GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis

Viruses have evolved multiple strategies to manipulate their host’s translational machinery for the synthesis of viral proteins. A common viral target is the alpha subunit of eukaryotic initiation factor 2 (eIF2α). In this study, we show that global protein synthesis was increased but the eIF2α phosphorylation level was markedly decreased in porcine kidney 15 (PK15) cells infected with pseudorabies virus (PRV), a swine herpesvirus. An increase in the eIF2α phosphorylation level by salubrinal treatment or transfection of constructs expressing wild-type eIF2α or an eIF2α phosphomimetic [eIF2α(S51D)] attenuated global protein synthesis and suppressed PRV replication. To explore the mechanism involved in the inhibition of eIF2α phosphorylation during PRV infection, we examined the phosphorylation status of protein kinase R-like endoplasmic reticulum kinase (PERK) and double-stranded RNA-dependent protein kinase R (PKR), two kinases that regulate eIF2α phosphorylation during infection with numerous viruses. We found that the level of neither phosphorylated (p)-PERK nor p-PKR was altered in PRV-infected cells or the lungs of infected mice. However, the expression of growth arrest and DNA damage-inducible protein 34 (GADD34), which promotes eIF2α dephosphorylation by recruiting protein phosphatase 1 (PP1), was significantly induced both in vivo and in vitro. Knockdown of GADD34 and inhibition of PP1 activity by okadaic acid treatment led to increased eIF2α phosphorylation but significantly suppressed global protein synthesis and inhibited PRV replication. Collectively, these results demonstrated that PRV induces GADD34 expression to promote eIF2α dephosphorylation, thereby maintaining de novo protein synthesis and facilitating viral replication.

Biochemical Basis for the Time-of-Day Effect on Glufosinate Efficacy against Amaranthus palmeri

Glufosinate, a glutamine synthetase (GS) inhibitor, often provides variable weed control depending on environmental conditions such as light, temperature and humidity at the time of application. Midday applications normally provide improved efficacy compared to applications at dawn or dusk. We investigated the biochemical basis for the time-of-day effect on glufosinate efficacy in Amaranthus palmeri. GS1/GS2 gene expression and GS1/GS2 protein abundance were assessed in different parts (young leaves, old leaves, and roots) of plants incubated in the dark compared to those in the light. The turnover of GS total activity was also evaluated overtime following glufosinate treatment at midday compared to dusk application. The results suggest that GS in A. palmeri is less expressed and less abundant in the dark compared to in the light. Midday application of glufosinate under intense light conditions following application provide full control of A. palmeri plants. Consequently, these plants are unable to recover GS activity by de novo protein synthesis. Full activity of GS is required for complete inhibition by the irreversible inhibitor glufosinate. Therefore, glufosinate applications should always be performed in the middle of the day when sunlight is intense, to prevent weed escapes from the herbicide treatment.

Biochemical Basis for the Time-of-day Effect on Glufosinate Efficacy Against Amaranthus palmeri

Glufosinate, a glutamine synthetase (GS) inhibitor, often provides variable weed control depending on environmental conditions such as light, temperature and humidity at the time of application. Midday applications normally provide improved efficacy compared to applications at dawn or dusk. We investigated the physiological, molecular, and biochemical basis for the time-of-day effect on glufosinate efficacy in Amaranthus palmeri. GS1 and GS2 gene expression and protein abundance were assessed in different parts (young leaves, old leaves, and roots) of plants incubated in the dark compared to those in the light. The turnover of GS total activity was also evaluated overtime following glufosinate treatment at midday compared to dusk application. The results suggest that GS in A. palmeri is less expressed and less abundant in the dark compared to in the light. Midday application of glufosinate under intense light conditions in the hours following application provide full control of A. palmeri plants. Consequently, these plants are unable to recover GS activity by de novo protein synthesis. Full activity of GS is required for complete inhibition by the irreversible inhibitor glufosinate. Therefore, glufosinate applications should always be performed in the middle of the day when sunlight is intense, to prevent weed escapes from the herbicide treatment.

Abstract 17411: De Novo Protein Synthesis of Alpha-Toxin Activated Platelets

Introduction: Endovascular infections with bacteria are often devastating with subsequent high morbidity and mortality. Exo- and or endotoxins of bacteria can activate endothelial cells, leukocytes and platelets. Platelets are first line defence they accumulate at sites of vascular injury or infection. Platelet activation is a necessary step in thrombus formation. Nevertheless, stimulation of platelets will result in de novo protein synthesis despite missing nucleus since platelets armed with translational equipment. Methods: In the present study we determined the effect of staphylococcus aureus α-toxin on platelet activation and de novo protein synthesis analysed with 2-D gels, proteomics and phosphorylation analysis. Results: α-toxin induced platelet activation resulted in modulation of de novo protein synthesis of DJ-1 Protein, ras suppressor protein1, PLEK protein, fumaryl aceto acetase sowie das coronin actin binding protein. This synthesis was time- and concentration-dependent and was markedly increased when platelets adhered to collagen or fibrinogen and required ligation of α IIb β 3 . Accumulation of protein synthesis in platelets was blocked by global translational inhibitors and attenuated by inhibitors that regulate signalling through the mammalian Target of Rapamycin (mTOR). In addition with phosphorylation analysis we were able demonstrate modulation of threonine phosphorylation of fumaryl aceto acetase, phosphor threonin signal of coronin actin binding protein, phosphorylation of peroxiredoxin-6, phosphorylation of tropomyosin-2, phosphothreonin signal of H + transporting two sector ATPase upon α-toxin stimulation. Conclusion: Interactions with staphylococcus aureus α-toxin and platelets might lead to their activation and de novo protein synthesis. These results suggest that platelets have an important role in inflammation besides their aggregating duties in inflammatory disease.

De novo protein synthesis is necessary for priming in preclinical models of migraine

Background Migraine attacks are often triggered by normally innocuous stimuli, suggesting that sensitization within the nervous system is present. One mechanism that may contribute to neuronal sensitization in this context is translation regulation of new protein synthesis. The goal of this study was to determine whether protein synthesis contributes to behavioral responses and priming in preclinical models of migraine. Methods Mice received a dural injection of interleukin-6 in the absence or presence of the protein synthesis inhibitor anisomycin or the translation initiation inhibitor 4EGI-1 and were tested for facial hypersensitivity. Upon returning to baseline, mice were given a second, non-noxious dural injection of pH 7.0 to test for priming. Additionally, eIF4ES209Amice lacking phosphorylation of mRNA cap-binding protein eIF4E received dural interleukin-6 or were subjected to repeated restraint stress and then tested for facial hypersensitivity. After returning to baseline, mice were given either dural pH 7.0 or a systemic sub-threshold dose of the nitric oxide donor sodium nitroprusside and tested for priming. Results Dural injection of interleukin-6 in the presence of anisomycin or 4EGI-1 or in eIF4ES209Amice resulted in the partial attenuation of acute facial hypersensitivity and complete block of hyperalgesic priming. Additionally, hyperalgesic priming following repeated restraint stress was blocked in eIF4ES209Amice. Conclusions These studies show that de novo protein synthesis regulated by activity-dependent translation is critical to the development of priming in two preclinical models of migraine. This suggests that targeting the regulation of protein synthesis may be a novel approach for new migraine treatment strategies.

eIF2α Phosphorylation by GCN2 Is Induced in the Presence of Chitin and Plays an Important Role in Plant Defense against B. cinerea Infection

Translation plays an important role in plant adaptation to different abiotic and biotic stresses; however, the mechanisms involved in translational regulation during each specific response and their effect in translation are poorly understood in plants. In this work, we show that GCN2 promotes eIF2α phosphorylation upon contact with Botrytis cinerea spores, and that this phosphorylation is required for the proper establishment of plant defense against the fungus. In fact, independent gcn2 mutants display an enhanced susceptibility to B. cinerea infection, which is highlighted by an increased cell death and reduced expression of ethylene- and jasmonic-related genes in the gcn2 mutants. eIF2α phosphorylation is not only triggered in the presence of the fungus, but interestingly, is also achieved in the sole presence of the microbe-associated molecular pattern (MAMP) chitin. Moreover, analysis of de novo protein synthesis by 35SMet–35SCys incorporation indicates that chitin treatment promotes a global inhibition of translation. Taken together, these results suggest that eIF2α phosphorylation by GCN2 is promoted in the presence of chitin and plays an important role in plant defense against B. cinerea infection.