Recovery Of Protein Synthesis Research Articles

Transient Inhibition of Translation Improves Cardiac Function After Ischemia/Reperfusion by Attenuating the Inflammatory Response.

The myocardium adapts to ischemia/reperfusion (I/R) by changes in gene expression, determining the cardiac response to reperfusion. mRNA translation is a key component of gene expression. It is largely unknown how regulation of mRNA translation contributes to cardiac gene expression and inflammation in response to reperfusion and whether it can be targeted to mitigate I/R injury. To examine translation and its impact on gene expression in response to I/R, we measured protein synthesis after reperfusion in vitro and in vivo. Underlying mechanisms of translational control were examined by pharmacological and genetic targeting of translation initiation in mice. Cell type-specific ribosome profiling was performed in mice that had been subjected to I/R to determine the impact of mRNA translation on the regulation of gene expression in cardiomyocytes. Translational regulation of inflammation was studied by quantification of immune cell infiltration, inflammatory gene expression, and cardiac function after short-term inhibition of translation initiation. Reperfusion induced a rapid recovery of translational activity that exceeds baseline levels in the infarct and border zone and is mediated by translation initiation through the mTORC1 (mechanistic target of rapamycin complex 1)-4EBP1 (eIF4E-binding protein 1)-eIF (eukaryotic initiation factor) 4F axis. Cardiomyocyte-specific ribosome profiling identified that I/R increased translation of mRNA networks associated with cardiac inflammation and cell infiltration. Short-term inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory cytokines such as Ccl2 (C-C motif chemokine ligand 2) of border zone cardiomyocytes, thereby attenuating Ly6Chi monocyte infiltration and myocardial inflammation. In addition, we identified a systemic immunosuppressive effect of eIF4F translation inhibitors on circulating monocytes, directly inhibiting monocyte infiltration. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin attenuated translation, reduced infarct size, and improved cardiac function after myocardial infarction. Global protein synthesis is inhibited during ischemia and shortly after reperfusion, followed by a recovery of protein synthesis that exceeds baseline levels in the border and infarct zones. Activation of mRNA translation after reperfusion is driven by mTORC1/eIF4F-mediated regulation of initiation and mediates an mRNA network that controls inflammation and monocyte infiltration to the myocardium. Transient inhibition of the mTORC1-/eIF4F axis inhibits translation and attenuates Ly6Chi monocyte infiltration by inhibiting a proinflammatory response at the site of injury and of circulating monocytes.

Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis

Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic cell death mechanism characterized by endoplasmic reticulum and mitochondria dilation. Intriguingly, oncogenic HRas sensitizes non-transformed cells to VCP inhibition-mediated paraptosis. The susceptibility of cancer cells to VCP inhibition is attributed to the non-attenuation and recovery of protein synthesis under proteotoxic stress. Mechanistically, mTORC2/Akt activation and eIF3d-dependent translation contribute to translational rebound and amplification of proteotoxic stress. Furthermore, the ATF4/DDIT4 axis augments VCP inhibition-mediated paraptosis by activating Akt. Given that hyperactive Akt counteracts chemotherapeutic-induced apoptosis, VCP inhibition presents a promising therapeutic avenue to exploit Akt-associated vulnerabilities in cancer cells by triggering paraptosis while safeguarding normal cells.

Recovery of protein synthesis to assay DNA repair activity in transcribed genes in living cells and tissues.

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that protects against the negative effects of transcription-blocking DNA lesions. Hereditary TC-NER deficiencies cause pleiotropic and often severe neurodegenerative and progeroid symptoms. While multiple assays have been developed to determine TC-NER activity for clinical and research purposes, monitoring TC-NER is hampered by the low frequency of repair events occurring in transcribed DNA. 'Recovery of RNA Synthesis' is widely used as indirect TC-NER assay based on the notion that lesion-blocked transcription only resumes after successful TC-NER. Here, we show that measuring novel synthesis of a protein after its compound-induced degradation prior to DNA damage induction is an equally effective but more versatile manner to indirectly monitor DNA repair activity in transcribed genes. This 'Recovery of Protein Synthesis' (RPS) assay can be adapted to various degradable proteins and readouts, including imaging and immunoblotting. Moreover, RPS allows real-time monitoring of TC-NER activity in various living cells types and even in differentiated tissues of living organisms. To illustrate its utility, we show that DNA repair in transcribed genes declines in aging muscle tissue of C. elegans. Therefore, the RPS assay constitutes an important novel clinical and research tool to investigate transcription-coupled DNA repair.

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response.

Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of newly synthesized RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.

NUPR1 interacts with eIF2α and is required for resolution of the ER stress response in pancreatic tissue.

Nuclear protein 1 (NUPR1) is a stress response protein overexpressed upon cell injury in virtually all organs including the exocrine pancreas. Despite NUPR1's well-established role in the response to cell stress, the molecular and structural machineries triggered by NUPR1 activation remain largely debated. In this study, we uncover a new role for NUPR1, participating in the unfolded protein response (UPR) and the integrated stress response. Biochemical results and ultrastructural morphological observations revealed alterations in the UPR of acinar cells of germline-deleted NUPR1 murine models, consistent with the inability to restore general protein synthesis after stress induction. Bioinformatic analysis of NUPR1-interacting partners showed significant enrichment in translation initiation factors, including eukaryotic initiation factor (eIF) 2α. Co-immunoprecipitation and proximity ligation assays confirmed the interaction between NUPR1 and eIF2α and its phosphorylated form (p-eIF2α). Furthermore, our data suggest loss of NUPR1 in cells results in maintained eIF2α phosphorylation and evaluation of nascent proteins by click chemistry revealed that NUPR1-depleted PANC-1 cells displayed a slower poststress protein synthesis recovery when compared to wild-type. Combined, these data propose a novel role for NUPR1 in the integrated stress response pathway, at least partially through promoting efficient PERK branch activity and resolution through a unique interaction with eIF2α.

Hibernation as a Stage of Ribosome Functioning.

In response to stress, eubacteria reduce the level of protein synthesis and either disassemble ribosomes into the 30S and 50S subunits or turn them into translationally inactive 70S and 100S complexes. This helps the cell to solve two principal tasks: (i) to reduce the cost of protein biosynthesis under unfavorable conditions, and (ii) to preserve functional ribosomes for rapid recovery of protein synthesis until favorable conditions are restored. All known genes for ribosome silencing factors and hibernation proteins are located in the operons associated with the response to starvation as one of the stress factors, which helps the cells to coordinate the slowdown of protein synthesis with the overall stress response. It is possible that hibernation systems work as regulators that coordinate the intensity of protein synthesis with the energy state of bacterial cell. Taking into account the limited amount of nutrients in natural conditions and constant pressure of other stress factors, bacterial ribosome should remain most of time in a complex with the silencing/hibernation proteins. Therefore, hibernation is an additional stage between the ribosome recycling and translation initiation, at which the ribosome is maintained in a "preserved" state in the form of separate subunits, non-translating 70S particles, or 100S dimers. The evolution of the ribosome hibernation has occurred within a very long period of time; ribosome hibernation is a conserved mechanism that is essential for maintaining the energy- and resource-consuming process of protein biosynthesis in organisms living in changing environment under stress conditions.

The protective role of m1A during stress-induced granulation.

Post-transcriptional methylation of N6-adenine and N1-adenine can affect transcriptome turnover and translation. Furthermore, the regulatory function of N6-methyladenine (m6A) during heat shock has been uncovered, including the enhancement of the phase separation potential of RNAs. In response to acute stress, e.g. heat shock, the orderly sequestration of mRNAs in stress granules (SGs) is considered important to protect transcripts from the irreversible aggregation. Until recently, the role of N1-methyladenine (m1A) on mRNAs during acute stress response remains largely unknown. Here we show that the methyltransferase complex TRMT6/61A, which generates the m1A tag, is involved in transcriptome protection during heat shock. Our bioinformatics analysis indicates that occurrence of the m1A motif is increased in mRNAs known to be enriched in SGs. Accordingly, the m1A-generating methyltransferase TRMT6/61A accumulated in SGs and mass spectrometry confirmed enrichment of m1A in the SG RNAs. The insertion of a single methylation motif in the untranslated region of a reporter RNA leads to more efficient recovery of protein synthesis from that transcript after the return to normal temperature. Our results demonstrate far-reaching functional consequences of a minimal RNA modification on N1-adenine during acute proteostasis stress.

ATF4-Dependent NRF2 Transcriptional Regulation Promotes Antioxidant Protection during Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress generates reactive oxygen species (ROS) that induce apoptosis if left unabated. To limit oxidative insults, the ER stress PKR-like endoplasmic reticulum Kinase (PERK) has been reported to phosphorylate and activate nuclear factor erythroid 2-related factor 2 (NRF2). Here, we uncover an alternative mechanism for PERK-mediated NRF2 regulation in human cells that does not require direct phosphorylation. We show that the activation of the PERK pathway rapidly stimulates the expression of NRF2 through activating transcription factor 4 (ATF4). In addition, NRF2 activation is late and largely driven by reactive oxygen species (ROS) generated during late protein synthesis recovery, contributing to protecting against cell death. Thus, PERK-mediated NRF2 activation encompasses a PERK-ATF4-dependent control of NRF2 expression that contributes to the NRF2 protective response engaged during ER stress-induced ROS production.

KMT2A-Rearranged Infant Acute Lymphoblastic Leukemia Cells Undergo ER-Stress-Induced Apoptosis Following Exposure to Proteasome Inhibitors

Infants diagnosed with KMT2A-rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) have a poor prognosis with an event free survival of 23-44%. To identify new treatment approaches we previously performed in vitro and in vivo assays to evaluate the activity of FDA approved compounds in 15 primary KMT2Ar infant leukemia samples. Three classes of agents were found to be active in these assays: proteasome inhibitors, anthracyclines, and histone deacetylase inhibitors (HDACi). KMT2Ar infant leukemia samples were exquisitely sensitive to the proteasome inhibitor bortezomib, requiring 10-100 fold less drug to achieve 50% toxicity when compared to non-KMT2Ar childhood ALL. Bortezomib is FDA approved for multiple myeloma and laboratory studies using this model system have previously demonstrated responses to be mediated through several mechanisms including NFKB inhibition, stabilization of cell cycle regulatory proteins, and perhaps most importantly the induction of an unfolded protein response (UPR) and endoplasmic reticulum (ER)-stress-induced apoptosis. To evaluate global protein dynamics in KMT2Ar ALL cells treated with bortezomib, we performed tandem mass tag (TMT) quantitative mass spectrometry on synchronized SEM cells exposed to either 50nM of bortezomib or DMSO at 0 hours (hr), 6hr, 12hr, 16hr, and 20hr. Applying pairwise comparison for 9232 unique proteins measured over the time course compared to untreated controls, we identified 1593 proteins with a log2 fold change >1.5 in bortezomib treated cells compared to 101 proteins in the DMSO control (FDR<0.01). Several proteins associated with ER-stress-induced apoptosis including ATF4, DDIT4, ATF3, TSC22D3 (GILZ), and PMAIP1 (NOXA) were upregulated more than 3-fold between 6 and 20hr, suggesting this pathway may play a role in bortezomib induced apoptosis of KMT2Ar cells (p<0.05 and log2 fold change of +/- 0.58). To validate this finding and further understand the role of the UPR and ER-stress-induced apoptosis, we evaluated seven key mediators of this pathway by western blot following bortezomib exposure on synchronized SEM cells over a 12 hour time course including ATF4, ATF6, CHOP, PERK, GADD34, CReP, and eIF2α as well as phosphorylated PERK (p-PERK) and eIF2α (p-eIF2α). This demonstrated a critical time point at 6hr where an increase in ATF4 (3.5 fold), CHOP (1.6 fold), and CReP (2.9 fold) protein levels was accompanied by a decrease in p-PERK (0.7 fold), and p-eIF2α (0.8 fold) whereas GADD34 levels remained constant. Although full-sized ATF6 (ATF6a) protein showed a considerable increase (1.9 fold), the levels of cleaved ATF6 (ATF6f) were only slightly increased (1.2 fold) consistent with ATF4-mediated upregulation of CHOP leading to increased protein synthesis along with ATP depletion, oxidative stress, and cell death. While GADD34 has been shown to be the main phosphatase that functions in a negative feedback loop to resolve cell stress, recent data suggests that stabilization of CReP mRNA by ER stress is able to reverse eIF2α phosphorylation at later stages of UPR leading to re-expression of key UPR proteins. Further, p-eIF2α-attenuated protein synthesis, and not ATF4 mRNA translation has been shown to promote cell survival. Our data support a model whereby the UPR and ER-stress in KMT2Ar ALL cells is induced upon exposure to bortezomib leading to increased levels of ATF4 and CHOP. Attenuation of p-eIF2α by CReP further contributes to cell death through the recovery of protein synthesis in a setting of limited protein folding capacity. These results support the use of proteasome inhibitors in KMT2Ar leukemia which is currently being formally evaluated in a Phase II clinical trial for newly diagnosed patients with infant ALL (NCT02553460). Disclosures Gruber: Bristol-Myers Squibb: Consultancy.

Pharmacological inhibition of spinal cord injury-stimulated ribosomal biogenesis does not affect locomotor outcome

After unresolved endoplasmic reticulum stress, recovery of protein synthesis including increased expression of ribosomal components and translation factors may induce cell death. Using a mouse model of moderate contusive spinal cord injury (SCI) at the T9 level, upregulation of ribosomal biogenesis was observed in the injury epicenter at 24h after trauma. Such upregulation coincided with endoplasmic reticulum stress response as previously reported in this model. It was also accompanied by changes in expression of many other genes associated with translational regulation. Systemic treatment with a pharmacological inhibitor of RNA-Polymerase-1, BMH-21 reduced rRNA transcription in the spinal cord. Moreover, in the injury epicenter, treatment with BMH-21 increased expression of oligodendrocyte-specific transcripts including Mbp and Cldn11 at 3days post injury. Although such findings may suggest at least transient reduction of oligodendrocyte death, locomotor outcome was mostly unaffected except slightly accelerated recovery of hindlimb function at week 2 post-injury. Therefore, at least in mice, RNA-Polymerase-1 does not appear to be a robust target for therapies to protect spinal cord tissue after contusion. However, these findings raise an interesting possibility that altered rate of ribosomal biogenesis contributes to the apparent translational reprogramming after contusive SCI. Such a reprogramming could be a major regulator of SCI-induced gene expression.

Protein synthesis inhibition and GADD34 control IFN-β heterogeneous expression in response todsRNA.

In innate immune responses, induction of type-I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type-I IFN production is linked to cell's ability to enter dsRNA-activated PKR-dependent translational arrest and then overcome this inhibition by decreasing eIF2α phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression. GADD34 expression, shown here to be dependent on the IRF3 transcription factor, is responsible for a biochemical cycle permitting pulse of IFN synthesis to occur in cells undergoing protein synthesis inhibition. Translation arrest is further demonstrated to be key for anti-viral response by acting synergistically with MAVS activation to amplify TBK1 signaling and IFN-β mRNA transcription, while GADD34-dependent protein synthesis recovery contributes to the heterogeneous expression of IFN observed in dsRNA-activated cells.

Creatine kinase in ischemic and inflammatory disorders.

The creatine/phosphocreatine pathway plays a conserved and central role in energy metabolism. Compartmentalization of specific creatine kinase enzymes permits buffering of local high energy phosphates in a thermodynamically favorable manner, enabling both rapid energy storage and energy transfer within the cell. Augmentation of this metabolic pathway by nutritional creatine supplementation has been shown to elicit beneficial effects in a number of diverse pathologies, particularly those that incur tissue ischemia, hypoxia or oxidative stress. In these settings, creatine and phosphocreatine prevent depletion of intracellular ATP and internal acidification, enhance post-ischemic recovery of protein synthesis and promote free radical scavenging and stabilization of cellular membranes. The creatine kinase energy system is itself further regulated by hypoxic signaling, highlighting the existence of endogenous mechanisms in mammals that can enhance creatine metabolism during oxygen deprivation to promote tissue resolution and homeostasis. Here, we review recent insights into the creatine kinase pathway, and provide rationale for dietary creatine supplementation in human ischemic and inflammatory pathologies.

Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis.

Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity.

Creatine supplementation during pregnancy: summary of experimental studies suggesting a treatment to improve fetal and neonatal morbidity and reduce mortality in high-risk human pregnancy

While the use of creatine in human pregnancy is yet to be fully evaluated, its long-term use in healthy adults appears to be safe, and its well documented neuroprotective properties have recently been extended by demonstrations that creatine improves cognitive function in normal and elderly people, and motor skills in sleep-deprived subjects. Creatine has many actions likely to benefit the fetus and newborn, because pregnancy is a state of heightened metabolic activity, and the placenta is a key source of free radicals of oxygen and nitrogen. The multiple benefits of supplementary creatine arise from the fact that the creatine-phosphocreatine [PCr] system has physiologically important roles that include maintenance of intracellular ATP and acid–base balance, post-ischaemic recovery of protein synthesis, cerebral vasodilation, antioxidant actions, and stabilisation of lipid membranes. In the brain, creatine not only reduces lipid peroxidation and improves cerebral perfusion, its interaction with the benzodiazepine site of the GABAA receptor is likely to counteract the effects of glutamate excitotoxicity – actions that may protect the preterm and term fetal brain from the effects of birth hypoxia. In this review we discuss the development of creatine synthesis during fetal life, the transfer of creatine from mother to fetus, and propose that creatine supplementation during pregnancy may have benefits for the fetus and neonate whenever oxidative stress or feto-placental hypoxia arise, as in cases of fetal growth restriction, premature birth, or when parturition is delayed or complicated by oxygen deprivation of the newborn.

Effect of L-carnitine on postischemic inhibition of protein synthesis in the rat brain

The purpose of this study was to investigate effects of carnitine administration on protein synthesis recovery after transient cerebral ischemia. Rats received L-carnitine in two doses of 16 mmol/kg i.p. 15 min before ischemia and just on the onset of reperfusion. Transient forebrain ischemia was induced by 4-vessel occlusion for 15 min, followed by 30 min or 7 days of reperfusion. Protein synthesis rate, reinitiation ability and neurodegeneration in the frontal cortex and hippocampus were measured by the incorporation of radioactively labelled leucine into polypeptide chains in postmitochondrial supernatants and by Fluoro-Jade B staining. A protective effect was observed, on protein synthesis as well as the number of surviving neurons, in the L-carnitine-treated groups. Our results indicate that L-carnitine can exert a protective effect in the development of reperfusion-induced injury. L-carnitine significantly reduced the ischemia/reperfusion-induced inhibition of translation and neurodegeneration in the neocortex as well as in the highly sensitive hippocampus and dorsolateral striatum. We expect that the ability of L-carnitine to keep translational machinery on facilitates efficacy of postischemic remodulation of gene expression.

Irreversible Translation Arrest in the Reperfused Brain

Irreversible translation arrest occurs in reperfused neurons that will die by delayed neuronal death. It is now recognized that suppression of protein synthesis is a general response of eukaryotic cells to exogenous stressors. Indeed, stress-induced translation arrest can be viewed as a component of cell stress responses, and consists of initiation, maintenance, and termination phases that work in concert with stress-induced transcriptional mechanisms. Within this framework, we review translation arrest in reperfused neurons. This framework provides a basis to recognize that phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is the initiator of translation arrest, and a key marker indicating activation of neuronal stress responses. However, eIF2 alpha phosphorylation is reversible. Other phases of stress-induced translation arrest appear to contribute to irreversible translation arrest specifically in ischemic vulnerable neuron populations. We detail two lines of evidence supporting this view. First, ischemia, as a stress stimulus, induces irreversible co-translational protein misfolding and aggregation after 4 to 6 h of reperfusion, trapping protein synthesis machinery into functionally inactive protein aggregates. Second, ischemia and reperfusion leads to modifications of stress granules (SGs) that sequester functionally inactive 48S preinitiation complexes to maintain translation arrest. At later reperfusion durations, these mechanisms may converge such that SGs become sequestered in protein aggregates. These mechanisms result in elimination of functionally active ribosomes and preclude recovery of protein synthesis in selectively vulnerable neurons. Thus, recognizing translation arrest as a component of endogenous cellular stress response pathways will aid in making sense of the complexities of postischemic translation arrest.

Regulation of translation factors eIF4GI and 4E-BP1 during recovery of protein synthesis from inhibition by p53

Activation of the tumour suppressor protein p53 rapidly inhibits protein synthesis. This is associated with dephosphorylation and cleavage of initiation factor eIF4GI and the eIF4E-binding protein 4E-BP1. When the activation of p53 is reversed within 16 h 4E-BP1 becomes rephosphorylated, the level of intact eIF4GI slowly increases and protein synthesis gradually recovers. The recovery of protein synthesis is partially blocked by rapamycin and wortmannin but not by the protein kinase inhibitors PD98059 and CGP74514A. Both rapamycin and wortmannin, but not PD98059 or CGP74514A, delay the reappearance of eIF4GI. In contrast, full-length 4E-BP1 rapidly becomes rephosphorylated and this process is partially inhibited by rapamycin, PD98059 and CGP74514A. Thus, activation of p53 results in the inhibition of distinct rapamycin- and wortmannin-sensitive pathways that target eIF4GI, and rapamycin-sensitive and -insensitive pathways that target 4E-BP1. Following inactivation of p53 the gradual recovery is determined largely by the kinetics of restoration of eIF4GI rather than by the rephosphorylation of full-length 4E-BP1. These findings suggest that the ability of cells to rephosphorylate 4E-BP1, resynthesise eIF4GI and restore the rate of protein synthesis after inactivation of p53 is an important aspect of recovery following the relief of physiological stress.

Nck in a Complex Containing the Catalytic Subunit of Protein Phosphatase 1 Regulates Eukaryotic Initiation Factor 2α Signaling and Cell Survival to Endoplasmic Reticulum Stress

Stress imposed on the endoplasmic reticulum (ER) induces the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2) on Ser51. This results in transient inhibition of general translation initiation while concomitantly activating a signaling pathway that promotes the expression of genes whose products improve ER function. Conversely, dephosphorylation of eIF2alphaSer51 is accomplished by protein phosphatase 1 (PP1c) complexes containing either the protein CReP or GADD34, which target PP1c to eIF2. Here, we demonstrate that the Src homology (SH) domain-containing adaptor Nck is a key component of a molecular complex that controls eIF2alpha phosphorylation and signaling in response to ER stress. We show that overexpression of Nck decreases basal and ER stress-induced eIF2alpha phosphorylation and the attendant induction of ATF4 and CHOP. In contrast, we demonstrate that the mouse embryonic fibroblasts lacking both isoforms of Nck (Nck1-/-Nck2-/-) show higher levels of eIF2alpha phosphorylation and premature induction of ATF4, CHOP, and GADD34 in response to ER stress and finally, are more resistant to cell death induced by prolonged ER stress conditions. We establish that a significant amount of Nck protein localizes at the ER and is in a complex with eIF2 subunits. Further analysis of this complex revealed that it also contains the Ser/Thr phosphatase PP1c, its regulatory subunit CReP, and dephosphorylates eIF2alpha on Ser51 in vitro. Overall, we demonstrate that Nck as a component of the CReP/PP1c holophosphatase complex contributes to maintain eIF2alpha in a hypophosphorylated state. In this manner, Nck modulates translation and eIF2alpha signaling in response to ER stress.

Mechanisms of the post-antibiotic effects induced by rifampicin and gentamicin in Escherichia coli

The mechanisms by which antibiotics induce a post-antibiotic effect in susceptible bacteria are poorly understood. To explore the mechanisms more fully we examined the recovery of macromolecular synthesis in Escherichia coli during gentamicin- and rifampicin-induced post-antibiotic effects. E. coli ATCC 25922 was exposed to rifampicin and to gentamicin at 5x MIC for 60 min to induce post-antibiotic effects. The antibiotics were then removed from the culture medium by washing the cells. The rates of DNA, RNA and protein synthesis during the post-antibiotic effect and recovery periods were subsequently determined by measuring the incorporation of radiolabelled uridine, thymidine and leucine into trichloroacetic acid precipitable material. Recovery of E. coli ATCC 25922 from the rifampicin-induced post-antibiotic effect coincided with the recovery of RNA and protein synthesis. Recovery from the gentamicin-induced post-antibiotic effect coincided with the recovery of protein synthesis. These data support the hypothesis that antibiotic molecules retained in the cell mediate the post-antibiotic effect by suppressing the biochemical activity of their molecular targets.

The effects of normovolemic hemodilution on protein synthesis recovery following postischemic reperfusion in the rat brain.

Normovolemic hemodilution is a possible way to improve the brain recovery after ischemia and reperfusion. Therefore we have decided to examine how this process may affect the post-ischemic protein synthesis machinery. We analysed rat brains after 4-vessel-occlusion and different time intervals of reperfusion using normovolemic hemodilution. We achieved an important increase of [4,5-3H]leucine incorporation into polypeptides in vitro in the rat brain neocortex 30 minutes after ischemia, but concurrently there was no significant change in the hippocampus and striatum. By extending the time course of reperfusion we did not observe any important deviation of in vitro [4,5-3H]leucine incorporation in the studied brain areas. Thus, although hemodilution increased protein synthesis in selective vulnerable regions after ischemia, this improvement is not of significant importance.