Decreased Rate Of Protein Synthesis Research Articles

Life after transcription: control of the fate of neurons

The neocortex is a complex structure responsible for higher-order cognitive abilities in mammals. It consists of six cell layers, each comprised of various subtypes of excitatory and inhibitory neurons. These neurons project their axons to specific targets within each layer. All neocortical projection neurons are generated by neural progenitor cells located in the proliferative ventricular zone. In recent decades, significant strides have been made in comprehending the transcriptional programs governing neuronal cell fate specification. Nonetheless, posttranscriptional mechanisms involved in this process remain largely unexplored. Recently, we found that TrkC-T1, an isoform of the TrkC receptor lacking the kinase domain, determines the fate of corticofugal projection neurons (CFuPN). Our study reveals that the balance between TrkC-T1 and TrkC-TK+, a more widely recognized isoform containing the kinase domain, depends on the type of cell within the developing cortex. Additionally, we demonstrate that two RNA-binding proteins, Srsf1 and Elavl1, work in opposition to establish this balance. Additionally, our data suggests that Srsf1 stimulates the CFuPN fate while Elavl1 stimulates the callosal projection neuron (CPN) fate in vivo by regulating the different TrkC-T1 to TrkC-TK+ ratios. In this study, we identified a protein translation-dependent mechanism that governs the cell fate switch in the developing neocortex. Our results demonstrate that Ire1α, the Inositol-Requiring Enzyme 1α, regulates global translation rates in the developing neocortex through its dynamic interaction with the ribosome, as well as the regulation of expression of translation elongation factors eIF4A1 and eEF-2. Inactivation of Ire1α leads to decreased protein synthesis rates that are associated with stalled ribosomes and a reduced number of sites for translation initiation. We demonstrate the distinctive sensitivity of neurons determined for the upper layer to translation rates. While eEF-2 is necessary for cortical lamination, eIF4A1 regulates the attainment of upper layer fate in a mechanism of translational control that is dependent on structural elements embedded in the 5’UTR of genes responsible for determining fate downstream of Ire1α. Our findings reveal the developmental control of ribosome dynamics as post-transcriptional mechanisms that coordinate the establishment of neuronal diversity and the assembly of cortical layers.

Rates of Synthesis of Individual Proteins in Muscles of Old Mice: A Heavy Water Approach.

Age‐associated loss of muscle mass and function is a major factor in the development of frailty, but the mechanisms responsible for this loss are unknown. Unquestionably, muscle atrophy is the consequence of changes in the balance between protein synthesis and breakdown. However, the nature and extent of these changes in old age is unclear, with some studies reporting reduced basal muscle protein synthetic rate in the aged compared with younger people and others suggesting no difference. Whole organism heavy water ([2H2]O) dynamic labelling of proteins, followed by LC‐MSMS, provides a significant improvement in analysis of protein turnover, allowing the rates of synthesis and degradation of both slow and fast turnover muscle proteins to be accurately modelled. The aim of this study was to use a [2H2]O labelling approach to determine the synthesis rates of individual proteins in the gastrocnemius muscles of adult and old mice.Ten adult and ten old male mice were primed with an intraperitoneal bolus injection of saline, prepared with 99% [2H2]O and subsequently, provided with 8% (v/v) [2H2]O in the drinking water, for up to 60 days. The sampling interval was distributed to obtain sufficient data points to cover both high and low turnover proteins. Muscle proteins were extracted and digested using standard procedures for analysis on a high‐resolution mass spectrometer (Thermo Q‐Exactive) and the raw data files were processed to allow protein identification using Crux‐tide ( McIlwain et al, 2014). The software RIAna (: https://github.com/Molecular‐Proteomics/riana) was then used to track time dependent incorporation of [2H] into tryptic peptides, yielding synthesis rates of proteins to be determined.Muscle mass was significantly reduced in the old mice and preliminary data demonstrated little change in the rate of synthesis of a large proportion of identified muscle proteins between adult and old mice. Several individual proteins showed a marked decrease in protein synthesis rates in muscles of old compared with adult mice, particularly structural proteins. Other proteins, particularly redox‐responsive proteins demonstrated an increased synthesis in muscles of old mice. Proteomic analysis of unlabelled muscle protein pools and RNASeq data will be integrated with the turnover data to further interpret these findings.This study was supported by the University of Liverpool, MRC and NIA (AG051442).

Age-related differences in sleep disturbances in rat models of preclinical Parkinson's disease

To describe the changes in temporal characteristics of sleep-wake cycle, which can serve as non-motor manifestations of an early stage of Parkinson's disease (PD), using the model of preclinical PD in rats of two age groups. A prolonged (up to 21 days) model of preclinical PD in middle-aged (7-8 month) and aged (19-20 month) rats was created. The model was based on cumulative inhibition of proteasomal system of the brain caused by intranasal administration of lactacystin, a specific proteasome inhibitor. Polysomnographic data were recorded daily using telemetric Dataquest A.R.T. System (DSI, USA) in unrestrained animals. Aging was accompanied with increased sleepiness during the active (dark) phase of the day (as was implied by a two-fold increase in the total time of drowsiness) and with 1.5-fold growth of light sleep during the inactive phase of the day. A common feature of sleep disturbances in the model of preclinical PD in both middle-aged and aged rats was hypersomnia during the active phase of the day. It was suggested to be similar to the excessive daytime sleepiness in humans. Hypersomnolence was more pronounced in aged rats because it added to sleepiness developing with aging. In both age groups, the model of preclinical PD was also associated with a decrease in EEG delta power during slow-wave sleep. It is considered dangerous because it might represent the decrease in protein synthesis rate and the weakening of restorative processes in neurons, occurring with the prolonged inhibition of proteasomal system of the brain. Sleep disturbances, identified the model of preclinical PD in rats of different age, may be recommended for clinical validation as low-cost early signs indicating the initial stage of PD.

EIF3f depletion impedes mouse embryonic development, reduces adult skeletal muscle mass and amplifies muscle loss during disuse.

In muscular cells, eukaryotic initiation factor subunit f (eIF3f) activates protein synthesis by allowing physical interaction between mechanistic target of rapamycin complex 1 (MTORC1) and ribosomal protein S6 kinase 1 (S6K1), although its physiological role in animals is unknown. A knockout approach suggests that homozygous mice carrying a null mutation of the eIF3f gene fail to develop and consequently die at early embryonic stage, whereas heterozygous mice associated with a partial depletion of eIF3f gene grow normally and are phenotypically indistinguishable from wild-type mice. Heterozygous mice express reduced eIF3f mRNA and protein levels in skeletal muscles and show diminished muscle mass associated with a decrease in the protein synthesis rate and an inhibition of the MTORC1 pathway. During hindlimb immobilization, heterozygous eIF3f mice display an exacerbated immobilization-induced muscle atrophy associated with reduced protein synthesis. These results highlight the essential role of eIF3f during embryonic development and its involvement in muscular homeostasis via protein synthesis regulation. Eukaryotic translation initiation factor 3, subunit F (eIF3f), a component of eIF3 complex, plays an important role in protein synthesis regulation, although its physiological functions are unknown. We generated and analysed mice carrying a null mutation in the eIF3f gene. We showed that homozygous eIF3f knockout fail to develop and that eIF3f-/- embryos die at an early stage of development but after the pre-implantation stage. However, disrupting one eIF3f allele does not affect growth, viability and fertility of heterozygous mice but, instead, reduces eIF3f mRNA and protein levels in all tissues examined. Although heterozygous mice are phenotypically indistinguishable from wild-type mice, they present a diminished body weight and a lean mass reduction associated with normal body size. Interestingly, skeletal muscles are mainly affected and display an altered cell size without modification of fibre number. Skeletal muscles of heterozygous mice show a deficiency in polysome content, a decrease in protein synthesis rate and an inhibition of the mechanistic target of rapamycin (MTOR) pathway. We then studied the effects of hindlimb immobilization that mimic muscle disuse on heterozygous mice aiming to further explore the involvement of eIF3f in protein synthesis. We found that eIF3f partial depletion amplifies muscle atrophy compared to wild-type mice. Mass and cross-sectional area decreases were associated with reduced MTOR pathway activation and protein synthesis rate. Taken together, our data indicate that eIF3f is essential for mice embryonic development and controls adult skeletal muscle mass via protein synthesis regulation in a MTOR-dependent manner.

Relationship between Sarcopenia and mTOR Pathway in Patients with Colorectal Cancer: Preliminary Report

Sarcopenia is a syndrome characterized by loss of muscle mass and strength that impacts clinical outcomes and mortality in cancer patients. Although the molecular pathways involved in sarcopenia are not fully elucidated, the decrease in protein synthesis rate appears to be one of the most important events. The objective of this study was to investigate the relationship between sarcopenia and mTOR signaling pathway in patients undergoing colorectal resection surgery. Three groups of patients were assessed: 1) the control group (no cancer, no sarcopenia), 2) the cancer non-sarcopenic group and 3) the cancer sarcopenic group. All individuals were evaluated in relation to presence of sarcopenia and mTOR signaling pathway. Sarcopenia was evaluated by the combination of low muscle mass and low muscle strength, measured using computerized tomography images, and hand grip strength, respectively. Rectus abdominis muscle biopsy was performed at the time of surgery. mTOR pathway was analyzed by MILLIPLEX Map Kit Phospho/total mTOR 2-Plex Magnetic Bead Panel. Results were presented by phosphor/total mTOR ratio. Independent T test, Kruskal-Wallis test, and Dunn-Bonferroni post hoc were performed for statistical analysis and P < 0.05 was considered. Thirty-six patients and five controls were evaluated. A total of 13 cancer patients (36.1%) had sarcopenia. The phospho/total mTOR ratio was different between the control group (0.167 MFI) and the cancer non-sarcopenic group (0.055 MFI) (P = 0.026) as well as between the control group (0.167 MFI) and the cancer sarcopenic group (0.0049 MFI) (P = 0.041). No difference was observed on the median phospho/total mTOR ratio between the cancer groups (P > 0.05). More research is needed to extrapolate these results.

Tribbles 3 Regulates Skeletal Muscle Mass in Fasting‐induced Atrophy

Tribbles homolog 3 (TRB3) is a pseudokinase that has been found in multiple tissues, including skeletal muscle, in response to various stress stimuli, such as insulin resistance, nutrient deprivation, and endoplasmic reticulum stress. TRB3 has been mainly known as a negative regulator of Akt, which is an essential protein kinase to control protein synthesis and breakdown. Recently, our lab has demonstrated that TRB3 decreases protein synthesis and increases protein degradation in mouse skeletal muscle at basal state. However, it still remains to be determined whether TRB3 regulates skeletal muscle mass under atrophic conditions. Here, we hypothesized that TRB3 has a detrimental effect on skeletal muscle mass under 48hr fasting‐induced atrophy. In order to test the hypothesis, we utilized C57BL/6 wild type (WT) mice and fasted them for 48hr to induce skeletal muscle atrophy. Our fasting strategy reduced body weight by 20% and effectively decreased skeletal muscle mass by 15–25% compared to fed controls in multiple muscles (TA; 26%, EDL; 26%, GAS;14%, all P&lt;0.05). In addition, TRB3 protein (60%) and mRNA (49%, P&lt;0.05) expression was significantly increased in fasted muscles, which was associated with elevated atrogin−1 (750%, P&lt;0.01) and MuRF‐1 (1840%, P&lt;0.001) mRNA expression. Next, we analyzed Akt and its downstream proteins associated with protein synthesis and degradation. Phosphorylation of Akt (T308) was significantly decreased after 48hr fasting (37%, P&lt;0.05) and FOXO1 (69%, P&lt;0.01) and FOXO3a (47%, P=0.10) were activated whereas protein synthesis, mTOR and p70S6K1, was not affected. Next, we fasted muscle‐specific TRB3 transgenic (TG) and WT mice for 48hr in order to determine if TRB3 overexpression worsens the fasting‐induced atrophy. Body weight was significantly reduced by 22% after 48hr fasting in both WT and TG groups. Interestingly, we found a significant reduction in muscle mass in TG mice compared to WT (WT vs. TG in EDL; P&lt;0.05, in GAS; P=0.07). Further, Akt and its downstream proteins were determined in EDL muscle. In TG mice, Akt phosphorylation (T308) was significantly decreased after fasting (18%, P&lt;0.05) in line with suppressed S6K1 (10%, P=0.065) and activated FOXO1 (76%, P&lt;0.001) and FOXO3a (32%, P=0.068) signaling. Additionally, protein synthesis rate was assessed by the incorporation of puromycin into nascent peptide chains. 48hr fasting significantly decreased protein synthesis rate in both WT and TG (P&lt;0.01) but the degree of reduction was prominent in TG mice compared to WT (P=0.08). Lastly, we studied 48hr fasting‐induced atrophy in TRB3 knockout (KO) mice to determine if the deletion of TRB3 could prevent fasting‐induced atrophy. Body weight was significantly decreased by 20% in both WT and KO groups. Although both genotypes significantly reduced body weight, KO mice interestingly preserved ~10% more muscle mass in fasted TA, EDL, and SOL compared to fasted WT muscles (TA; 8.5%, EDL;15%, P&lt;0.05, SOL;3.6%). These data suggest that TRB3 could be a key regulator of protein synthesis and breakdown through the Akt/mTOR/FOXO signaling under 48hr fasting‐induced atrophy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Eukaryotic Translation Initiation Factor 2A (eIF2A) Protects Pancreatic Beta Cells During ER Stress-Induced ApoptosisImage 6

Unfolded protein response (UPR) induced in response to endoplasmic reticulum (ER) stress is a well-known mechanism mediating beta cell dysfunction and death in diabetes. Inhibition of general protein translation via PERK-dependent phosphorylation of eIF2α is key event during UPR, which if unresolved induces apoptosis. eIF2A has been implicated in the translation of specific mRNAs during viral infection, when translation is similarly suppressed. We hypothesized that eIF2A plays a key role in translation of specific mRNAs during UPR in beta cells. We found that UPR inducers thapsigargin (1 µmol/L) and palmitate (0.5 mmol/L in BSA) induced mRNA (3.5±0.4-fold and 2.0±0.3) and protein (2.2±0.2-fold and 2.1±0.2) expression of eIF2A in mouse and human islets. We found 84.7±6.9% and 63.7±7.8% reductions in thapsigargin- and palmitate-induced apoptosis in mouse islet cells overexpressing eIF2A (3-fold), respectively. To elucidate molecular mechanism, we used a stable MIN6 cell line overexpressing eIF2A. Reduced cell death was associated with 88.3±8.4% decrease in translation of CHOP mRNA. MIN6 cells showed decreased protein synthesis rate by 80.2±10% after 2 hours of thapsigargin treatment. However, the protein synthesis rate was unchanged in cells overexpressing eIF2A. Immunoprecipitation of eIF2A showed increase in the interaction of eIF2A with eIF2α during UPR in MIN6 cells with the maximum of 5.9±1.1-fold increase 24 hours after thapsigargin treatment. We conclude that eIF2A suppresses UPR-induced apoptosis in beta cells via inhibition of translational repression. We identified a novel protective role for eIF2A in the context of ER-stressed beta cells via the selective inhibition of CHOP mRNA translation.

Reduced insulin/insulin-like growth factor signaling decreases translation in Drosophila and mice.

Down-regulation of insulin/insulin-like growth factor signaling (IIS) can increase lifespan in C. elegans, Drosophila and mice. In C. elegans, reduced IIS results in down-regulation of translation, which itself can extend lifespan. However, the effect of reduced IIS on translation has yet to be determined in other multicellular organisms. Using two long-lived IIS models, namely Drosophila lacking three insulin-like peptides (dilp2-3,5−/−) and mice lacking insulin receptor substrate 1 (Irs1−/−), and two independent translation assays, polysome profiling and radiolabeled amino acid incorporation, we show that reduced IIS lowers translation in these organisms. In Drosophila, reduced IIS decreased polysome levels in fat body and gut, but reduced the rate of protein synthesis only in the fat body. Reduced IIS in mice decreased protein synthesis rate only in skeletal muscle, without reducing polysomes in any tissue. This lowered translation in muscle was independent of Irs1 loss in the muscle itself, but a secondary effect of Irs1 loss in the liver. In conclusion, down-regulation of translation is an evolutionarily conserved response to reduced IIS, but the tissues in which it occurs can vary between organisms. Furthermore, the mechanisms underlying lowered translation may differ in mice, possibly associated with the complexity of the regulatory processes.

Abstract LB-077: T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy

Abstract The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ T cells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of strategies to increase ACT responses in cancer. Citation Format: Patrick Raber, Rosa A. Sierra, Shuzhong Zhang, Paul Thevenot, Paulo C. Rodriguez. T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-077.

T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy.

The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ Tcells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of better strategies to increase ACT responses in cancer.

MTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for myogenic differentiation.

Proteolytic signaling mechanisms in skeletal muscles in patients with alcohol-induced muscle disease

Chronic alcoholic myopathy is one of most numerous and profound manifestations of chronic alcohol intoxication. This disease is characterized by the pronounced atrophy of the locomotor muscles, which involves predominantly those fibers expressing myosin isoforms of the I "fast" type. In early experiments with alcohol-fed rats and studies of patients it was shown the impairment of the anabolic intracellular signaling pathways and decrease in protein synthesis rate. We were the first to analyze the signaling pathways involved in the pathogenesis of alcoholic myopathy with different fiber atrophy levels. At the early stages of the pathogenesis we observed also the sufficient increase of mRNA of E3 ubiquitin ligases. However the ubiquitinylation level was not altered in patients as compared to the control subjects. This phenomenon could be associated with the increased expression of the heat-shock proteins, known for their protective action.

A Self-defeating Anabolic Program Leads to β-Cell Apoptosis in Endoplasmic Reticulum Stress-induced Diabetes via Regulation of Amino Acid Flux

Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing β-cells in type 2 diabetes mellitus. β-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in β-cells in vivo. Paradoxically, chronic ER stress in β-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in β-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.

Modulation of the gut microbiota with antibiotic treatment suppresses whole body urea production in neonatal pigs

We examined whether changes in the gut microbiota induced by clinically relevant interventions would impact the bioavailability of dietary amino acids in neonates. We tested the hypothesis that modulation of the gut microbiota in neonatal pigs receiving no treatment (control), intravenously administered antibiotics, or probiotics affects whole body nitrogen and amino acid turnover. We quantified whole body urea kinetics, threonine fluxes, and threonine disposal into protein, oxidation, and tissue protein synthesis with stable isotope techniques. Compared with controls, antibiotics reduced the number and diversity of bacterial species in the distal small intestine (SI) and colon. Antibiotics decreased plasma urea concentrations via decreased urea synthesis. Antibiotics elevated threonine plasma concentrations and turnover, as well as whole body protein synthesis and proteolysis. Antibiotics decreased protein synthesis rate in the proximal SI and liver but did not affect the distal SI, colon, or muscle. Probiotics induced a bifidogenic microbiota and decreased plasma urea concentrations but did not affect whole body threonine or protein metabolism. Probiotics decreased protein synthesis in the proximal SI but not in other tissues. In conclusion, modulation of the gut microbiota by antibiotics and probiotics reduced hepatic ureagenesis and intestinal protein synthesis, but neither altered whole body net threonine balance. These findings suggest that changes in amino acid and nitrogen metabolism resulting from antibiotic- or probiotic-induced shifts in the microbiota are localized to the gut and liver and have limited impact on whole body growth and anabolism in neonatal piglets.

Endotoxin transiently inhibits protein synthesis through Akt and MAPK mediating pathways in C2C12 myotubes

In this study, the effect of lipopolysaccharide (LPS) on protein synthesis (PS) and intracellular signaling factors that regulate it have been investigated in C2C12 murine-derived myotubes. In particular, the role of Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinases (MAPKs) [p38 and extracelluar regulated protein kinase (ERK1/2)] have been examined. The direct effect of LPS on PS was measured at 3 and 18 h. LPS significantly decreased PS at 3 h but not at the 18-h time point. This effect was preceded by decreased Akt phosphorylation at 5 and 30 min after LPS administration. The mTOR phosphorylation exhibited a long time dose-dependent increase at all the time points. Similarly, the activity-related phosphorylation of p38 and ERK1/2 significantly increased in a time- and dose-dependent manner at all the time points. Polymyxin B abolished the LPS-induced decrease in PS rate. The phosphatidylinositol 3-kinase inhibitor LY-0294002 in combination with LPS significantly decreased the rate of PS by 81% and alone by 66%, respectively, for the 3- and 18-h time points, whereas p38 and ERK inhibitors in combination with LPS significantly decreased the rate PS rate at the 18-h time point by 41% and 59%, respectively, compared with control cells. In conclusion, LPS alone transiently decreased the rate of PS by 50% at 3 h; this effect is most likely mediated via the Toll-like receptor 4 (TLR4)-Akt/mTOR pathway, and both p38 and ERK when inhibited in the presence of LPS at 3 h have a similar effect in preventing the LPS-induced reduction in PS.

Phosphorylation of eIF2α in response to 26S proteasome inhibition is mediated by the haem-regulated inhibitor (HRI) kinase

The present study demonstrates that even brief inhibition of degradation by the 26S proteasome inhibits global protein synthesis, mediated through increased phosphorylation of eIF2alpha (eukaryotic translational initiation factor 2alpha) by the HRI (haem-regulated inhibitor) kinase. Exposure of COS-7 cells to the proteasome inhibitor MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) for 4 h resulted in a 55-60% decrease in protein synthesis rate compared with control cells. This repression of protein synthesis after treatment with MG-132 is not due to induction of apoptosis, which is known to occur after longer periods of 26S inhibition. Instead, we observed a significantly increased phosphorylation of eIF2alpha, which is known to repress global protein synthesis. In three MEF (mouse embryonic fibroblast) knockout cell lines lacking one of the four kinases known to phosphorylate eIF2alpha, increased phosphorylation of eIF2alpha still occurred after inhibition of the 26S proteasome. These three cell lines included a deletion of the PKR (double-stranded-RNA-dependent protein kinase); a deletion of the PERK (PKR-like endoplasmic reticulum resident kinase); or a deletion of the GCN2 (positive general control of transcription-2) kinase, indicating that none of these kinases was primarily responsible for the observed phosphorylation of eIF2alpha. In contrast, in a fourth MEF knockout cell line, HRI(-/-) cells lacking the HRI kinase failed to increase eIF2alpha phosphorylation upon proteasome inhibitor treatment (MG-132 or various doses of Bortezomib), indicating that the HRI kinase is the primary kinase activated by brief treatment of MEFs with 26S proteasome inhibitors.

Effects of environmental hypercapnia on animal physiology: A 13C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus

Global climate change is associated with a progressive rise in ocean CO 2 concentrations (hypercapnia) and, consequently, a drop in seawater pH. However, a comprehensive picture of the physiological mechanisms affected by chronic CO 2 stress in marine biota is still lacking. Here we present an analysis of protein biosynthesis rates in isolated muscle of the marine invertebrate Sipunculus nudus, a sediment dwelling worm living at various water depths. We followed the incorporation of 13C-labelled phenylalanine into muscular protein via high-resolution NMR spectroscopy. Protein synthesis decreased by about 60% at a medium pH of 6.70 and a consequently lowered intracellular pH (pHi). The decrease in protein synthesis rates is much stronger than the concomitant suppression of protein degradation (60% versus 10–15%) possibly posing a threat to the cellular homeostasis of structural as well as functional proteins. Considering the progressive rise in ocean CO 2 concentrations, permanent disturbances of cellular protein turnover might seriously affect growth and reproductive performance in many marine organisms with as yet unexplored impacts on species density and composition in marine ecosystems.

Comprehensive analysis of differential gene expression profiles on carbon tetrachloride-induced rat liver injury and regeneration

Microarray analysis of RNA from carbon tetrachloride (CCl 4)-administered rat livers was performed at various time points to establish a global gene expression profile during injury and regeneration stages. A single dose of 1 ml/kg of CCl 4 was given by ip injection, and the liver samples were obtained after 6, 24, 48 h, and 2 weeks. Histopathologic, biochemical, and immunohistochemical studies enabled the classification of the CCl 4 effect into injury (6 and 24 h) and regeneration (48 h and 2 weeks) stages. The expression levels of 5180 clones on a custom rat gene microarray were analyzed and 587 clones yielded changeable gene expression on at least single time point. One hundred seventy-nine clones were classified as injury-specific clones, while 38 clones as regeneration-specific clones. Characteristic gene expression profiles could be associated with CCl 4-induced gene expression with the disruption of lipid metabolism, which is known to cause the fatty liver induced by CCl 4 treatment. In addition, induction of the transcripts for many ribosomal proteins was detected during the injury stage, particularly at the 24-h time point, despite the previous report of decreased protein synthesis rate upon CCl 4 treatment. Several genes with known functions were also identified as CCl 4-regulated genes. In conclusion, we established a global gene expression profile utilizing microarray analysis in rat liver upon acute CCl 4 administration with a full chronological profile that not only covers injury stage but also later points of regeneration stage.

The proteasome inhibitor, MG132, promotes the reprogramming of translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complex.

The eukaryotic translation initiation factor (eIF) 4E, is regulated by modulating both its phosphorylation and its availability to interact with the scaffold protein, eIF4G, to form the mature eIF4F complex. Here we show that treatment of C2C12 myoblasts with the proteasomal inhibitor, MG132 (N-carbobenzoxyl-Leu-Leu-leucinal), resulted in an early decrease in protein synthesis rates followed by a partial recovery, reflecting the reprogramming of translation. The early inhibition of protein synthesis was preceded by a transient increase in eIF2alpha phosphorylation, followed by a sustained increase in eIF4E phosphorylation. Inhibition of eIF4E phosphorylation with CGP57380 failed to prevent translational reprogramming or the moderate decrease in eIF4F complexes at later times. Prolonged incubation with MG132 resulted in the increased expression of heat shock protein (hsp)25, alphaB-crystallin and hsp70, with a population of hsp25 associating with the eIF4F complex in a p38 mitogen-activated protein kinase-dependent manner. Under these conditions, eIF4GI, and to a lesser extent eIF4E, re-localized from a predominantly cytoplasmic distribution to a more perinuclear and granular staining. Although MG132 had little effect on the colocalization of eIF4E and eIF4GI, it promoted the SB203580-sensitive association of eIF4GI and hsp25, an effect not observed with alphaB-crystallin. Addition of recombinant hsp25 to an in vitro translation assay resulted in stimulation of on-going translation and a moderate decrease in de novo translation, indicating that this modified eIF4F complex containing hsp25 has a role to play in recovery of mRNA translation following cellular stress.

Translational regulation by p38 mitogen-activated protein kinase signaling during human cholangiocarcinoma growth

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is aberrantly expressed and maintains transformed cell growth in malignant human cholangiocytes. Because cell growth requires and is intimately related to protein synthesis, our aims were to assess the effect of p38 MAPK signaling on protein synthesis during growth of malignant human cholangiocytes. Inhibition of p38 MAPK activity during mitogenic stimulation decreased protein synthesis rates and tumor cell xenograft growth in nude mice. Altered protein synthesis resulted from decreased translational efficiency with impaired initiation of translation. Mitogenic stimulation resulted in phosphorylation of the eukaryotic initiation factor (eIF)-4E. Inhibition of p38 MAPK signaling or functional dysregulation of translation by small interfering double-stranded RNA (siRNA) to eIF-4E decreased anchorage-independent growth of malignant cholangiocytes. In conclusion, these studies identify a relationship between p38 MAPK activity and the regulation of protein synthesis during human cholangiocarcinoma growth. As protein synthesis is intimately linked to cell growth, dysregulation of translation initiation is a mechanism by which cellular p38 MAPK signaling participates in growth regulation of malignant cholangiocytes. (H epatology 2003;38:158-166.)