Double-stranded RNA-activated Protein Kinase Research Articles

C16, a PKR inhibitor, suppresses cell proliferation by regulating the cell cycle via p21 in colorectal cancer

Double-stranded RNA-activated protein kinase R (PKR) is highly expressed in colorectal cancer (CRC). However, the role of PKR in CRC remains unclear. The aim of this study was to clarify whether C16 (a PKR inhibitor) exhibits antitumor effects and to identify its target pathway in CRC. We evaluated the effects of C16 on CRC cell lines using the MTS assay. Enrichment analysis was performed to identify the target pathway of C16. The cell cycle was analyzed using flow cytometry. Finally, we used immunohistochemistry to examine human CRC specimens. C16 suppressed the proliferation of CRC cells. Gene Ontology (GO) analysis revealed that the cell cycle-related GO category was substantially enriched in CRC cells treated with C16. C16 treatment resulted in G1 arrest and increased p21 protein and mRNA expression. Moreover, p21 expression was associated with CRC development as observed using immunohistochemical analysis of human CRC tissues. C16 upregulates p21 expression in CRC cells to regulate cell cycle and suppress tumor growth. Thus, PKR inhibitors may serve as a new treatment option for patients with CRC.

TRIM21 of Micropterus salmoides exerts antiviral roles against largemouth bass ulcer syndrome virus

Tripartite motif 21 (TRIM21), a member of the TRIM family, plays an important role in apoptosis, autophagy and ubiquitination in human, and has been proven to play antiviral roles in different organisms. In this study, the TRIM21 gene of Micropterus salmoides (MsTRIM21) was cloned, and it encoded 376 amino acids, which showed 89.3% similarity with Micropterus dolomieu and 38.3% with homo sapiens. Bioinformatics analysis revealed MsTRIM21 contained four domains: C4HC3-type RING-variant (RINGv), coiled coil, PRY and SPRY. The high expression level of MsTRIM21 could be detected in liver, stomach and muscle of healthy Micropterus salmoides, and it was significantly upregulated in head kidney, muscle, gill and brain and significantly down-regulated in the stomach of Micropterus salmoides infected with largemouth bass ulcer syndrome virus (LBUSV). The overexpression of MsTRIM21 could significantly inhibit the viral replication in vitro, evidenced by the reduction of CPE severity and the downregulation of the viral gene transcription. In addition, the overexpression of MsTRIM21 could significantly increase the expression level of interferon regulatory factor (IRF) 3, IRF7, myxovirus resistance 1 (Mx1), interferon stimulated gene 15 (ISG15), double-stranded RNA-activated protein kinase (PKR) and tumor necrosis factor α (TNF-α) in vitro, indicating the enhancement of innate immune response and inflammatory response, which may directly affect the replication of LBUSV. Thus, these results provide new lights on the roles of fish TRIM21 in innate immune response against iridovirus.

Maternal obstructive sleep apnea aggravates metabolic dysfunction-associated fatty liver disease via HMGB1-TLR4 signaling-mediated endoplasmic reticulum stress in male offspring rats

Aims/hypothesisMaternal obstructive sleep apnea (MOSA) may inflict long-term metabolic effects on offspring. We hypothesize that MOSA increases the propensity for metabolic dysregulation in offspring and thus facilitates the development of metabolic dysfunction-associated fatty liver disease (MAFLD). This study aims to test the hypothesis and explore the underlying mechanism. MethodsThe MOSA rat model of upper airway obstruction was established and fecundated. The postweaning male offspring (n = 171) from both the control group and MOSA group were randomly fed the normal chow diet (NCD, n = 89) or high-fat diet (HFD, n = 82) for the next 5 months. Liver function, lipid profile, glucose, and insulin levels were measured. Expression levels of fibrosis-related proteins and endoplasmic reticulum (ER) stress-related proteins in liver tissues were assessed using immunohistochemistry and western blotting. ResultsMOSA increased body and liver weight in male offspring, along with augmented liver organ coefficient. Serum levels of aminotransferases, low-density lipoprotein, high-density lipoprotein, triglycerides, total cholesterol, total bile acid, fasting glucose, and insulin increased significantly. MOSA exacerbated HFD-induced hepatic steatosis and fibrosis. These effects were driven by the overactivated double-stranded RNA-activated protein kinase (PKR)-like eukaryotic initiation factor 2(PERK)-activating transcription factor (ATF)4-C/EBP homologous protein (CHOP) signaling pathway-induced ER stress, and hyperacetylation and release of high mobility group box-1(HMGB1) elicited above signaling in a TLR4-dependent manner. ConclusionsThese findings indicate that MOSA can exert prolonged adverse effects manifested as metabolic dysfunction in male offspring. Therefore, surveillance and management of OSA during pregnancy may be necessary to prevent and alleviate MAFLD in offspring.

Smooth Muscle Cell Phenotypic Switch Induced by Traditional Cigarette Smoke Condensate: A Holistic Overview.

Cigarette smoke (CS) is a risk factor for inflammatory diseases, such as atherosclerosis. CS condensate (CSC) contains lipophilic components that may represent a systemic cardiac risk factor. To better understand CSC effects, we incubated mouse and human aortic smooth muscle cells (SMCs) with CSC. We evaluated specific markers for contractile [i.e., actin, aortic smooth muscle (ACTA2), calponin-1 (CNN1), the Kruppel-like factor 4 (KLF4), and myocardin (MYOCD) genes] and inflammatory [i.e., IL-1β, and IL-6, IL-8, and galectin-3 (LGALS-3) genes] phenotypes. CSC increased the expression of inflammatory markers and reduced the contractile ones in both cell types, with KLF4 modulating the SMC phenotypic switch. Next, we performed a mass spectrometry-based differential proteomic approach on human SMCs and could show 11 proteins were significantly affected by exposition to CSC (FC ≥ 2.7, p ≤ 0.05). These proteins are active in signaling pathways related to expression of pro-inflammatory cytokines and IFN, inflammasome assembly and activation, cytoskeleton regulation and SMC contraction, mitochondrial integrity and cellular response to oxidative stress, proteostasis control via ubiquitination, and cell proliferation and epithelial-to-mesenchymal transition. Through specific bioinformatics resources, we showed their tight functional correlation in a close interaction niche mainly orchestrated by the interferon-induced double-stranded RNA-activated protein kinase (alternative name: protein kinase RNA-activated; PKR) (EIF2AK2/PKR). Finally, by combining gene expression and protein abundance data we obtained a hybrid network showing reciprocal integration of the CSC-deregulated factors and indicating KLF4 and PKR as the most relevant factors.

Chemotherapy-Induced Senescence Reprogramming Promotes Nasopharyngeal Carcinoma Metastasis by circRNA-Mediated PKR Activation.

Senescence is associated with tumor metastasis and chemotherapy resistance, yet the mechanisms remain elusive. Here, it is identified that nasopharyngeal carcinoma (NPC) patients who developed distant metastasis are characterized by senescence phenotypes, in which circWDR37 is a key regulator. CircWDR37 deficiency limits cisplatin or gemcitabine-induced senescent NPC cells from proliferation, migration, and invasion. Mechanistically, circWDR37 binds to and dimerizes double-stranded RNA-activated protein kinase R (PKR) to initiate PKR autophosphorylation and activation. Independent of its kinase activity, phosphorylated PKR induces I-kappaB kinase beta (IKKβ) phosphorylation, binds to and releases RELA from NF-κB inhibitor alpha (IκBα) to trigger nuclear factor kappa B (NF-κB) activation, thereby stimulating cyclin D1 (CCND1)and senescence-associated secretory phenotype component gene transcription in a circWDR37-dependent manner. Low circWDR37 levels correlate with chemotherapy response and favorable survival in NPC patients treated with gemcitabine or cisplatin induction chemotherapy. This study uncovers a new mechanism of circWDR37 activated PKR in senescence-driven metastasis and provides appealing therapeutic targets in NPC.

ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response

The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated, and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1’s kinase substrate(s) and their functions are largely unknown. Here, we discovered that sphingosine 1-phosphate (S1P) lyase (SPL), the enzyme that degrades S1P, is a substrate for the mammalian IRE1 kinase. Our data show that IRE1-dependent SPL phosphorylation inhibits SPL’s enzymatic activity, resulting in increased intracellular S1P levels. S1P has previously been shown to induce the activation of mitochondrial UPR (UPRmt) in nematodes. We determined that IRE1 kinase-dependent S1P induction during ER stress potentiates UPRmt signaling in mammalian cells. Phosphorylation of eukaryotic translation initiation factor 2α (eif2α) is recognized as a critical molecular event for UPRmt activation in mammalian cells. Our data further demonstrate that inhibition of the IRE1-SPL axis abrogates the activation of two eif2α kinases, namely double-stranded RNA-activated protein kinase (PKR) and PKR–like ER kinase upon ER stress. These findings show that the IRE1-SPL axis plays a central role in coordinating the adaptive responses of ER and mitochondria to ER stress in mammalian cells.

PKR Binds Enterovirus IRESs, Displaces Host Translation Factors, and Impairs Viral Translation to Enable Innate Antiviral Signaling.

ABSTRACTFor RNA virus families except Picornaviridae, viral RNA sensing includes Toll-like receptors and/or RIG-I. Picornavirus RNAs, whose 5′ termini are shielded by a genome-linked protein, are predominately recognized by MDA5. This has important ramifications for adaptive immunity, as MDA5-specific patterns of type-I interferon (IFN) release are optimal for CD4+T cell TH1 polarization and CD8+T cell priming. We are exploiting this principle for cancer immunotherapy with recombinant poliovirus (PV), PVSRIPO, the type 1 (Sabin) PV vaccine containing a rhinovirus type 2 internal ribosomal entry site (IRES). Here we show that PVSRIPO-elicited MDA5 signaling is preceded by early sensing of the IRES by the double-stranded (ds)RNA-activated protein kinase (PKR). PKR binding to IRES stem-loop domains 5–6 led to dimerization and autoactivation, displaced host translation initiation factors, and suppressed viral protein synthesis. Early PKR-mediated antiviral responses tempered incipient viral translation and the activity of cytopathogenic viral proteinases, setting up accentuated MDA5 innate inflammation in response to PVSRIPO infection.

Activation of Double-Stranded RNA-Activated Protein Kinase in the Dorsal Root Ganglia and Spinal Dorsal Horn Regulates Neuropathic Pain Following Peripheral Nerve Injury in Rats.

Double-stranded RNA (dsRNA)-activated kinase (PKR) is an important component in inflammation and immune dysfunction. However, the role of PKR in neuropathic pain remains unclear. Here, we showed that lumbar 5 spinal nerve ligation (SNL) led to a significant increase in the level of phosphorylated PKR (p-PKR) in both the dorsal root ganglia (DRG) and spinal dorsal horn. Images of double immunofluorescence staining revealed that p-PKR was expressed in myelinated A-fibers, unmyelinated C-fibers, and satellite glial cells in the DRG. In the dorsal horn, p-PKR was located in neuronal cells, astrocytes, and microglia. Data from behavioral tests showed that intrathecal (i.t.) injection of 2-aminopurine (2-AP), a specific inhibitor of PKR activation, and PKR siRNA prevented the reductions in PWT and PWL following SNL. Established neuropathic pain was also attenuated by i.t. injection of 2-AP and PKR siRNA, which started on day 7 after SNL. Prior repeated i.t. injections of PKR siRNA prevented the SNL-induced degradation of IκBα and IκBβ in the cytosol and the nuclear translocation of nuclear factor κB (NF-κB) p65 in both the DRG and dorsal horn. Moreover, the SNL-induced increase in interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) production was diminished by this treatment. Collectively, these results suggest that peripheral nerve injury-induced PKR activation via NF-κB signaling-regulated expression of proinflammatory cytokines in the DRG and dorsal horn contributes to the pathogenesis of neuropathic pain. Our findings suggest that pharmacologically targeting PKR might be an effective therapeutic strategy for the treatment of neuropathic pain.

NMDA receptor antagonists engender neuroprotection against gp120-induced cognitive dysfunction in rats through modulation of PKR activation, oxidative stress, ER stress and IRE1α signal pathway.

It is widely accepted that the surface glycoprotein (gp120) of human immunodeficiency virus-1 (HIV-1) plays an important role in HIV-1-induced nerve damage and pathogenesis of HIV-associated neurocognitive disorders (HAND). Our previous work has demonstrated that gp120 enhanced excitatory postsynaptic currents (EPSCs) mediated by N-methyl-d-aspartate receptors (NMDARs) and caused neural injury. However, the relationship between gp120, NMDARs and HAND is still unclear. Several lines of evidence indicate that double-stranded RNA-activated protein kinase (PKR) is involved in NMDA-induced cerebral ischaemia and retinal damage, but because its role in neuropathology is still debated, we examined whether PKR links oxidative stress and endoplasmic reticulum (ER) stress to exert a deleterious role in the rat model with gp120-induced dementia. In this study, we found that NMDAR antagonist memantine or PKR inhibitor C16 improved gp120-induced learning and memory impairment and inhibited gp120-induced PKR activity. Furthermore, memantine or C16 was found to attenuate gp120-induced neuroinflammation, oxidative stress, ER stress and its downstream IRE1α/JNK pathway. Additionally, memantine or C16 evidently inhibited apoptotic pathways by reducing the Bax and caspase-3, -8, -9 expressions and increasing Bcl-2 expression. So the NMDA receptor antagonists could alleviate HIV/gp120-induced dementia in the rat model by altering PKR level. In conclusion, this study demonstrates that NMDARs play a key role in HIV/gp120-induced hippocampal damage and cognitive dysfunction through PKR-mediated oxidative stress, ER stress, and IRE1α/JNK signalling pathway in rats, and implicating PKR inhibitors could provide a novel neuroprotective strategy for HAND via inhibiting ER stress and its downstream IRE1α signalling pathway.

DYT-PRKRA Mutation P222L Enhances PACT's Stimulatory Activity on Type I Interferon Induction.

DYT-PRKRA (dystonia 16 or DYT-PRKRA) is caused by mutations in the PRKRA gene that encodes PACT, the protein activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR). PACT participates in several cellular pathways, of which its role as a PKR activator protein during integrated stress response (ISR) is the best characterized. Previously, we have established that the DYT-PRKRA mutations cause enhanced activation of PKR during ISR to sensitize DYT-PRKRA cells to apoptosis. In this study, we evaluate if the most prevalent substitution mutation reported in DYT-PRKRA patients alters PACT’s functional role in induction of type I IFNs via the retinoic acid-inducible gene I (RIG-I) signaling. Our results indicate that the P222L mutation augments PACT’s ability to induce IFN β in response to dsRNA and the basal expression of IFN β and IFN-stimulated genes (ISGs) is higher in DYT-PRKRA patient cells compared to cells from the unaffected controls. Additionally, IFN β and ISGs are also induced at higher levels in DYT-PRKRA cells in response to dsRNA. These results offer a new avenue for investigations directed towards understanding the underlying molecular pathomechanisms in DYT-PRKRA.

Pretreatment with Propofol Reduces Pulmonary Injury in a Pig Model of Intestinal Ischemia-Reperfusion via Suppressing the High-Mobility Group Box 1 Protein (HMGB1)/Toll-Like Receptor 4 (TLR4)/Protein Kinase R (PKR) Signaling Pathway.

BackgroundPropofol improves rodent pulmonary injury after intestinal ischemia-reperfusion (IIR). However, its effect and underlying mechanisms in large animals remain unclear. Here, we examined whether pretreatment with propofol could relieve lung injury during IIR in pigs, then investigated the underlying mechanism.Material/MethodsA porcine model of IIR-induced lung injury was built by clamping the super mesenteric artery for 2 h and loosening the clamp for 4 h. Randomized grouping was used, and pigs were assigned to a sham-operated group, an IIR with saline pretreatment group, and an IIR with propofol pretreatment group. Pulmonary histopathologic changes, permeability, and oxygenation were assessed to evaluate the effect of propofol. We assessed levels of methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD), myeloperoxidase (MPO), high-mobility group box 1 protein (HMGB1), Toll-like receptor 4 (TLR4), and double-stranded RNA activated protein kinase R (PKR) to investigate the underlying mechanism.ResultsIIR caused severe lung damage, including morphological changes, high permeability, airway resistance, low static compliance, hypoxemia, and acidemia. Pulmonary and plasma MDA content and MPO activity increased, whereas SOD activity decreased. The HMGB1/TLR4/PKR signaling pathway was activated following IIR. Pretreatment with propofol markedly attenuated lung injury (such as reducing the lung edema and permeability), increased MDA content and MPO activity, and restored SOD activity induced by IIR, accompanied by inhibiting the effect of the HMGB1/TLR4/PKR signaling pathway.ConclusionsIIR caused acute lung injury in pigs. Pretreatment with propofol alleviated the lung injury, which was related to its suppression of the HMGB1/TLR4/PKR signaling pathway.

TRIM28 functions as a negative regulator of aggresome formation

ABSTRACT Selective recognition and elimination of misfolded polypeptides are crucial for protein homeostasis. When the ubiquitin-proteasome system is impaired, misfolded polypeptides tend to form small cytosolic aggregates and are transported to the aggresome and eventually eliminated by the autophagy pathway. Despite the importance of this process, the regulation of aggresome formation remains poorly understood. Here, we identify TRIM28/TIF1β/KAP1 (tripartite motif containing 28) as a negative regulator of aggresome formation. Direct interaction between TRIM28 and CTIF (cap binding complex dependent translation initiation factor) leads to inefficient aggresomal targeting of misfolded polypeptides. We also find that either treatment of cells with poly I:C or infection of the cells by influenza A viruses triggers the phosphorylation of TRIM28 at S473 in a way that depends on double-stranded RNA-activated protein kinase. The phosphorylation promotes association of TRIM28 with CTIF, inhibits aggresome formation, and consequently suppresses viral proliferation. Collectively, our data provide compelling evidence that TRIM28 is a negative regulator of aggresome formation. Abbreviations: BAG3: BCL2-associated athanogene 3; CTIF: CBC-dependent translation initiation factor; CED: CTIF-EEF1A1-DCTN1; DCTN1: dynactin subunit 1; EEF1A1: eukaryotic translation elongation factor 1 alpha 1; EIF2AK2: eukaryotic translation initiation factor 2 alpha kinase 2; HDAC6: histone deacetylase 6; IAV: influenza A virus; IP: immunoprecipitation; PLA: proximity ligation assay; polypeptidyl-puro: polypeptidyl-puromycin; qRT-PCR: quantitative reverse-transcription PCR; siRNA: small interfering RNA

DIM-C-pPhtBu induces lysosomal dysfunction and unfolded protein response - mediated cell death via excessive mitophagy.

Despite technological advances in cancer treatment, the survival rate of patients with head and neck cancer (HNC) has not improved significantly. Many studies have shown that endoplasmic reticulum (ER) stress-related signals are associated with mitochondrial damage and that these signals determine whether cells maintain homeostasis or activate cell death programs. The unfolded protein response (UPR) is regulated by ER membrane proteins such as double-stranded RNA-activated protein kinase R(PKR)-like ER kinase (PERK), which directly activate transcription of chaperones or genes that function in redox homeostasis, protein secretion, or cell death programs. In this study, we focused on the role of mitophagy and ER stress-mediated cell death induced by DIM-C-pPhtBu in HNC cancer. We found that DIM-C-pPhtBu, a compound that activates ER stress in many cancers, induced lysosomal dysfunction, excessive mitophagy, and cell death in HNC cells. Moreover, DIM-C-pPhtBu strongly inhibited HNC progression in a xenograft model by altering mitophagy related protein expression. Taken together, the results demonstrate that DIM-C-pPhtBu induces excessive mitophagy and eventually UPR-mediated cell death in HNC cells, suggesting that new anti-cancer drugs could be developed based on the connection between mitophagy and cancer cell death.

Deoxynivalenol Induces Inflammation in the Small Intestine of Weaned Rabbits by Activating Mitogen-Activated Protein Kinase Signaling.

Deoxynivalenol (DON) can activate related signaling pathways and induce gastrointestinal disorders. Based on the results of previous studies, this study tried to explore the relationship between DON-induced intestinal inflammation of weaned rabbits and the ERK-p38 signaling pathway. Forty-five weaned rabbits were divided into three treatments: control, LD and HD group. All rabbits were treated with diet containing a same nutrient content, but animals in the LD and HD groups were additionally administered DON via drinking water at 0.5 and 1.5 mg/kg b.w./d, respectively. The protocol consisted of a total feeding period of 31 days, including a pre-feeding period of 7 days. Western blotting, qRT-PCR, and immunohistochemistry were applied for analysis the expression of protein and mRNA of extracellular signal-regulated kinase (ERK), p38, double-stranded RNA-activated protein kinase (PKR), and hematopoietic cell kinase (Hck) in the duodenum, jejunum, and ileum of rabbits, as well as the distribution of positive reactants. The results proved that DON intake could enhance the levels of inflammatory factors in serum and damage the intestinal structure barrier of rabbits. Meanwhile, DON addition can stimulate the protein and mRNA expression for ERK, p38, PKR, and Hck in the intestine of rabbits, especially in the duodenum, as well as expand the distribution of positive reactants, in a dose-dependent manner.

Dicer represses the interferon response and the double-stranded RNA-activated protein kinase pathway in mouse embryonic stem cells

Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D−/−ESCs) as expected. Surprisingly, D−/−ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D−/−ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D−/−ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D−/−ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D−/−ESCs. Treatment of D−/−ESCs with a PKR inhibitor and IFNβ-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs.

Dystonia 16 (DYT16) mutations in PACT cause dysregulated PKR activation and eIF2α signaling leading to a compromised stress response

Dystonia 16 (DYT16) is caused by mutations in PACT, the protein activator of interferon-induced double-stranded RNA-activated protein kinase (PKR). PKR regulates the integrated stress response (ISR) via phosphorylation of the translation initiation factor eIF2α. This post-translational modification attenuates general protein synthesis while concomitantly triggering enhanced translation of a few specific transcripts leading either to recovery and homeostasis or cellular apoptosis depending on the intensity and duration of stress signals. PKR plays a regulatory role in determining the cellular response to viral infections, oxidative stress, endoplasmic reticulum (ER) stress, and growth factor deprivation. In the absence of stress, both PACT and PKR are bound by their inhibitor transactivation RNA-binding protein (TRBP) thereby keeping PKR inactive. Under conditions of cellular stress these inhibitory interactions dissociate facilitating PACT-PACT interactions critical for PKR activation. While both PACT-TRBP and PKR-TRBP interactions are pro-survival, PACT-PACT and PACT-PKR interactions are pro-apoptotic. In this study we evaluate if five DYT16 substitution mutations alter PKR activation and ISR. Our results indicate that the mutant DYT16 proteins show stronger PACT-PACT interactions and enhanced PKR activation. In DYT16 patient derived lymphoblasts the enhanced PACT-PKR interactions and heightened PKR activation leads to a dysregulation of ISR and increased apoptosis. More importantly, this enhanced sensitivity to ER stress can be rescued by luteolin, which disrupts PACT-PKR interactions. Our results not only demonstrate the impact of DYT16 mutations on regulation of ISR and DYT16 etiology but indicate that therapeutic interventions could be possible after a further evaluation of such strategies.

Pathogenesis and promising therapeutics of Alzheimer disease through eIF2α pathway and correspondent kinases.

Eukaryotic initiation factor 2 (eIF2α) pathway is overactivated in Alzheimer disease and is probably associated with synaptic and memory deficiencies. EIF2α protein is principally in charge of the regulation of protein synthesis in eukaryotic cells. Four kinases responsible for eIF2α phosphorylation at ser-51 are: General control non-derepressible-2 kinase (GCN2), double-stranded RNA-activated protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase (HRI) are the four kinases. They lead to reduced levels of general translation and paradoxical increase of stress-responsive mRNAs expression including the B-secretase (BACE1) and the transcriptional modulator activating transcription factor 4 (ATF4), which in turn accelerates the beta-amyloidogenesis, tau phosphorylation, proapoptotic pathway induction and autophagy elements formation leading to the main pathological hallmarks of AD. Findings suggest that genetic or pharmacological inhibition of correspondent kinases can restore memory and prevent neurodegeneration. This implies that inhibition of eIF2α phosphorylation through respondent kinases is indeed a feasible prospect of clinical application. This review discusses recent therapeutic approaches targeting eIF2α pathway and provides an overview of the links between correspondent kinases overactivation with neurodegeneration in AD.

Pseudorabies virus infection inhibits stress granules formation via dephosphorylating eIF2α

Pseudorabies virus (PRV) is one of the most notorious pathogens in the global pig industry. During infection, viruses may evolve various strategies, such as modulating stress granules (SGs) formation, to create an optimal surroundings for viral replication. However, the interplay between PRV infection and SGs formation remains largely unknown. Here we showed that PRV infection markedly blocked SGs formation induced by sodium arsenate (AS) and DL-Dithiothreitol (DTT). Accordantly, the phosphorylation of eIF2α was markedly inhibited in PRV-infected cells, although two eIF2α kinases double-stranded RNA-activated protein kinase (PKR) and PKR-like ER kinase (PERK) were activated during PRV infection. Furthermore, we also found that the dephosphorylation of eIF2α occurred at the early stage of virus infection but without the elevated production of GADD34 and PP1. Moreover, inhibition of PP1 activity by salubrinal could counteract PRV-mediated eIF2α dephosphorylation partially and inhibit virus replication. Our results revealed that, on the one hand, PRV infection activated eIF2α kinases PKR (latter inhibited) and PERK, and on the other hand, PRV encoded-functions dephosphorylated eIF2α and inhibited SGs formation to facilitate virus replication.

Chikungunya virus infection induces differential inflammatory and antiviral responses in human monocytes and monocyte-derived macrophages

Chikungunya virus (CHIKV) is a zoonotic arthropod-borne virus that has caused several outbreaks in tropical and subtropical areas worldwide during the last 50 years. The virus is known to target different human cell types throughout the course of infection including epithelial and endothelial cells, fibroblasts, primary monocytes and monocyte-derived macrophages (MDMs). The two latter are phagocytic cell populations of the innate immune system which are involved in some aspects of CHIKV pathogenesis. However, monocytes and macrophages also potentially contribute to the control of viral replication through the expression of different pattern recognition receptors sensing viral pathogens and subsequently, inducing an type I interferone (IFN-I)-dependent antiviral immune response. The aim of this study was to determine the modulation of the expression of Toll-like receptors (TLRs), cytokine secretion capabilities and antiviral factor production in monocytes and MDMs following infection with CHIKV. Moreover, we sought to determine the replication kinetics of CHIKV in these two cell populations. We found that the maximum peak of CHIKV replication was observed between 18- and 24-hours post-infection (hpi), while after that the is strongly reduced. Furthermore, CHIKV infection induced the pro-inflammatory cytokine production starting from the first 6 hpi in both monocytes and MDMs, with similar kinetics but different protein levels. In contrast, the kinetics of transcriptional expression of some TLRs were different between both cell types. In addition, IFN-I, 2′,5′-oligoadenylate synthetase 1 (OAS1), and double-stranded RNA-activated protein kinase R (PKR) mRNA levels were detected in response to CHIKV infection of monocytes and MDMs, resulting the highest expression levels at 48 hpi. In conclusion, our data provides evidence that CHIKV infection activates the TLR pathways in primary monocytes and MDMs, which play a crucial role in CHIKV pathogenesis and/or host defense, differentially. However, additional studies are required to determine the functional role of TLRs in monocytes and MDMs.

Grouper PKR activation inhibits red-spotted grouper nervous necrosis virus (RGNNV) replication in infected cells

The double-stranded RNA-activated protein kinase (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, PKR inhibits or promotes viral replication. In the present study, PKR homologues of orange-spotted grouper (Epinephelus coioides) (EcPKR) were cloned and the involvement of EcPKR during Red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. EcPKR encodes a 621-amino acid polypeptide that is closely related to the equivalent protein in Larimichthys crocea. EcPKR encoded two dsRNA binding domains and a Serine/Threonine protein kinase domain. Quantitative real-time PCR (qRT-PCR) analysis indicated that EcPKR was present in all examined tissues, with higher expression in spleen, intestine and gill. When stimulated with poly(I:C), the expression of EcPKR in the grouper spleen was increased, with highest expression 12 h post stimulation. EcPKR concentration was significantly increased in RGNNV-infected cells, with highest expression at 36 h post stimulation. EcPKR is mainly present in the cytoplasm. Overexpression of EcPKR in grouper spleen (GS) cells inhibits the transcription of the RGNNV genes. Furthermore, our results show that EcPKR overexpression significantly enhances the immune response of interferon and the activation of interferon-beta (IFN-β), interferon stimulated response element (ISRE) and nuclear factor-kappa B (NF-κB). Taken together, these results are important for better understanding of the function of PKR in fish and reveal its involvement in host response to immune challenges in RGNNV.