scholarly journals The generation of persister cells is regulated at the initiation of translation by (p)ppGpp

2020 ◽  
Author(s):  
Roberto C. Molina-Quiroz ◽  
Andrew Camilli
Keyword(s):  
Protein Synthesis ◽  
Protein Translation ◽  
Synthesis Process ◽  
Phenotypic Trait ◽  
Dependent Manner ◽  
Bacterial Survival ◽  
Persister Cells ◽  
Initiation Of Translation ◽  
Persister Cell

AbstractBacterial persistence is a non-heritable phenotypic trait characterized by a dormant state that leads to tolerance to different antibiotics. Several mechanisms contributing to persister cells generation have been identified. Among these, is the signaling molecule (p)ppGpp, but knowledge of how this molecule regulates persister generation is incomplete. Here, we show an increase of the persister fraction of uropathogenic Escherichia coli (UPEC) that correlates with the time of protein synthesis inhibition and a decrease in the availability of antibiotic target. Specifically, the arrest of translation initiation induces bacterial survival to ampicillin and ciprofloxacin in a (p)ppGpp-dependent manner. These findings support a global mechanism of persister cell generation and establish a regulatory role of the (p)ppGpp molecule in this phenomenon.ImportanceThe study of persister cell formation is relevant because this bacterial subpopulation is involved in the emergence of antibiotic resistance and the generation of chronic infections. A role of the (p)ppGpp molecule in the generation of the persister fraction has been described, but the identification of the regulatory mechanism mediated by this alarmone during protein translation and its contribution to persistence has not been described to date. In this work, we show that (p)ppGpp regulates the generation of persister cells at the initiation of the protein synthesis process in UPEC. Our results also suggest that a (p)ppGpp-dependent regulation of translation, might be a global mechanism for the generation of the persister fraction.

Oxygen reactive radicals production in cell culture by okadaic acid and their implication in protein synthesis inhibition

1999 ◽  
Vol 18 (10) ◽  
pp. 634-639 ◽  
Author(s):  
W G Matias ◽  
A Traore ◽  
M Bonini ◽  
A Sanni ◽  
E E Creppy
Keyword(s):  
Lipid Peroxidation ◽  
Protein Synthesis ◽  
Okadaic Acid ◽  
Synthesis Process ◽  
Dependent Manner ◽  
Protein Synthesis Inhibition ◽  
Free System ◽  
Synthesis Inhibition ◽  
Inhibition Of Protein Synthesis ◽  
Reactive Radicals

Okadaic acid (OA), a diarrhetic shellfish toxin is a potent promoter of tumours in mouse skin and a specific inhibitor of protein phosphatases 1 and 2A. Recently it has been shown that OA inhibited protein synthesis in a cell-free system, with 50% inhibitory concentration of 6.3610712 M but the mechanism whereby this inhibition is mediated was still unclear. In the present study, the effect of OA on protein synthesis in Vero cell cultures was investigated. Protein synthesis was inhibited by OA alone in Vero cells in a concentration-dependent manner (IC50=27 ng/ml i.e. 3.361078 M). Since OA also induced lipid peroxidation and likely oxygen reactive radicals, it was interesting to know whether these radicals impair the protein synthesis process. Therefore, SOD+catalase known as scavenger of active oxygen radicals were added in the culture medium in the presence of OA and labelled leucine. These enzymes partially prevented the inhibition of protein synthesis induced by OA, indicating that the formation of high reactive oxygen free radicals could be one of the pathways this marine toxin induces its toxicity. Since the prevention by SOD+catalase was only partial (the IC50 increased from 27 ng/ml to 48 ng/ml i.e. 3.361078 M to 5.961078 M)itwas speculated that the production of oxygen reactive radical scavengered by SOD+catalase is not the main mechanism whereby OA induces its cytotoxicity. Vitamins E and C completely prevent the lipid peroxidation induced by OA in cells, but failed to reduce the inhibition of protein synthesis to the same level, indicating that a more specific mechanism might be responsible for protein synthesis inhibition. That is the hyperphosphorylation of elongation factor EF-2 in the protein synthesis machinery. However our results pointed to lipid peroxidation being a precocious phenomenon following the OA exposure, since a concentrationwithenhancedMDAproductionwaslower than that inducing significant cellular protein synthesis inhibition.

scholarly journals Functional Characterization of the Canine Heme-Regulated eIF2αKinase: Regulation of Protein Synthesis

2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Kimon C. Kanelakis ◽  
Jayashree Pyati ◽  
Pamela C. Wagaman ◽  
Jui Chang Chuang ◽  
Young Yang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Expression System ◽  
Functional Characterization ◽  
Protein Translation ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Suitable Model ◽  
Dependent Manner

The heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α(eIF2α) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

scholarly journals Role of Global Regulators and Nucleotide Metabolism in Antibiotic Tolerance in Escherichia coli

2008 ◽  
Vol 52 (8) ◽  
pp. 2718-2726 ◽  
Author(s):  
Sonja Hansen ◽  
Kim Lewis ◽  
Marin Vulić
Keyword(s):  
Escherichia Coli ◽  
Cell Formation ◽  
Nucleotide Metabolism ◽  
Flavin Mononucleotide ◽  
Formation Mechanisms ◽  
Antibiotic Tolerance ◽  
Persister Cells ◽  
Global Regulators ◽  
Persister Cell

ABSTRACT Bacterial populations produce a small number of persister cells that exhibit multidrug tolerance. Persister cells are largely responsible for the antibiotic recalcitrance of biofilm infections. The mechanism of persister cell formation largely remains unknown due to the challenges in identifying persister genes. We screened an ordered comprehensive library of 3,985 Escherichia coli knockout strains to identify mutants with altered antibiotic tolerance. Stationary-state cultures in 96-well plates were exposed to ofloxacin at a concentration which allows only tolerant persister cells to survive. The persister cell level of each culture was determined. A total of 150 mutants with decreased persistence were identified in the initial screen, and subsequent validation confirmed that neither the growth rate nor the ofloxacin MIC was affected for 10 of them. The genes affected in these strains were dnaJ and dnaK (chaperones), apaH (diadenosine tetraphosphatase), surA (peptidyl-prolyl cis-trans isomerase), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (transcriptional regulator of rRNA transcription), ygfA (5-formyl-tetrahydrofolate cyclo-ligase), and yigB (flavin mononucleotide [FMN] phosphatase). The prominent presence of global regulators among these strains pointed to the likely redundancy of persister cell formation mechanisms: the elimination of a regulator controlling several redundant persister genes would be expected to produce a phenotype. This observation is consistent with previous findings for a possible role of redundant genes such as toxin/antitoxin modules in persister cell formation. ygfA and yigB were of special interest. The mammalian homolog of YgfA (methenyltetrahydrofolate synthetase) catalyzes the conversion of 5-formyl-tetrahydrofolate (THF) into the rapidly degraded 5,10-methenyl-THF, depleting the folate pool. The YigB protein is a phosphatase of FMN which would deplete the pool of this cofactor. Stochastic overexpression of these genes could lead to dormancy and, hence, tolerance by depleting the folate and FMN pools, respectively. Consistent with this scenario, the overexpression of both genes produced increased tolerance to ofloxacin.

scholarly journals Role of PKR in the Inhibition of Proliferation and Translation by Polycystin-1

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yan Tang ◽  
Guang Shi ◽  
JungWoo Yang ◽  
Wang Zheng ◽  
Jingfeng Tang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Synthesis ◽  
Kinase Activity ◽  
Protein Translation ◽  
Dominant Negative ◽  
Inhibition Of Proliferation ◽  
Proliferation And Apoptosis ◽  
Autosomal Dominant Polycystic Kidney ◽  
Underlying Mechanisms

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations in the PKD1 (~85%) or PKD2 (~15%) gene which, respectively, encode polycystin-1 (PC1) and polycystin-2 (PC2). How PC1 regulates cell proliferation and apoptosis has been studied for decades but the underlying mechanisms remain controversial. Protein kinase RNA-activated (PKR) is activated by interferons or double-stranded RNAs, inhibits protein translation, and induces cell apoptosis. In a previous study, we found that PC1 reduces apoptosis through suppressing the PKR/eIF2α signaling. Whether and how PKR is involved in PC1-inhibited proliferation and protein synthesis remains unknown. Here we found that knockdown of PKR abolishes PC1-inhibited proliferation and translation. Because suppressed PKR-eIF2α signaling/activity by PC1 would stimulate, rather than inhibit, the proliferation and translation, we examined the effect of dominant negative PKR mutant K296R that has no kinase activity and found that it enhances the inhibition of proliferation and translation by PC1. Thus, our study showed that inhibition of cell proliferation and protein synthesis by PC1 is mediated by the total expression but not the kinase activity of PKR, possibly through physical association.

scholarly journals Endurance exercise decreases protein synthesis and ER-mitochondria contacts in mouse skeletal muscle

2019 ◽  
Vol 127 (5) ◽  
pp. 1297-1306 ◽  
Author(s):  
Audrey Merle ◽  
Maxence Jollet ◽  
Florian A. Britto ◽  
Bénédicte Goustard ◽  
Nadia Bendridi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Dna Damage ◽  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Translation Initiation ◽  
Energy Demand ◽  
Protein Translation ◽  
Strong Increase ◽  
Hindlimb Muscles

Exercise is important to maintain skeletal muscle mass through stimulation of protein synthesis, which is a major ATP-consuming process for cells. However, muscle cells have to face high energy demand during contraction. The present study aimed to investigate protein synthesis regulation during aerobic exercise in mouse hindlimb muscles. Male C57Bl/6J mice ran at 12 m/min for 45 min or at 12 m/min for the first 25 min followed by a progressive increase in velocity up to 20 m/min for the last 20 min. Animals were injected intraperitoneally with 40 nmol/g of body weight of puromycin and euthanized by cervical dislocation immediately after exercise cessation. Analysis of gastrocnemius, plantaris, quadriceps, soleus, and tibialis anterior muscles revealed a decrease in protein translation assessed by puromycin incorporation, without significant differences among muscles or running intensities. The reduction of protein synthesis was associated with a marked inhibition of mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1, a mechanism consistent with reduced translation initiation. A slight activation of AMP-activated protein kinase consecutive to the running session was measured but did not correlate with mTORC1 inhibition. More importantly, exercise resulted in a strong upregulation of regulated in development and DNA damage 1 (REDD1) protein and gene expressions, whereas transcriptional regulation of other recognized exercise-induced genes ( IL-6, kruppel-like factor 15, and regulator of calcineurin 1) did not change. Consistently with the recently discovered role of REDD1 on mitochondria-associated membranes, we observed a decrease in mitochondria-endoplasmic reticulum interaction following exercise. Collectively, these data raise questions concerning the role of mitochondria-associated endoplasmic reticulum membrane disruption in the regulation of muscle proteostasis during exercise and, more generally, in cell adaptation to metabolic stress. NEW & NOTEWORTHY How muscles regulate protein synthesis to cope with the energy demand during contraction is poorly documented. Moreover, it is unknown whether protein translation is differentially affected among mouse hindlimb muscles under different physiological exercise modalities. We showed here that 45 min of running decreases puromycin incorporation similarly in 5 different mouse muscles. This decrease was associated with a strong increase in regulated in development and DNA damage 1 protein expression and a significant disruption of the mitochondria and sarcoplasmic reticulum interaction.

scholarly journals Protein synthesis regulation by leucine

2010 ◽  
Vol 46 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Daiana Vianna ◽  
Gabriela Fullin Resende Teodoro ◽  
Francisco Leonardo Torres-Leal ◽  
Julio Tirapegui
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mrna Translation ◽  
Protein Translation ◽  
Cellular Effects ◽  
Cellular Processes ◽  
High Protein Diets

In vivo and in vitro studies have demonstrated that high protein diets affect both protein synthesis and regulation of several cellular processes. The role of amino acids as substrate for protein synthesis has been established in the literature. However, the mechanism by which these amino acids modulate transcription and regulate the mRNA translation via mTOR-dependent signaling pathway has yet to be fully determined. It has been verified that mTOR is a protein responsible for activating a cascade of biochemical intracellular events which result in the activation of the protein translation process. Of the aminoacids, leucine is the most effective in stimulating protein synthesis and reducing proteolysis. Therefore, it promotes a positive nitrogen balance, possibly by favoring the activation of this protein. This amino acid also directly and indirectly stimulates the synthesis and secretion of insulin, enhancing its anabolic cellular effects. Therefore, this review aimed to identify the role of leucine in protein synthesis modulation and to discuss the metabolic aspects related to this aminoacid.

scholarly journals Identification of a secretion-enhancing cis regulatory targeting element (SECReTE) involved in mRNA localization and protein synthesis

2018 ◽  
Author(s):  
Osnat Cohen-Zontag ◽  
Lisha Qiu Jin Lim ◽  
Dvir Dahary ◽  
Tsviya Olender ◽  
Yitzhak Pilpel ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Protein Translation ◽  
Active Role ◽  
Mrna Localization ◽  
Dependent Manner ◽  
Translation Product ◽  
Synonymous Mutations ◽  
Regulatory Processes

AbstractEarlier dogma states that mRNAs encoding secreted and membrane protein (mSMPs) reach the ER in a translation-dependent manner through the signal recognition particle (SRP) pathway. In this pathway, the signal sequence of the translation product is recognized by SRP and the mRNA-ribosome-nascent-chain-SRP complex is recruited to the ER via the interaction with an endoplasmic reticulum (ER)-localized SRP receptor. This model suggests that the translation product dictates the delivery of mRNAs to the ER and that the mRNA is a passive passenger. However, new evidence challenges this model and implies the existence of both translation - and SRP-independent mRNA localization to the ER, raising the possibility that mRNAs have an active role in determining their localization to the ER.Besides serving as a template for protein translation, mRNAs carry information required for other regulatory processes such as mRNA processing, translation and transcription efficiency, degradation and localization. In yeast, mRNA localization governed by cis-acting sequence elements has been characterized for asymmetrically (e.g. bud) localized mRNAs that localize to, and are transported with, cortical ER. Now, we identify a cis motif in mSMPs that targets mRNAs mainly to the nuclear ER in yeast and increases both protein synthesis and secretion. Termed SECReTE, for secretion-enhancing cis regulatory targeting element, this motif was identified by computational analysis of genes encoding secretome proteins. SECReTE consists of ≥10 repetitive triplets enriched with pyrimidines (i.e. C’s and U’s) every third base (i.e. NNY, N - any nucleotide, Y - pyrimidine), and is found particularly in mRNAs coding for cell wall proteins. To study the physiological relevance of SECReTE, we introduced synonymous mutations that either elevate or decrease its overall score in genes coding for secreted proteins, without changing the protein sequence, and examined the physiological effects in yeast. An increase in the SECReTE score elevated the synthesis and secretion of endogenous proteins while, in contrast, a reduction led to less secretion and physiological defects. Importantly, the addition of SECReTE to the 3’UTR of an exogenous protein (e.g. SS-GFP) led to its increased secretion from yeast. SECReTE is present all through evolution and, thus, constitutes a novel RNA targeting motif found in both prokaryotes and eukaryotes.

scholarly journals Transient Hsp90 suppression promotes a heritable change in protein translation

2018 ◽  
Author(s):  
Peter Tsvetkov ◽  
Zarina Brune ◽  
Timothy J. Eisen ◽  
Sven Heinrich ◽  
Greg A. Newby ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Heat Shock Protein 90 ◽  
Protein Translation ◽  
Translation Regulation ◽  
Hsp90 Inhibition ◽  
Transient Loss ◽  
Hsp90 Chaperone ◽  
Physical Interactions ◽  
Heritable Change

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a unique role promoting the evolution of new heritable traits. To investigate the role of Hsp90 in modulating protein synthesis, we screened more than 1200 proteins involved in mRNA regulation for physical interactions with Hsp90 in human cells. Among the top hits was CPEB2, which strongly binds Hsp90 via its prion domain, reminiscent of the prion-like regulation of translation of Aplysia CPEB. In a yeast model of CPEB prion-dependent translation regulation, transient inhibition of Hsp90 amplified CPEB2 prion activity and resulted in persistent translation of the CPEB reporter. Remarkably, inhibition of Hsp90 was sufficient to induce a heritable change in protein translation that persisted for 30 generations, even in the absence of exogenous CPEB. Although we identified a variety of perturbations that enhanced translation of the reporter, only Hsp90 inhibition led to persistent activation. Thus, transient loss of Hsp90 function leads to the non-genetic inheritance of a novel translational state. We propose that, in addition to sculpting the conformational landscape of the proteome, Hsp90 promotes phenotypic variation by modulating protein synthesis.

Inhibition of MAP Kinase-Interacting Kinase-1 (Mnk1) Regulates Platelet Functional Responses and Protein Synthesis in Megakaryocytes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 711-711
Author(s):  
Bhanu Kanth Manne ◽  
Rachit Badolia ◽  
Robert A. Campbell ◽  
Satya P. Kunapuli ◽  
Andrew S. Weyrich ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Arachidonic Acid ◽  
Translational Control ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Protein Translation ◽  
Dependent Manner ◽  
Functional Responses ◽  
Proplatelet Formation

Abstract MAPK-interacting kinases (Mnks) are a subfamily of serine/threonine kinases with two isoforms: Mnk1 and Mnk2. Mnks control protein synthesis in neurons and neutrophils, in part through Erk or p38 MAPK phosphorylation. Dysregulated translational control mechanisms are implicated in disorders of cell growth and proliferation. Nevertheless, the expression and function of Mnks in megakaryocytes and platelets is not known. In this study,we investigated the Mnk1 pathway in megakaryocytes and platelets. Mnk1, but not Mnk2, is robustly expressed in human and murine megakaryocytes and platelets (Figure A). Megakaryocytes in their final stages of proplatelet formation expressed activated Mnk1. Interestingly, inhibition of Mnk1 in megakaryocytes with ETP-45835, a specific Mnk1 pharmacological inhibitor, reduced proplatelet formation. Since protein synthesis is critical in the final stages of proplatelet formation, we next sought to determine if Mnk1 regulated translation in megakaryocyte. In neurons, Mnk1 is known to regulate protein translation by releasing CYFIP1 and FMR1 from eIF4E, allowing for the formation of the translational machinery. However, this pathway has not been examined in megakaryocytes. We observed, for the first time, that CYFIP1 and FMR1 are expressed in megakaryocytes and activation of Mnk1 in megakaryocytes resulted in eIF4E phosphorylation and Mnk1-eIF4E complex formation. Importantly, Mnk1 inhibition significantly reduced (~30%, p<0.05) protein synthesis (Figure A). However, the level of inhibition was not as great as with global translation inhibitors, suggesting Mnk1 regulated translation of a subset of mRNAs. We next sought to determine the role of Mnk1 in circulating platelets. Stimulation of washed platelets with thrombin, collagen, and 2MeS-ADP caused Mnk1 activation in a time-dependent manner. Mnk1 activation was abolished in the presence of U0126, an Erk1/2 specific inhibitor, suggesting that Erk regulates Mnk1 activation in platelets. Inhibiting Mnk1 activation ex vivo with ETP-45385 blocked agonist-dependent platelet aggregation, granule secretion, and thromboxane generation (Figure B). The addition of arachidonic acid to ETP45385-treated platelets fully rescued platelet functional responses, demonstrating that Mnk1 activation in platelets regulates the arachidonic acid pathway. Blocking Mnk1 activation with ETP-45385 also markedly inhibited platelet adhesion to collagen under flow conditions (Figure B). Mnk1 inhibition in vivo with ETP-45385 protected mice from collagen and epinephrine-induced pulmonary embolism. Taken together, our findings demonstrate that Mnk1 a canonical translation regulatory protein, mediates megakaryocyte proplatelet formation and protein translation, but it also responsible for reducing susceptibility to in vivo thrombosis through thromboxane dependent mechanism. We hypothesize that Mnk1 and its downstream targets represent new regulatory mechanisms for translational control in megakaryocytes and platelets. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals An RNA Interference Screen Identifies a Novel Regulator of Target of Rapamycin That Mediates Hypoxia Suppression of Translation in Drosophila S2 Cells

2008 ◽  
Vol 19 (10) ◽  
pp. 4051-4061 ◽  
Author(s):  
Soo-Jung Lee ◽  
Renny Feldman ◽  
Patrick H. O'Farrell
Keyword(s):  
Protein Synthesis ◽  
Rna Interference ◽  
Fluorescent Protein ◽  
Tyrosine Phosphatase ◽  
Protein Translation ◽  
Target Of Rapamycin ◽  
Dependent Manner ◽  
S2 Cells ◽  
A Genome ◽  
Drosophila S2 Cells

In addition to its central role in energy production, oxygen has pervasive regulatory actions. Hypoxia (oxygen limitation) triggers the shutdown of major cellular processes, including gene expression. We carried out a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells for functions required to down-regulate translation during hypoxia. RNAi knockdown of specific genes allowed induction of a green fluorescent protein (GFP) reporter gene and continued protein synthesis during hypoxia. Among the identified genes, Tsc1 and Tsc2, which together form the tuberose sclerosis complex that negatively regulates target of rapamycin (TOR) kinase, gave an especially strong effect. This finding is consistent with the involvement of TOR in promoting translation. Another gene required for efficient inhibition of protein translation during hypoxia, the protein tyrosine phosphatase 61F (Ptp61F), down-regulates TOR activity under hypoxia. Lack of Ptp61F or Tsc2 improves cell survival under prolonged hypoxia in a TOR-dependent manner. Our results identify Ptp61F as a novel modulator of TOR activity and suggest that its function during hypoxia contributes to the down-regulation of protein synthesis.

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