Depletion of tryptophan and the accumulation of tryptophan metabolites mediated by the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1), trigger immune cells to undergo apoptosis. ...However, cancer cells in the same microenvironment appear not to be affected. Mechanisms whereby cancer cells resist accelerated tryptophan degradation are not completely understood. We hypothesize that cancer cells co-opt IMPACT (the product of IMPrinted and AnCienT gene), to withstand periods of tryptophan deficiency.
A range of bioinformatic techniques including correlation and gene set variation analyses was applied to genomic datasets of cancer (The Cancer Genome Atlas) and normal (Genotype Tissue Expression Project) tissues to investigate IMPACT's role in cancer. Survival of IMPACT-overexpressing GL261 glioma cells and their wild type counterparts cultured in low tryptophan media was assessed using fluorescence microscopy and MTT bio-reduction assay. Expression of the Integrated Stress Response proteins was measured using Western blotting.
We found IMPACT to be upregulated and frequently amplified in a broad range of clinical cancers relative to their non-malignant tissue counterparts. In a subset of clinical cancers, high IMPACT expression associated with decreased activity of pathways and genes involved in stress response and with increased activity of translational regulation such as the mTOR pathway. Experimental studies using the GL261 glioma line showed that cells engineered to overexpress IMPACT, gained a survival advantage over wild-type lines when cultured under limiting tryptophan concentrations. No significant difference in the expression of proteins in the Integrated Stress Response pathway was detected in tryptophan-deprived GL261 IMPACT-overexpressors compared to that in wild-type cells. IMPACT-overexpressing GL261 cells but not their wild-type counterparts, showed marked enlargement of their nuclei and cytoplasmic area when stressed by tryptophan deprivation.
The bioinformatics data together with our laboratory studies, support the hypothesis that IMPACT mediates a protective mechanism allowing cancer cells to overcome microenvironmental stresses such as tryptophan deficiency.
The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. ...Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.
Translational control directed by the eukaryotic translation initiation factor 2 α-subunit (eIF2α) kinase GCN2 is important
for coordinating gene expression programs in response to nutritional ...deprivation. The GCN2 stress response, conserved from
yeast to mammals, is critical for resistance to nutritional deficiencies and for the control of feeding behaviors in rodents.
The mouse protein IMPACT has sequence similarities to the yeast YIH1 protein, an inhibitor of GCN2. YIH1 competes with GCN2
for binding to a positive regulator, GCN1. Here, we present evidence that IMPACT is the functional counterpart of YIH1. Overexpression
of IMPACT in yeast lowered both basal and amino acid starvation-induced levels of phosphorylated eIF2α, as described for YIH1
( 31 ). Overexpression of IMPACT in mouse embryonic fibroblasts inhibited phosphorylation of eIF2α by GCN2 under leucine starvation
conditions, abolishing expression of its downstream target genes, ATF4 ( CREB-2 ) and CHOP ( GADD153 ). IMPACT bound to the minimal yeast GCN1 segment required for interaction with yeast GCN2 and YIH1 and to native mouse GCN1.
At the protein level, IMPACT was detected mainly in the brain. IMPACT was found to be abundant in the majority of hypothalamic
neurons. Scattered neurons expressing this protein at higher levels were detected in other regions such as the hippocampus
and piriform cortex. The abundance of IMPACT correlated inversely with phosphorylated eIF2α levels in different brain areas.
These results suggest that IMPACT ensures constant high levels of translation and low levels of ATF4 and CHOP in specific
neuronal cells under amino acid starvation conditions.
Raw (fresh) and frozen poultry products are frequently associated with Staphylococcus aureus contamination. New Zealand is among the developed countries with high incidences of staphylococcal food ...poisoning. The study investigated the S. aureus isolates obtained from various stages of poultry production, to determine the primary source of contamination. Viable cell counts of S. aureus were enumerated using Petrifilm™ Staph Express Count Plates, and the isolates were confirmed by Gram-stain and coagulase-positive test. Sixty S. aureus isolates were further confirmed by PCR. The PCR analysis used primers that specifically amplifies a fragment of the femA gene, unique to S. aureus. The confirmed S. aureus strains were further examined for enterotoxigenicity by PCR. Multilocus Sequence Typing (MLST) was then used to identify sequence types (STs) of the sixty isolates of S. aureus. The relatedness of the sequence types was investigated by eBURST. In this study, it was observed that all samples from the processing plant and live chickens at the farm were contaminated by S. aureus. Fifty-nine (59) of the 60 isolates were enterotoxigenic carrying enterotoxin genes: seg, sei, seh, sek, sel, sem, sen, or seo. The sixty isolates were categorised into six different sequence types: ST5, ST2594, ST101, ST83, ST398, ST1; where ST5, ST83 and ST2594 belonged to the Clonal Complex (CC) 5 with ST5 being the clonal ancestor. The sources of S. aureus contamination in the final poultry products were linked to fresh mechanically separated meat, fresh skin, fresh skin-on-breast fillet, rubber fingers on mechanical pluckers, and live chickens at the farm. The skin of live chickens at the farm was most likely the origin of S. aureus contamination on equipment and final products. Not all identified S. aureus strains at the farm were observed in the final products. Therefore, further investigation on other potential contamination sources such as gloves and knives used at the processing plant, and feeders and drinkers at the farm level is recommended.
•Swab samples collected from the processing plant and live chickens at the farm were contaminated by S. aureus.•Of the 60 S. aureus isolates from the main sampling sites, 59 were enterotoxic.•S. aureus ST5 was identified in microbiological samples collected in the processing plant and live chickens at the farm.•A major clonal complex CC5 was identified.•The source of S. aureus contamination in the final products was linked to the live chickens at the farm.
Genetic and pharmacological interventions in yeast and mammalian cells have suggested a cross-talk between the actin cytoskeleton and protein synthesis. Regulation of the activity of the translation ...initiation factor 2 (eIF2) is a paramount mechanism for cells to rapidly adjust the rate of protein synthesis and to trigger reprogramming of gene expression in response to internal and external cues. Here, we show that disruption of F-actin in mammalian cells inhibits translation in a GCN2-dependent manner, correlating with increased levels of uncharged tRNA. GCN2 activation increased phosphorylation of its substrate eIF2α and the induction of the integrated stress response master regulator, ATF4. GCN2 activation by latrunculin-B is dependent on GCN1 and inhibited by IMPACT. Our data suggest that GCN2 occurs in two different complexes, GCN2-eEF1A and GCN2-GCN1. Depolymerization of F-actin shifts GCN2 to favor the complex with GCN1, concomitant with GCN1 being released from its binding to IMPACT, which is sequestered by G-actin. These events might further contribute to GCN2 activation. Our findings indicate that GCN2 is an important sensor of the state of the actin cytoskeleton.
The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the α subunit
of translation initiation factor 2 (eIF2α) kinase GCN2 and consequent ...induction of GCN4, a transcriptional activator of amino
acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in
starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn â phenotype) by suppressing eIF2α phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex
formation, and, consistent with this, the Gcn â phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259
in GCN1 in vitro and in full-length GCN1 in vivo , as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2α phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor
of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a
genetic reduction in actin levels decreases the GAAC response. This Gcn â phenotype was partially suppressed by deletion of YIH1 , consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin
complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in
a restricted cellular compartment in which YIH1 is displaced from monomeric actin.
Intensive poultry production due to public demand raises the risk of contamination, creating potential foodborne hazards to consumers. The prevalence and microbial load of the pathogens ...Campylobacter, Salmonella, Staphylococcus aureus, and Escherichia coli was determined by standard methods at the farm level. After disinfection, swab samples collected from wall crevices, drinkers, and vents were heavily contaminated, as accumulated organic matter and dust likely protected the pathogens from the disinfectants used. The annex floor also showed high microbial concentrations, suggesting the introduction of pathogens from external environments, highlighting the importance of erecting hygiene barriers at the entrance of the main shed. Therefore, pathogen control measures and proper application of disinfectants are recommended as intervention strategies. Additionally, quantitative polymerase chain reaction (qPCR) was evaluated as a quantification tool. qPCR showed limitations with samples containing low microbial counts because of the low detection limit of the method. Thus, bacterial pre-enrichment of test samples may be necessary to improve the detection of pathogens by qPCR.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase ...Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1‐9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1‐9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation‐inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin–Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.
Adjusting protein synthesis by phosphorylating eukaryotic translation initiation factor 2 (eIF2α) is a major mechanism by which eukaryotes adapt to and overcome stress. The eIF2α kinase Gcn2 is ...essential for overcoming amino acid starvation in all eukaryotes. We have shown that to sense starvation, the Gcn2 RWD domain must directly contact its effector protein, Gcn1, and both must bind to the ribosome, suggesting that starvation is sensed within a Gcn1–Gcn2–ribosome complex. The mammalian protein IMPACT, highly expressed in neurons, and its yeast orthologue yeast IMPACT homologue (Yih1) harbour an RWD domain with Gcn1‐binding activity. We have shown that Yih1 downregulates Gcn2 by competing with Gcn2 for Gcn1‐binding. Here, we provide evidence that Yih1 forms a complex with ribosomes. In velocity sedimentation assays, overexpressed glutathione S‐transferase (GST)‐tagged Yih1 cosedimented with polyribosomes independently of Gcn1. Reduction of polyribosomes to monosomes concomitantly decreased GST–Yih1 sedimentation in the heavy fractions where polyribosomes are normally found. Furthermore, GST–Yih1 coprecipitated large ribosomal protein Rpl39 independently of Gcn1. GST–Yih1 overexpression did not significantly affect Gcn1–ribosome or Gcn2–ribosome cosedimentation. myc‐tagged Yih1 expressed from its own promoter cosedimented with polyribosomes independently of Gcn1, indicating that Yih1–ribosome interaction occurs under physiological conditions. GST–IMPACT cosedimented with yeast ribosomes and coprecipitated Rpl39 in a Gcn1‐independent fashion, suggesting that Yih1/IMPACT–ribosome association is evolutionarily conserved. Moreover, GST–IMPACT coprecipitated actin as found for GST–Yih1. Taken together, our findings strongly suggest that IMPACT/Yih1 associates with ribosomes and that these ribosomes may simultaneously carry Gcn1 and Gcn2. Close physical proximity of Yih1 to the Gcn1–Gcn2–ribosome complex would allow cells to quickly inhibit Gcn2 whenever or wherever necessary.
Structured digital
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Polyribosomes physically interact with YIH1, GCN1, GCN2, eEF3, eEF1A, GIR2, RPL39, RPS22, by co‐sedimentationassays (Viewinteraction)
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YIH1 physicallyinteracts with RPL39 by cross‐linkingstudy (Viewinteraction)
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IMPACT physicallyinteracts with GCN1, actin, RPS22 and RPL39 by pulldown (Viewinteraction)
For sensing amino acid starvation the protein kinase Gcn2 must bind to Gcn1, and both must contact ribosomes. Yih1, or its mammalian ortholog IMPACT, inhibit Gcn2 by disrupting Gcn1‐Gcn2 interaction. Here we provide evidence that Yih1/IMPACT is in a complex with ribosomes, raising the possibility that close physical proximity of Yih1/IMPACT to the Gcn1‐Gcn2‐ribosome complex allows quick Gcn2 inhibition.
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•A synthesis of the current knowledge on GCN2′s relevance in viral defence.•GCN2 can sense viral infections through various avenues.•Viruses utilise multiple avenues to counteract or ...exploit GCN2 and the GCN2 pathway.•Knowledge on molecular mechanisms holds promise for new anti-viral measures.•Viral mechanisms may be exploited for non-communicable disease treatment and more.
The recent SARS-CoV-2 pandemic and associated COVID19 disease illustrates the important role of viral defence mechanisms in ensuring survival and recovery of the host or patient. Viruses absolutely depend on the host’s protein synthesis machinery to replicate, meaning that impeding translation is a powerful way to counteract viruses. One major approach used by cells to obstruct protein synthesis is to phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Mammals possess four different eIF2α-kinases: PKR, HRI, PEK/PERK, and GCN2. While PKR is currently considered the principal eIF2α-kinase involved in viral defence, the other eIF2α-kinases have also been found to play significant roles. Unsurprisingly, viruses have developed mechanisms to counteract the actions of eIF2α-kinases, or even to exploit them to their benefit. While some of these virulence factors are specific to one eIF2α-kinase, such as GCN2, others target all eIF2α-kinases. This review critically evaluates the current knowledge of viral mechanisms targeting the eIF2α-kinase GCN2. A detailed and in-depth understanding of the molecular mechanisms by which viruses evade host defence mechanisms will help to inform the development of powerful anti-viral measures.
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