overexpression of the Q525X mutant did not produce similar reductions. Meanwhile, neither wild type nor Q525X mutation of GADD34 affected the GSK3β phosphorylation status. GADD34 also did not affect the canonical Wnt signaling pathway downstream of GSK3β. Cell proliferation rates were higher, while expression levels of the cyclin-dependent kinase inhibitor p21 were lower in CHO-K1-G34M cells compared to the CHO-K1-normal cells. The GADD34 Q525X mutant had a reduced ability to inhibit cell proliferation and enhance p21 expression of the CHO-K1-normal cells compared to the wild-type GADD34 protein. These results suggest that the GADD34 protein C-terminal plays important roles in regulating not only eIF2α dephosphorylation but also cell proliferation in CHO-K1 cells.

Gadd45基因表达调控对肿瘤发生的影响

恶性肿瘤发生发展机制是生命科学研究的热点方向。研究发现，Gadd45蛋白是重要的致癌应激反应因子，肿瘤细胞的存活受Gadd45α、Gadd45β和Gadd45γ蛋白的调节。另外，Gadd45蛋白也参加由环境和生理损伤引起的细胞周期紊乱、DNA损伤修复、细胞凋亡过程，同时在细胞发育过程中也起重要的作用。本文综述了Gadd45基因表达调控对肿瘤发生的影响。 The hot direction of life science is to explore the development mechanism of malignant tumor. Recent studies have shown that, in tumor formation, whether in vivo or in vitro, Gadd45 protein is an important factor which calls monogenic stress response. Tumor cell survival relies on Gadd45α, Gadd45β and Gadd45γ. Gadd45 protein also participates in cell cycle stagnation, DNA repair and apoptosis, which were caused by environmental and physiological damage, meanwhile plays an important role in the development process. This paper reviews the effect of Gadd45 gene expres-sion regulation in tumor genesis.

All roads go to Salubrinal: endoplasmic reticulum stress, neuroprotection and glial scar formation.

All roads go to Salubrinal: endoplasmic reticulum stress, neuroprotection and glial scar formation.