Search Results (92)

Background: Protein kinase R (PKR) inhibits general mRNA translation by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). PKR also modulates NF-κB signaling during viral infections, but comparative studies of PKR-mediated NF-κB responses across mammalian species and their regulation by viral inhibitors remain largely unexplored. This study aimed to characterize the conserved antiviral and inflammatory roles of mammalian PKR orthologs and investigate their modulation by poxviral inhibitors. Methods: Using reporter gene assays and quantitative RT-PCR, we assessed the impact of 17 mammalian PKR orthologs on general translation inhibition, stress-responsive translation, and NF-κB-dependent induction of target genes. Congenic human and rabbit cell lines infected with a myxoma virus strain lacking PKR inhibitors were used to compare the effects of human and rabbit PKR on viral replication and inflammatory responses. Site-directed mutagenesis was employed to determine key residues responsible for differential sensitivity to the viral inhibitor M156. Results: All 17 mammalian PKR orthologs significantly inhibited general translation, strongly activated stress-responsive ATF4 translation, and robustly induced NF-κB target genes. Inhibition of these responses was specifically mediated by poxviral K3 orthologs that effectively suppressed PKR activation. Comparative analyses showed human and rabbit PKRs similarly inhibited virus replication and induced cytokine transcripts. Amino acid swaps between rabbit PKRs reversed their sensitivity to viral inhibitor M156 and NF-κB activation. Conclusions: Our data show that the tested PKR orthologs exhibit conserved dual antiviral and inflammatory regulatory roles, which can be antagonized by poxviral K3 orthologs that exploit eIF2α mimicry to modulate the PKR-NF-κB axis. Full article

Glycine has the greatest rate of deposition in whole-body proteins among all amino acids in neonates, but its provision from sow’s milk meets only 20% of the requirement of suckling piglets. The results of our recent studies indicate that piglets with intrauterine growth restriction (IUGR) have a reduced ability to synthesize glycine. The present study determined the role of glycine in the growth of sow-reared IUGR piglets. In Experiment 1, 56 newborn piglets (postnatal day 0) with a low birth weight (<1.10 kg) were selected from 14 litters, providing 4 IUGR piglets/litter that were allotted randomly into one of four treatment groups (14 piglets/group). Piglets received oral administration of either 0, 0.1, 0.2 or 0.4 g glycine/kg body weight (BW) twice daily (i.e., 0, 0.2, 0.4 or 0.8 g glycine/kg BW/day) between 0 and 14 days of age. L-Alanine was used as the isonitrogenous control. The BWs of all piglets were recorded each week during the experiment. Two weeks after the initiation of glycine supplementation, blood and tissue samples were collected for biochemical analyses. In Experiment 2, rates of muscle protein synthesis in tissues were determined on day 14 using the ³H-phenylalanine flooding dose technique. Compared with piglets in the control group, oral administration of 0.2, 0.4 and 0.8 g glycine/kg BW/day did not affect their milk intake (p > 0.05) but increased (p < 0.05) concentrations of glycine in plasma by 1.52-, 1.94-, and 2.34-fold, respectively, and body weight by 20%, 37%, and 34%, respectively. The dose of 0.4 g glycine/kg BW/day was the most cost-effective. Consistent with its growth-promoting effect, glycine supplementation stimulated (p < 0.05) the phosphorylation of mechanistic target of rapamycin (MTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and ribosomal protein S6 kinase beta-1 (p70^S6K) as well as protein synthesis in skeletal muscle, compared with the control group. Collectively, oral administration of glycine activated the MTOR signaling pathway in skeletal muscle and enhanced the growth performance of IUGR piglets. These results indicate that endogenous synthesis of glycine is inadequate to meet the needs of IUGR piglets during the suckling period and that oral supplementation with glycine to these compromized neonates can improve their growth performance. Full article

This study utilized MAC-T cells cultured in vitro as a model to investigate the effects of varying concentrations of valine on α-casein synthesis and its underlying regulatory mechanisms. In this experiment, MAC-T cells were subjected to a 12 h starvation period, followed by the addition of valine in a range of concentrations (a total of seven concentrations: 0.000, 1.596, 3.192, 6.384, 12.768, 25.536, and 51.072 mM, as well as in 10% Fetal Bovine Serum). The suitable range of valine concentrations was determined using enzyme-linked immunosorbent assays (ELISAs). Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot analyses were employed to evaluate the expression levels and phosphorylation states of the casein alpha s1 gene (CSN1S1), casein alpha s2 gene (CSN1S2) and mTOR signaling pathway-related genes. The functionality of the mTOR signaling pathway was further validated through rapamycin (100.000 nM) inhibition experiments. Results indicated that 1× Val (6.384 mM), 2× Val (12.768 mM), 4× Val (25.536 mM), and 8× Val (51.072 mM) significantly enhanced α-casein synthesis (p < 0.01). Within this concentration range, valine significantly upregulated the expression of CSN1S1, CSN1S2, and mTOR signaling pathway-related genes including the RagA gene (RRAGA), RagB gene (RRAGB), RagC gene (RRAGC), RagD gene (RRAGD), mTOR, raptor gene (RPTOR), and 4EBP1 gene (EIF4EBP1), eukaryotic initiation factor 4E (EIF4E), and S6 Kinase 1 (S6K1) (p < 0.01). Notably, the expression of the eukaryotic elongation factor 2 (EEF2) gene peaked at 1× Val (6.384 mM), while the expression of other genes reached their maximum at 4× Val (25.536 mM). Additionally, valine significantly increased the phosphorylation levels of mTOR, S6K1, 4E-binding protein-1 (4EBP1), ribosomal protein S6 (RPS6), and eEF2 (p < 0.01), with the highest phosphorylation levels of mTOR, S6K1, and RPS6 observed at 4× Val (25.536 mM). Rapamycin treatment significantly inhibited mTOR phosphorylation and α-casein synthesis (p < 0.01); however, the addition of 4× Val (25.536 mM) partially mitigated this inhibitory effect. In conclusion, valine promotes α-casein synthesis by activating the mTOR signaling pathway, with an optimal concentration of 4× Val (25.536 mM). Full article

Under conditions of amino acid deficiency, mammalian cells activate a nutrient-sensing kinase known as general control nonderepressible 2 (GCN2). The activation of GCN2 results in the phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α), which can be phosphorylated by three other three integrated stress response (ISR) kinases, reducing overall protein synthesis. GCN2 activation also promotes the translation of specific mRNAs, some of which encode transcription factors that enhance the transcription of genes involved in the synthesis, transport, and metabolism of amino acids to restore cellular homeostasis. The phosphorylation of eIF2α has been shown to protect oligodendrocytes, the cells responsible for producing myelin in the central nervous system during remyelination. Here, we explore the potential role of the kinase GCN2 in the myelination process. We challenged mice deficient in the GCN2-encoding gene with a pharmacological demyelinating stimulus (cuprizone) and evaluated the recovery of myelin as well as ISR activation through the levels of eIF2α phosphorylation. Our findings indicate that GCN2 controls the establishment of myelin by fine-tuning its abundance and morphology in the central nervous system. We also found that GCN2 is essential for remyelination. Surprisingly, we discovered that GCN2 is necessary to maintain eIF2α levels during remyelination. Full article

Oxidative stress, endoplasmic reticulum (ER) stress, and alterations in autophagy activity have been described as prominent factors mediating many pathological processes in chronic kidney disease (CKD). The accumulation of misfolded proteins in the ER may stimulate the unfolded protein response (UPR). The interplay between autophagy and UPR in hemodialysis (HD) patients remains unclear. The aim of the present study was to explore the associations between serum oxidative stress markers, autophagy activity, and ER stress markers in the peripheral blood mononuclear cells (PBMCs) of patients on HD. Autophagy and ER stress-related protein expression levels in PBMCs were measured using western blotting. The redox state of human serum albumin was measured via high-performance liquid chromatography. Levels of the microtubule associated protein light chain 3 (LC3)-II, BECLIN1, and p62/SQSTM1 proteins were significantly increased in PBMCs of HD patients compared to healthy subjects. The PBMCs in HD patients also displayed augmented glucose-regulated protein 78 kDa (GRP78), phosphorylated eukaryotic translation initiation factor 2, subunit 1 alpha (p-eIF2α), and activating transcription factor 6 (ATF6) levels (p < 0.05). Additionally, nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2) levels were elevated in the PBMCs of HD patients, compared to those of healthy subjects. Correlation analysis showed that the redox status of albumin was significantly correlated with the p62 protein level in PBMCs. Compared to healthy controls, we found elevated autophagosome formation in HD patients. Increased expression of ER stress markers was also observed in HD patients. Furthermore, increased p62 expression was positively correlated with the protein expression of NRF2, as well as a reduced form of serum albumin (human mercaptalbumin; HMA), in HD patients. Full article

Phenylalanine (Phe) is a potentially limiting amino acid for lactating cows. The mechanism by which Phe regulates milk protein synthesis remains unclear. The present study elucidates the mechanisms by which phenylalanine affects milk protein synthesis, amino acid utilization, and related signaling pathways in bovine mammary epithelial cells (BMECs). The BMECs were treated with five concentrations (0, 0.22, 0.44, 0.88, 1.76 mM, and serum free). Rapamycin inhibitors and RNA interference (RNAi) were used to inhibit the phosphorylation of the mammalian target of rapamycin (mTOR) signaling pathway and the expression of relevant amino acid transporters, respectively. The results showed that 4×Phe (0.88 mM) significantly increased (p < 0.05) both the mRNA and protein expression of α-casein (CSN1S1), β-casein (CSN2), and κ-casein (CSN3), as well as L-type amino acid transporter-1 (LAT1) mRNA expression. Protein expression and modification assays of mTOR-related proteins showed that 4×Phe could increase (p < 0.05) the expression of α-casein and eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and tended to increase the expression of ribosomal protein S6 protein kinase (S6K1, p = 0.054). The general control nonderepressible 2 (GCN2) signaling pathway factor, eukaryotic initiation factor 2 (eIF2α), was downregulated by 4×Phe treatment (p < 0.05). The rapamycin inhibition test showed that Phe regulated casein synthesis via the mTOR signaling pathway. RNAi experiments showed that LAT1 mediated the entry of Phe into cells. Moreover, 4×Phe treatment tended to decrease (0.05 < p < 0.10) the consumption of valine, leucine, histidine, tyrosine, cysteine, alanine, asparagine, and serine in the medium. Collectively, phenylalanine enhanced α-casein synthesis by regulating the phosphorylation of 4EBP1 and eIF2α and promoting the formation of the mTOR-centered casein translation initiation complex. Full article

We live in a society where extending one’s healthy lifespan is becoming increasingly important. Momordica charantia (MC) extract contains many bioactive substances, such as vitamin D, phytosterols, glycosides, saponins, alkaloids, and triterpenes, and has various health-promoting effects, but its effect on extending a healthy lifespan is unknown. This study investigated the effects of MC extract on a healthy lifespan, focusing on bitter taste receptors and the mammalian target of rapamycin (mTOR). Male and female mice from the Institute of Cancer Research (ICR) were divided into control and MC-extract-treated groups, with the latter receiving oral doses of MC extract three times a week for two years. In aged male mice, MC extract increased the muscle mass and grip strength and prolonged the time to exhaustion. MC extract also enhanced the signaling from taste receptor type 2 member 1 (T2R1) to mTOR in muscle in both sexes, elevating the ribosomal protein S6 kinase beta-1 and ribosomal protein S6 levels. This T2R1/mTOR pathway works in protein synthesis and is important for increasing muscle mass. Conversely, the levels of eukaryotic translation initiation factor 4E-binding protein 1 and microtubule-associated protein light chain 3 decreased in both aged male and female mice after MC extract administration. These findings suggest that the administration of MC extract may extend the healthy lifespan of male mice, with bitter taste receptors and mTOR signaling playing key roles in this process. Full article

Transglutaminase type 2 (TG2) is the most ubiquitously expressed and well characterized member of the transglutaminase family. It is a ubiquitous multifunctional enzyme implicated in the regulation of several cellular pathways that support the survival, death, and general homeostasis of eukaryotic cells. Due to its multiple localizations both inside and outside the cell, TG2 participates in the regulation of many crucial intracellular signaling cascades in a tissue- and cell-specific manner, making this enzyme an important player in disease development and progression. Moreover, TG2 is capable of modulating the tumor microenvironment, a process of dynamic tissue remodeling and biomechanical events, resulting in changes which influence tumor initiation, growth, and metastasis. Even if generally related to the Ca²⁺-dependent post-translational modification of proteins, a number of different biological functions have been ascribed to TG2, like those of a peptide isomerase, protein kinase, guanine nucleotide binder, and cytosolic–nuclear translocator. With respect to cancer, TG2′s role is controversial and highly debated; it has been described both as an anti- and pro-apoptotic factor and is linked to all the processes of tumorigenesis. However, numerous pieces of evidence support a tissue-specific role of TG2 so that it can assume both oncogenic and tumor-suppressive roles. Full article

Viruses evolve many strategies to ensure the efficient synthesis of their proteins. One such strategy is the inhibition of the integrated stress response—the mechanism through which infected cells arrest translation through the phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α). We have recently shown that the human common cold betacoronavirus OC43 actively inhibits eIF2α phosphorylation in response to sodium arsenite, a potent inducer of oxidative stress. In this work, we examined the modulation of integrated stress responses by OC43 and demonstrated that the negative feedback regulator of eIF2α phosphorylation GADD34 is strongly induced in infected cells. However, the upregulation of GADD34 expression induced by OC43 was independent from the activation of the integrated stress response and was not required for the inhibition of eIF2α phosphorylation in virus-infected cells. Our work reveals a complex interplay between the common cold coronavirus and the integrated stress response, in which efficient viral protein synthesis is ensured by the inhibition of eIF2α phosphorylation but the GADD34 negative feedback loop is disrupted. Full article

This study aimed to assess the impact of different resistance training (RT) loads and repetition on muscle damage, intramuscular anabolic signaling, and maximal muscle strength (MMS) in weightlifters. Eighteen male weightlifters were randomly assigned to 8 weeks of supervised RT regimes: high-load, low-repetition (HL), low-load, high-repetition (LH), and combination of HL and LH (COMBI). All groups exhibited a significant increase in skeletal muscle mass (SMM) and growth hormone levels, which ultimately contributed to improvement in MMS as indicated by 1-repetition maximum in the back squat and back muscle strength. Notably, while there were no significant changes in the mTOR protein, the phosphorylation of phosphorylation of p70 ribosomal protein S6 kinase 1 (p70S6K1), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and eukaryotic elongation factor 2 (eEF2), which are involved in muscle cell growth, was significantly affected by the different training regimens. More importantly, LH-RT led to a significant reduction in muscle damage markers, creatine kinase (CK) and lactate dehydrogenase (LDH), suggesting reduced recovery time and fatigue. Our results demonstrated that the LH-RT paradigm could be a viable alternative for weightlifters to enhance MMS and muscle hypertrophy similar to HL-RT, while reducing RT-induced muscle damage, ultimately contributing to the enhancement of exercise performance. Full article

Seneca Valley Virus (SVV), a member of the Picornaviridae family, is an emerging porcine virus that can cause vesicular disease in pigs. However, the immune evasion mechanism of SVV remains unclear, as does its interaction with other pathways. STING (Stimulator of interferon genes) is typically recognized as a critical factor in innate immune responses to DNA virus infection, but its role during SVV infection remains poorly understood. In the present study, we observed that STING was degraded in SVV-infected PK-15 cells, and SVV replication in the cells was affected when STING was knockdown or overexpressed. The STING degradation observed was blocked when the SVV-induced autophagy was inhibited by using autophagy inhibitors (Chloroquine, Bafilomycin A1) or knockdown of autophagy related gene 5 (ATG5), suggesting that SVV-induced autophagy is responsible for STING degradation. Furthermore, the STING degradation was inhibited when reticulophagy regulator 1 (FAM134B), a reticulophagy related receptor, was knocked down, indicating that SVV infection induces STING degradation via reticulophagy. Further study showed that in eukaryotic translation initiation factor 2 alpha kinase 3 (PERK)/activating transcription factor 6 (ATF6) deficient cells, SVV infection failed to induce reticulophagy-medaited STING degradation, indicating that SVV infection caused STING degradation via PERK/ATF6-mediated reticulophagy. Notably, blocking reticulophagy effectively hindered SVV replication. Overall, our study suggested that SVV infection resulted in STING degradation via PERK and ATF6-mediated reticulophagy, which may be an immune escape strategy of SVV. This finding improves the understanding of the intricate interplay between viruses and their hosts and provides a novel strategy for the development of novel antiviral drugs. Full article

Wolcott-Rallison syndrome is a rare cause of permanent neonatal diabetes mellitus caused by mutations in the eukaryotic translation initiation factor 2 alpha kinase 3 gene (EIF2AK3). Individuals affected by this disorder have severe hyperglycemia, pancreatic failure, and bone abnormalities and are prone to severe and life-threatening episodes of liver failure. This report illustrates the case of a 2-month-old infant with extreme hyperglycemia and severe diabetic ketoacidosis. Acute management was focused on correcting severe acidosis. Further management aimed to obtain stable blood glucose levels, balancing the patient’s need for comfort and lack of distress with the clinicians’ need for adequate information regarding the patient’s glycemic control. Genetic testing of the patient and his parents confirmed the diagnosis. The follow-up for 18 months after diagnosis is detailed, illustrating both the therapeutic success of subcutaneous insulin therapy and the ongoing complications that patients with Wolcott-Rallison syndrome are subject to. Full article

Previously, we identified a highly conserved, γ-shaped RNA element (γRE) from satellite RNAs of cucumber mosaic virus (CMV), and we determined γRE to be structurally required for satRNA survival and the inhibition of CMV replication. It remains unknown how γRE biologically functions. In this work, pull-down assays were used to screen candidates of host factors from Nicotiana benthamiana plants using biotin-labeled γRE as bait. Nine host factors were found to interact specifically with γRE. Then, all of these host factors were down-regulated individually in N. benthamiana plants via tobacco rattle virus-induced gene silencing and tested with infection by GFP-expressing CMV (CMV-gfp) and the isolate T1 of satRNA (sat-T1). Out of nine candidates, three host factors, namely histone H3, GTPase Ran3, and eukaryotic translation initiation factor 4A, were extremely important for infection by CMV-gfp and sat-T1. Moreover, we found that cytosolic glyceraldehyde-3-phosphate dehydrogenase 2 contributed to the replication of CMV and sat-T1, but also negatively regulated CMV 2b activity. Collectively, our work provides essential clues for uncovering the mechanism by which satRNAs inhibit CMV replication. Full article

The long non-coding RNA (lncRNA) actin fiber-associated protein-1 antisense RNA 1 (AFAP1-AS1) exerted oncogenic activity in triple-negative breast cancer (TNBC). We designed this study and conducted it to investigate the upstream regulation mechanism of AFAP1-AS1 in TNBC tumorigenesis. In this work, we proved the localization of AFAP1-AS1 in the cytoplasm. We elucidated the mechanism by which the transcription factor specificity protein 1 (SP1) modulated AFAP1-AS1 in TNBC progression, which has yet to be thoroughly studied. Dual luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay revealed a strong affinity of SP1 toward the promoter regions P3 of AFAP1-AS1, proving the gene expression regulation of AFAP1-AS1 via SP1 in TNBC. Additionally, SP1 could facilitate the tumorigenesis of TNBC cells in vitro and in vivo by regulating the AFAP1-AS1 expression. Furthermore, silenced AFAP1-AS1 suppressed the expression of genes in the mTOR pathway, such as eukaryotic translation initiation factor 4B (EIF4B), mitogen-activated protein kinase-associated protein 1 (MAPKAP1), SEH1-like nucleoporin (SEH1L), serum/glucocorticoid regulated kinase 1 (SGK1), and its target NEDD4-like E3 ubiquitin protein ligase (NEDD4L), and promoted the gene expression of s-phase kinase-associated protein 2 (SKP2). Overall, this study emphasized the oncogenic role of SP1 and AFAP1-AS1 in TNBC and illustrated the AFAP1-AS1 upstream interaction with SP1 and the downstream modulatory of mTOR signaling, thus offering insights into the tumorigenesis mechanism in TNBC. Full article

Mitochondrial diseases are a group of heterogeneous disorders caused by dysfunctional mitochondria. Interestingly, a large proportion of mitochondrial diseases are caused by defects in genes associated with tRNA metabolism. We recently discovered that partial loss-of-function mutations in tRNA Nucleotidyl Transferase 1 (TRNT1), the nuclear gene encoding the CCA-adding enzyme essential for modifying both nuclear and mitochondrial tRNAs, causes a multisystemic and clinically heterogenous disease termed SIFD (sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay; SIFD). However, it is not clear how mutations in a general and essential protein like TRNT1 cause disease with such clinically broad but unique symptomatology and tissue involvement. Using biochemical, cell, and mass spectrometry approaches, we demonstrate that TRNT1 deficiency is associated with sensitivity to oxidative stress, which is due to exacerbated, angiogenin-dependent cleavage of tRNAs. Furthermore, reduced levels of TRNT1 lead to phosphorylation of Eukaryotic Translation Initiation Factor 2 Subunit Alpha (eIF2α), increased reactive oxygen species (ROS) production, and changes in the abundance of distinct proteins. Our data suggest that the observed variable SIFD phenotypes are likely due to dysregulation of tRNA maturation and abundance, which in turn negatively affects the translation of distinct proteins. Full article