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The objective of this study was to investigate the effects of supplementing with L-tryptophan (L-Trp) on milk protein synthesis using an immortalized bovine mammary epithelial (MAC-T) cell line. Cells were treated with 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM of supplemental L-Trp, and the most efficient time for protein synthesis was determined by measuring cell, medium, and total protein at 0, 24, 48, 72, and 96 h. Time and dose tests showed that the 48 h incubation time and a 0.9 mM dose of L-Trp were the optimal values. The mechanism of milk protein synthesis was elucidated through proteomic analysis to identify the metabolic pathway involved. When L-Trp was supplemented, extracellular protein (medium protein) reached its peak at 48 h, whereas intracellular cell protein reached its peak at 96 h with all L-Trp doses. β-casein mRNA gene expression and genes related to milk protein synthesis, such as mammalian target of rapamycin (mTOR) and ribosomal protein 6 (RPS6) genes, were also stimulated ( p < 0.05). Overall, there were 51 upregulated and 59 downregulated proteins, many of which are involved in protein synthesis. The results of protein pathway analysis showed that L-Trp stimulated glycolysis, the pentose phosphate pathway, and ATP synthesis, which are pathways involved in energy metabolism. Together, these results demonstrate that L-Trp supplementation, particularly at 0.9 mM, is an effective stimulus in β-casein synthesis by stimulating genes, proteins, and pathways related to protein and energy metabolism.

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z' values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

The EDR peptide (Glu-Asp-Arg) has been previously established to possess neuroprotective properties. It activates gene expression and synthesis of proteins, involved in maintaining the neuronal functional activity, and reduces the intensity of their apoptosis in in vitro and in vivo studies. The EDR peptide interferes with the elimination of dendritic spines in neuronal cultures obtained from mice with Alzheimer's (AD) and Huntington's diseases. The tripeptide promotes the activation of the antioxidant enzyme synthesis in the culture of cerebellum neurons in rats. The EDR peptide normalizes behavioral responses in animal studies and improves memory issues in elderly patients. The purpose of this review is to analyze the molecular and genetics aspects of the EDR peptide effect on gene expression and synthesis of proteins involved in the pathogenesis of AD. The EDR peptide is assumed to enter cells and bind to histone proteins and/or ribonucleic acids. Thus, the EDR peptide can change the activity of the MAPK/ERK signaling pathway, the synthesis of proapoptotic proteins (caspase-3, p53), proteins of the antioxidant system (SOD2, GPX1), transcription factors PPARA, PPARG, serotonin, calmodulin. The abovementioned signaling pathway and proteins are the components of pathogenesis in AD. The EDR peptide can be AD.

The emergence of genome-wide analyses to interrogate cellular DNA, RNA, and protein content has revolutionized the study of control networks that mediate cellular homeostasis. mRNA translation represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are orchestrated by nutrient signaling pathways. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during nutrient stress by translation of selective mRNAs. In this review, we describe recent advances in our understanding of translational control principles; nutrient-sensing mechanisms; and their dysregulation in human diseases such as diabetes, cancer, and aging. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health.

Alpha one antitrypsin (α1AT), a serine proteinase inhibitor primarily produced by the liver, protects pulmonary tissue from neutrophil elastase digestion. Mutations of the SERPINA1 gene results in a misfolded α1AT protein which aggregates inside hepatocytes causing cellular damage. Therefore, inhibition of mutant α1AT production is one practical strategy to alleviate liver damage. Here we show that proteasome inhibitors can selectively downregulate α1AT expression in human hepatocytes by suppressing the translation of α1AT. Translational suppression of α1AT is mediated by phosphorylation of eukaryotic translation initiation factor 2α and increased association of RNA binding proteins, especially stress granule protein Ras GAP SH3 binding protein (G3BP1), with α1AT mRNA. Treatment of human-induced pluripotent stem cell-derived hepatocytes with a proteasome inhibitor also results in translational inhibition of mutant α1AT in a similar manner. Together we revealed a previously undocumented role of proteasome inhibitors in the regulation of α1AT translation.

Adherens junction protein p120 is thought to be crucial for maintaining vascular integrity, which is important in many pathologies and diseases including atherosclerosis, vascular malformations, hemorrhagic stroke, sepsis and others. However, the mechanisms responsible for this is not completely understood. In this study, using an unbiased proteomics approach, followed by other experimental techniques, we identified that in HUVECs p120 overexpression inhibits LPS/TNFα-induced angiopoietin-2 (Ang2) expression, a key switch of endothelial destabilization. Interestingly, p120 overexpression did not inhibit LPS/TNFα-induced expression of adhesion molecules/cytokines including VCAM-1, ICAM-1, E-selectin, MCP-1, IL-8 and IL-6 in our experimental system. Furthermore, this p120-mediated repression of Ang2 is in an NF-κB independent manner, possibly via transcription factor Ets1. Our results demonstrate that p120 influences vascular integrity by secreted signals, providing new insights into the mechanisms of p120-mediated vascular stability., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H 2 O 2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H 2 O 2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H 2 O 2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H 2 O 2 levels are cytoprotective. We identified p21 Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a "redox rheostat" in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

ROR family of nuclear receptor transcription factors forms nodes connecting metabolic and inflammatory signaling pathways. The RORα members of the family have intrinsic transcriptional activity and they are involved in both activation and repression of a wide range of genes. The role of RORα in control of inflammation has been extensively studied using animal models but its function in human cells is not as well understood. To address this shortcoming, we analyzed how RORα is shaping the inflammatory state of human macrophages. Using CRISPR-Cas9 system, we deleted RORA in THP-1 human monocytic cell line. In mutant cells we observed a dramatic increase in basal expression of a subset of NF-κB regulated genes, including TNF, IL-1β and IL-6, at both transcriptional and translational levels. Furthermore, RORA-deletion cells produced notable amounts of pro-IL-1β even in the absence of LPS stimulation. Subsequent LPS stimulation induced cleavage of pro-IL-1β to mature form. Our RNAseq analysis further confirmed the key role of RORA in setting the inflammatory state of macrophages and defined the set of differentially regulated genes. Overall, our data provides evidence supporting the anti-inflammatory function of RORα in human macrophages., Competing Interests: The authors have declared that no competing interests exist.

Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.

Transfer RNA (tRNA) plays a role in stress response programs involved in various pathological conditions including neurological diseases. Under cell stress conditions, intracellular tRNA is cleaved by a specific ribonuclease, angiogenin, generating tRNA-derived fragments or tRNA-derived stress-induced RNA (tiRNA). Generated tiRNA contributes to the cell stress response and has potential cell protective effects. However, tiRNA generation under stress conditions in neuronal cells has not been fully elucidated. To examine angiogenin-mediated tiRNA generation in neuronal cells, we used the rat neuronal cell line, PC12, in combination with analysis of SYBR staining and immuno-northern blotting using anti-1-methyladenosine antibody, which specifically and sensitively detects tiRNA. Oxidative stress induced by arsenite and hydrogen peroxide caused tRNA cleavage and tiRNA generation in PC12 cells. We also demonstrated that oxygen-glucose deprivation, which is an in vitro model of ischemic-reperfusion injury, induced tRNA cleavage and tiRNA generation. In these stress conditions, the amount of generated tiRNA was associated with the degree of morphological cell damage. Time course analysis indicated that generation of tiRNA was prior to severe cell damage and cell death. Angiogenin over-expression did not influence the amount of tiRNA in normal culture conditions; however, it significantly increased tiRNA generation induced by cell stress conditions. Our findings show that angiogenin-mediated tiRNA generation can be induced in neuronal cells by different cell stressors, including ischemia-reperfusion. Additionally, detection of tiRNA could be used as a potential cell damage marker in neuronal cells. Cover Image for this issue: doi: 10.1111/jnc.14191., (© 2018 International Society for Neurochemistry.)

Translation factor eIF5A participates in protein synthesis at the stage of polypeptide chain elongation. Two eIF5A isoforms are known that are encoded by related genes whose expression varies significantly in different tissues. The eIF5A1 isoform is a constitutively and ubiquitously expressed gene, while the eIF5A2 isoform is expressed in few normal tissues and is an oncogene by a number of parameters. Unique feature of eIF5A isoforms is that they are the only two proteins that contain amino acid hypusine. Modification with hypusine is critical requirement for eIF5A activity. Another distinctive feature of eIF5A is its involvement in the translation of only a subset of the total population of cell mRNAs. The genes for which mRNAs translation requires eIF5A are the members of certain functional groups and are involved in cell proliferation, apoptosis, inflammatory processes, and regulation of transcription and RNA metabolism. The involvement of eIF5A is necessary for the translation of proteins containing oligoproline fragments and some other structures. Modification of eIF5A by hypusine is implemented by two highly specialized enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH), which are not involved in other biochemical reactions. Intracellular activity of these enzymes is closely associated with systems of protein acetylation, polyamine metabolism and other biochemical processes. Inhibition of DHS and DOHH activity provides the possibility of pharmacological control of eIF5A activity and expression of eIF5A-dependent genes.

Angiotensin II (Ang II) is reported to be involved in the development of various cardiovascular diseases by disrupting microvessel permeability, however, the underlying mechanism remains to be elucidated. The present study aimed to investigate the mechanism by which Ang II disrupts microvascular permeability. Rat endothelial cells were subjected to primary culture and identification. Cells in passages 4‑7 were then used for the following experiments. The cells were divided into control, Ang II, and Ang II + valsartan groups, and reverse transcription‑quantitative polymerase chain reaction and western blot analyses were perform to evaluate the expression of zonula occludens‑1 (ZO‑1) and vascular endothelial (VE)‑cadherin in the cells. The distribution of ZO‑1 protein was also detected using immunofluorescence assays. It was found that, compared with the control group, lower expression levels of ZO‑1 and VE‑cadherin were present in the Ang II group (P<0.01). ZO‑1 was also irregularly distributed at the periphery of the cells. In addition, the overexpression of VE‑cadherin reversed the effect of Ang II on the expression and distribution of ZO‑1 in endothelial cells. Together, these results suggested that Ang II inhibited the protein expression of ZO‑1 in vascular endothelial cells by downregulating VE‑cadherin, thus destroying the tight junctions between endothelial cells, which may also be the mechanism by which Ang II is involved in the development of cardiovascular diseases.

The central dogma of molecular biology, that DNA is transcribed into RNA and RNA translated into protein, was coined in the early days of modern biology. Back in the 1950s and 1960s, bacterial genetics first opened the way toward understanding life as the genetically encoded interaction of macromolecules. As molecular biology progressed and our knowledge of gene control deepened, it became increasingly clear that expression relied on many more levels of regulation. In the process of dissecting mechanisms of gene expression, specific small-molecule inhibitors played an important role and became valuable tools of investigation. Small molecules offer significant advantages over genetic tools, as they allow inhibiting a process at any desired time point, whereas mutating or altering the gene of an important regulator would likely result in a dead organism. With the advent of modern sequencing technology, it has become possible to monitor global cellular effects of small-molecule treatment and thereby overcome the limitations of classical biochemistry, which usually looks at a biological system in isolation. This review focuses on several molecules, especially natural products, that have played an important role in dissecting gene expression and have opened up new fields of investigation as well as clinical venues for disease treatment.

Alternative splicing has been widely demonstrated to function as pivotal regulation in specifying cellular fates and biological functions. The relative expression or cellular localization of a splicing factor constitutes an important mechanism in spatiotemporal programming of cell- and stage-specific splicing profiles. In this study, results of deep RNA-sequencing (RNA-Seq) analyses first revealed the reprogrammed splicing profile and reduced expression of serine/arginine-rich splicing factor protein kinase 1 (SRPK1) throughout the development of brown adipose tissue (BAT). A gradual increase in the exon 10-skipped SRPK1 transcript, a potential target of a nonsense-mediated decay (NMD) mechanism, was noted during brown adipogenesis. Elevated RBM4a constituted the regulatory mechanism that led to skipping of SRPK1 exon 10. Moreover, brown adipogenesis-induced upregulation of microRNA (miR)-485 interfered with SRPK1 expression by targeting its 3'-untranslated region (UTR). Depletion of endogenous SRPK1 enhanced the development of C3H10T1/2 cells toward brown adipocytes. Taking our results together, multiple post-transcriptional regulations reduced SRPK1 expression, which subsequently affected brown adipogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chuvash polycythemia is an inherited disease caused by a homozygous germline VHLR200W mutation, which leads to impaired degradation of HIF2α, elevated levels of serum erythropoietin, and erythrocytosis/polycythemia. This phenotype is recapitulated by a mouse model bearing a homozygous VhlR200W mutation. We previously showed that iron-regulatory protein 1-knockout (Irp1-knockout) mice developed erythrocytosis/polycythemia through translational derepression of Hif2α, suggesting that IRP1 could be a therapeutic target to treat Chuvash polycythemia. Here, we fed VhlR200W mice supplemented with Tempol, a small, stable nitroxide molecule and observed that Tempol decreased erythropoietin production, corrected splenomegaly, normalized hematocrit levels, and increased the lifespans of these mice. We attribute the reversal of erythrocytosis/polycythemia to translational repression of Hif2α expression by Tempol-mediated increases in the IRE-binding activity of Irp1, as reversal of polycythemia was abrogated in VhlR200W mice in which Irp1 was genetically ablated. Thus, a new approach to the treatment of patients with Chuvash polycythemia may include dietary supplementation of Tempol, which decreased Hif2α expression and markedly reduced life-threatening erythrocytosis/polycythemia in the VhlR200W mice.

Our bioinformatics research shows that there are four G-rich sequences (S1-S4) in the upstream region of the transcription start site of c-Myb gene, and we have proved that these sequences have the ability to form G-quadruplex structures. This work mainly focuses on G-quadruplex function, recognition and transcription regulation in c-Myb gene, revealing a novel regulatory element in c-Myb proximal promoter region, and its transcription regulation by G-quadruplex binder. The research has identified that the enhancer effect in c-Myb transcription was primarily affected by the G-quadruplex formed by S1 sequence, and the up-regulation effect may due to the removal of repressive progress of MZF-1 by stabilizing G-quadruplex. Attentions were being paid to the development of G-quadruplex binders for selective recognition, and topotecan was found to have high binding affinity in vitro and could effectively affect the c-Myb transcription activities in cells. The regulation of G-quadruplex with binders in transcriptional, translational levels by Q-RT-PCR and western blot was in expectation of providing a strategy for gene expression modulation. In conclusion, our study revealed a G-quadruplex structure in c-Myb proximal promoter region, which was of great importance in the regulation of c-Myb function., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The conditional depletion of a protein of interest (POI) is useful not only for loss-of-function studies, but also for the modulation of biological pathways. Technologies that work at the level of DNA, mRNA, and protein are available for temporal protein depletion. Compared with technologies targeting the pretranslation steps, direct protein depletion (or protein knockdown approaches) is advantageous in terms of specificity, reversibility, and time required for depletion, which can be achieved by fusing a POI with a protein domain called a degron that induces rapid proteolysis of the fusion protein. Conditional degrons can be activated or inhibited by temperature, small molecules, light, or the expression of another protein. The conditional degron-based technologies currently available are described and discussed.

Occludin (OCLN) is an essential factor for HCV entry through interacting with other surface receptors. The aim of this study was to investigate the epigenetic regulation of Occludin expression and to study its impact on viral infectivity. microRNAs expression was assessed using qRT-PCR, while OCLN protein expression was investigated by indirect immunofluorescence and Western blotting. Viral infectivity was assessed by measuring viral-load using qRT-PCR. In silico analysis predicted that miR-200c targeted the OCLN 3'UTR, which was further experimentally confirmed. miR-122 was previously validated to target the 3'UTR of OCLN and was used as a control. We report a significant down-regulation of miR-200c in liver tissues of HCV-infected patients. Ectopic expression of both miR-122 and miR-200c in Huh7 cells reduced OCLN mRNA and protein levels. Viral infectivity was significantly reduced by miR-200c but enhanced by miR-122. This work sheds light on miR-200c as a novel regulator of HCV infectivity through the regulation of OCLN.

Alpha-ketoglutarate (AKG), a key intermediate in the Krebs cycle, has been reported to promote protein synthesis through activating mechanistic targeting of rapamycin (mTOR) in enterocytes. The study tested the hypothesis that AKG may enhance growth and milk protein synthesis in porcine mammary epithelial cells (PMECs). PMECs were cultured for 96 h in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing prolactin (2 µg/ml) and AKG (0 or 1.5 mM). At the end of 96-h culture, the abundance of apoptosis-related proteins (caspase-3, caspase-9), milk-specific proteins (α-lactalbumin and β-casein), mTOR signaling proteins (mTOR, p-mTOR, PERK, p-PERK, eIF2a, P70S6K and p-P70S6K), and endoplasmic reticulum stress (ERS)-associated proteins (BiP and CHOP) in PMEC were determined. Addition of AKG dose-dependently enhanced cell viability in the absence or presence of prolactin, with optimal concentrations of AKG being at 1.0 and 1.5 mM, respectively. In the presence of prolactin, addition of 1.5 mM AKG: (1) decreased (P < 0.05) the abundance of caspase-3 and caspase-9 by 21 and 39 %; (2) enhanced (P < 0.05) the phosphorylation of p-mTOR and p-P70S6K by 39 and 89 %, respectively; (3) increased (P < 0.05) the production of β-casein and α-lactalbumin by 16 and 20 %, respectively; (4) attenuated (P < 0.05) the expression of CHOP by 34 % but promoted (P < 0.05) the expression of BiP by 46 %; (5) increased (P < 0.05) the secretion of lactose by 15 %, when compared to the 0 mM AKG group. Rapamycin (50 nM; an inhibitor of mTOR) attenuated (P < 0.05) the stimulatory effect of AKG on mTOR signaling and syntheses of milk protein and lactose, while relieving (P < 0.05) an inhibitory effect of AKG on expression of proteins related to ERS. Collectively, our results indicate that AKG enhances milk protein production by modulating mTOR and ERS signaling pathways in PMECs.

Unlabelled: Cholinergic vulnerability, characterized by loss of acetylcholine (ACh), is one of the hallmarks of Alzheimer's disease (AD). Previous work has suggested that decreased ACh activity in AD may contribute to pathological changes through global alterations in alternative splicing. This occurs, at least partially, via the regulation of the expression of a critical protein family in RNA processing, heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins. These proteins regulate several steps of RNA metabolism, including alternative splicing, RNA trafficking, miRNA export, and gene expression, providing multilevel surveillance in RNA functions. To investigate the mechanism by which cholinergic tone regulates hnRNPA2/B1 expression, we used a combination of genetic mouse models and in vivo and in vitro techniques. Decreasing cholinergic tone reduced levels of hnRNPA2/B1, whereas increasing cholinergic signaling in vivo increased expression of hnRNPA2/B1. This effect was not due to decreased hnRNPA2/B1 mRNA expression, increased aggregation, or degradation of the protein, but rather to decreased mRNA translation by nonsense-mediated decay regulation of translation. Cell culture and knock-out mice experiments demonstrated that M1 muscarinic signaling is critical for cholinergic control of hnRNPA2/B1 protein levels. Our experiments suggest an intricate regulation of hnRNPA2/B1 levels by cholinergic activity that interferes with alternative splicing in targeted neurons mimicking deficits found in AD., Significance Statement: In Alzheimer's disease, degeneration of basal forebrain cholinergic neurons is an early event. These neurons communicate with target cells and regulate their long-term activity by poorly understood mechanisms. Recently, the splicing factor hnRNPA2/B, which is decreased in Alzheimer's disease, was implicated as a potential mediator of long-term cholinergic regulation. Here, we demonstrate a mechanism by which cholinergic signaling controls the translation of hnRNPA2/B1 mRNA by activation of M1 muscarinic type receptors. Loss of cholinergic activity can have profound effects in target cells by modulating hnRNPA2/B1 levels., (Copyright © 2016 the authors 0270-6474/16/366287-10$15.00/0.)

Type-I interferon (IFN)-induced activation of the mammalian target of rapamycin (mTOR) signaling pathway has been implicated in translational control of mRNAs encoding interferon-stimulated genes (ISGs). However, mTOR-sensitive translatomes commonly include mRNAs with a 5' terminal oligopyrimidine tract (TOP), such as those encoding ribosomal proteins, but not ISGs. Because these translatomes were obtained under conditions when ISG expression is not induced, we examined the mTOR-sensitive translatome in human WISH cells stimulated with IFN β. The mTOR inhibitor Torin1 resulted in a repression of global protein synthesis, including that of ISG products, and translation of all but 3 ISG mRNAs (TLR3, NT5C3A, and RNF19B) was not selectively more sensitive to mTOR inhibition. Detailed studies of NT5C3A revealed an IFN-induced change in transcription start site resulting in a switch from a non-TOP to a TOP-like transcript variant and mTOR sensitive translation. Thus, we show that, in the cell model used, translation of the vast majority of ISG mRNAs is not selectively sensitive to mTOR activity and describe an uncharacterized mechanism wherein the 5'-UTR of an mRNA is altered in response to a cytokine, resulting in a shift from mTOR-insensitive to mTOR-sensitive translation.

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein-protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein-protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situ PLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein-protein interaction network of these proteins., (Copyright © 2014 Elsevier Inc. All rights reserved.)

TRPM7 is an essential and ubiquitous channel-kinase regulating cellular influx of Mg2+. Although TRPM7 mRNA is highly abundant, very small amount of the protein is detected in cells, suggesting post-transcriptional regulation of trpm7 gene expression. We found that TRPM7 mRNA 5'-leader contains two evolutionarily conserved upstream open reading frames that act together to drastically inhibit translation of the TRPM7 reading frame at high magnesium levels and ensure its optimal translation at low magnesium levels, when the activity of the channel-kinase is most required. The study provides the first example of magnesium channel synthesis being controlled by Mg2+ in higher eukaryotes., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Introduction: Some studies have shown that peptides have high treatment potential due to their biological activity, harmlessness, and tissue-specific action. Tetrapeptide Ala-Asp-Glu-Leu (ADEL) was effective on models of acute bacterial lung inflammation, fibrosis, and toxic lung damage in several studies., Methods: We measured Ki67, Mcl-1, p53, CD79, and NOS-3 protein levels in the 1st, 7th, and 14th passages of bronchoepithelial human embryonic cell cultures. Gene expression of NKX2-1, SCGB1A1, SCGB3A2, FOXA1, FOXA2, MUC4, MUC5AC, and SFTPA1 was measured by real-time polymerase chain reaction. Using the methods of spectrophotometry, viscometry, and circular dichroism, we studied the ADEL-DNA interaction in vitro., Results: Peptide ADEL regulates the levels of Ki67, Mcl-1, p53, CD79, and NOS-3 proteins in cell cultures of human bronchial epithelium in various passages. The strongest activating effect of peptide ADEL on bronchial epithelial cell proliferation through Ki67 and Mcl-1 was observed in "old" cell cultures. ADEL regulates the expression of genes involved in bronchial epithelium differentiation: NKX2-1, SCGB1A1, SCGB3A2, FOXA1, and FOXA2. ADEL also activates several genes, which reduced expression correlated with pathological lung development: MUC4, MUC5AC, and SFTPA1. Spectrophotometry, viscometry, and circular dichroism showed ADEL-DNA interaction, with a binding region in the major groove (N7 guanine)., Conclusions: ADEL can bind to specific DNA regions and regulate gene expression and synthesis of proteins involved in the differentiation and maintenance of functional activity of the bronchial epithelium. Through activation of some specific gene expression, peptide ADEL may protect the bronchial epithelium from pulmonary pathology. ADEL also may have a geroprotective effect on bronchial tissue.

The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is a basic helix-loop-helix transcription factor. An issue of whether SHARP-1 is an insulin-inducible transcription factor was examined. Insulin rapidly increased the level of SHARP-1 mRNA both in vivo and in vitro. Then, signaling pathways involved with the increase of SHARP-1 mRNA by insulin were determined in H4IIE rat hepatoma cells. Pretreatments with LY294002, wortmannin, and staurosporine completely blocked the induction effect, suggesting the involvement of both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) pathways. In fact, overexpression of a dominant negative form of atypical protein kinase C lambda (aPKCλ) significantly decreased the induction of the SHARP-1 mRNA. In addition, inhibitors for the small GTPase Rac or Jun N-terminal kinase (JNK) also blocked the induction of SHARP-1 mRNA by insulin. Overexpression of a dominant negative form of Rac1 prevented the activation by insulin. Furthermore, actinomycin D and cycloheximide completely blocked the induction of SHARP-1 mRNA by insulin. Finally, when a SHARP-1 expression plasmid was transiently transfected with various reporter plasmids into H4IIE cells, the promoter activity of PEPCK reporter plasmid was specifically decreased. Thus, we conclude that insulin induces the SHARP-1 gene expression at the transcription level via a both PI 3-K/aPKCλ/JNK- and a PI 3-K/Rac/JNK-signaling pathway; protein synthesis is required for this induction; and that SHARP-1 is a potential repressor of the PEPCK gene expression., (© Georg Thieme Verlag KG Stuttgart · New York.)

Dysregulation of the astroglial glutamate transporters GLAST and GLT-1 has been implicated in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) where a loss of GLT-1 protein expression and activity is reported. Furthermore, the two principal C-terminal splice variants of GLT-1 (namely GLT-1a and GLT-1b) show altered expression ratio in animal models of this disease. Considering the putative link between inflammation and excitotoxicity, we have here characterized the influence of TNF-α on glutamate transporters in cerebral cortical astrocyte cultures from wild-type rats and from a rat model of ALS (hSOD1G93A). Contrasting with the down-regulation of GLAST, a 72 h treatment with TNF-α substantially increased the expression of GLT-1a and GLT-1b in both astrocyte cultures. However, as the basal level of GLT-1a appeared considerably lower in hSOD1G93A astrocytes, its up-regulation by TNF-α was insufficient to recapitulate the expression observed in wild-type astrocytes. Also the glutamate uptake activity after TNF-α treatment was lower for hSOD1G93A astrocytes as compared to wild-type astrocytes. In the presence of the protein synthesis inhibitor cycloheximide, TNF-α did not influence GLT-1 isoform expression, suggesting an active role of dynamically regulated protein partners in the adaptation of astrocytes to the inflammatory environment. Confirming the influence of inflammation on the control of glutamate transmission by astrocytes, these results shed light on the regulation of glutamate transporter isoforms in neurodegenerative disorders.

Adiponectin has been shown to play a critical role in immunity. Recently, we reported that the adiponectin levels after allogeneic stem cell transplantation were higher in recipients with chronic graft-versus-host disease (cGVHD). However, the effects of adiponectin on extracellular matrix (ECM) and regulatory factors in dermal fibroblasts remain unclear. We compared the messenger RNA (mRNA) levels of collagen type1 (COL1A), fibronectin 1 (FN1), matrix metalloproteinase (MMP)1, MMP3, tissue inhibitor of metalloproteinase (TIMP)1, TIMP3, transforming growth factor-β (TGF-β), and TGF-β receptor 2 (TGF-βR2) in human normal dermal fibroblasts cultured with and without adiponectin, and we assessed the degree of synthesis of ECMs by immunofluorescent microscopy. Furthermore, we also assessed these mRNA levels after blocking of TGF-βR2. Adiponectin induced higher mRNA levels of FN1, MMP1, MMP3, TIMP1, TIMP3, and TGF-βR2 in a dose-dependent manner, but did not significantly affect COL1A or TGF-β. In addition, adiponectin was shown to upregulate FN1, MMPs, and TIMPs after blocking of TGF-βR2. Immunofluorescent microscopy revealed that adiponectin promoted a greater synthesis of ECMs than in the control in vitro. The finding that adiponectin upregulated ECM-associated factors might mean that high levels of adiponectin could modulate dermal fibrosis was observed in recipients with cGVHD. Further basic investigation is warranted to elucidate whether the adiponectin-pathway could be a target for the treatment of sclerotic cGVHD., (Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Tumor cells rely on aerobic glycolysis to maintain unconstrained cell growth and proliferation. Imiquimod (IMQ), a synthetic Toll-like receptor (TLR) 7/8 ligand, exerts anti-tumor effects directly by inducing cell death in cancer cells and/or indirectly by activating cellular immune responses against tumor cells. However, whether IMQ modulates glucose metabolism pathways remains unclear. In this study, we demonstrated that IMQ can enhance aerobic glycolysis by up-regulating HIF-1α expression at the transcriptional and translational levels via ROS mediated STAT3- and Akt-dependent pathways, independent of TLR7/8 signaling. The genetic silencing of HIF-1α not only repressed IMQ-induced aerobic glycolysis but also sensitized cells to IMQ-induced apoptosis due to faster ATP and Mcl-1 depletion. Moreover, the glucose analog 2-DG and the Hsp90 inhibitor 17-AAG, which destabilizes the HIF-1α protein, synergized with IMQ to induce tumor cell apoptosis in vitro and significantly inhibited tumor growth in vivo. Thus, we hypothesize that the IMQ-induced up-regulation of HIF-1α and aerobic glycolysis is a protective response to the metabolic stress generated by IMQ treatment, and thus, co-treatment with inhibitors of HIF-1α and/or glycolysis may be a useful therapeutic strategy to enhance the anti-tumor effects of IMQ in clinical settings.

Fragile X mental retardation protein (FMRP) is encoded by Fmr1 gene in which mutation is known to cause fragile X syndrome characterized by mental impairment and other psychiatric symptoms similar to autism spectrum disorders. FMRP plays important roles in cellular mRNA biology such as transport, stability, and translation as an RNA-binding protein. In the present study, we identified potential role of FMRP in the neural differentiation, using cortical neural progenitor cells from Sprague-Dawley rat. We newly found NeuroD1, an essential regulator of glutamatergic neuronal differentiation, as a new mRNA target interacting with FMRP in co-immunoprecipitation experiments. We also identified FMRP as a regulator of neuronal differentiation by modulating NeuroD1 expression. Down-regulation of FMRP by siRNA also increased NeuroD1 expression along with increased pre- and post-synaptic development of glutamatergic neuron, as evidenced by Western blot and immunocytochemistry. On the contrary, cells harboring FMRP over-expression construct showed decreased NeuroD1 expression. Treatment of cultured neural precursor cells with a histone deacetylase inhibitor, valproic acid known as an inducer of hyper-glutamatergic neuronal differentiation, down-regulated the expression of FMRP, and induced NeuroD1 expression. Our study suggests that modulation of FMRP expression regulates neuronal differentiation by interaction with its binding target mRNA, and provides an example of the gene and environmental interaction regulating glutamatergic neuronal differentiation.

Recent in vivo studies establish that osteopontin (OPN) expression is hydrogen peroxide (H2O2)-dependent. However, the mechanisms by which H2O2 increases OPN expression remain poorly defined. OPN protein expression increased in an unusual biphasic pattern in response to H2O2. To investigate whether these increases were mediated through transcriptional and/or translational regulation of OPN, smooth muscle cells stimulated with 50 μM H2O2 were used as an in vitro cell system. Early protein increases at 6 h were not preceded by increased mRNA, whereas later increases (18 h) were, suggesting multiple mechanisms of regulation by H2O2. Polyribosomal fractionation assays established that early increases (6 h) in OPN expression were due to increased translation. This increase in translation occurred through phosphorylation of 4E-BP1 at the reactive oxygen species-sensitive Ser-65, which allowed for release and activation of eukaryotic initiation factor eIF4E and subsequent OPN translation. This early increase (6 h) in OPN was blunted in cells expressing a phospho-deficient 4E-BP1 mutant. H2O2 stimulation increased rat OPN promoter activity at 8 and 18 h, and promoter truncation studies established that promoter region -2284 to -795 is crucial for H2O2-dependent OPN transcription. ChIP studies determined that H2O2-dependent transcription is mediated by the reactive oxygen species-sensitive transcription factors NF-κB and AP-1. In conclusion, H2O2 stimulates OPN expression in a unique biphasic pattern, where early increases are translational and late increases are transcriptional.

All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-messenger systems. More recent studies reveal that metabolites also affect post-transcriptional regulatory mechanisms. Here, we review the increasing number of connections between metabolism and post-transcriptional regulation in eukaryotic organisms. First, we present evidence that riboswitches, a common mechanism of metabolite sensing in bacteria, also function in eukaryotes. Next, we review an example of a double stranded RNA modifying enzyme that directly interacts with a metabolite, suggesting a link between RNA editing and metabolic state. Finally, we discuss work that shows some metabolic enzymes bind directly to RNA to affect mRNA stability or translation efficiency. These examples were discovered through gene-specific genetic, biochemical, and structural studies. A directed systems level approach will be necessary to determine whether they are anomalies of evolution or pioneer discoveries in what may be a broadly connected network of metabolism and post-transcriptional regulation., (Copyright © 2013 John Wiley & Sons, Ltd.)

The unfolded protein response (UPR) is an important regulatory network that responds to perturbations in protein homeostasis in the endoplasmic reticulum (ER). In mammalian cells, the UPR features translational and transcriptional mechanisms of gene expression aimed at restoring proteostatic control. A central feature of the UPR is phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF2) by PERK (EIF2AK3/PEK), which reduces the influx of nascent proteins into the ER by lowering global protein synthesis, coincident with preferential translation of key transcription activators of genes that function to expand the processing capacity of this secretory organelle. Upon ER stress, the apicomplexan parasite Toxoplasma gondii is known to induce phosphorylation of Toxoplasma eIF2α and lower translation initiation. To characterize the nature of the ensuing UPR in this parasite, we carried out microarray analyses to measure the changes in the transcriptome and in translational control during ER stress. We determined that a collection of transcripts linked with the secretory process are induced in response to ER stress, supporting the idea that a transcriptional induction phase of the UPR occurs in Toxoplasma. Furthermore, we determined that about 500 gene transcripts showed enhanced association with translating ribosomes during ER stress. Many of these target genes are suggested to be involved in gene expression, including JmjC5, which continues to be actively translated during ER stress. This study indicates that Toxoplasma triggers a UPR during ER stress that features both translational and transcriptional regulatory mechanisms, which is likely to be important for parasite invasion and development.

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Recently, we have demonstrated that 5-LO mRNA expression is regulated by alternative splicing and nonsense-mediated mRNA decay (NMD). In addition to this, 5-LO protein expression was reduced on translational level in UPF1 knockdown cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry-based proteomics study was performed to identify compartment-specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis revealed that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (~21%) but not in the cytosolic fraction (<1%). The results suggest that UPF1 is a critical gene expression regulator in a compartment-specific way. During differentiation by TGFβ and calcitriol, the majority of UPF1 regulated proteins were adjusted to normal level. This indicates that the translational regulation by UPF1 can potentially be cell differentiation-dependent.

When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms., (Copyright © 2013 American Society for Bone and Mineral Research.)

Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.

The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D2O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D2O on the kinetics of in-vitro GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D2O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D2O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D2O in a recombinant Escherichia coli strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent.

Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3'-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.

Endothelin-1 (ET-1) dose-dependently increased HIF1α expression in pulmonary artery smooth muscle cells (PASMCs). Inhibition of protein synthesis did not affect ET-1-induced HIF1α expression. The maximum effect of ET-1 was similar to that caused by proteasome inhibitor MG132. Further study indicates that ET-1 also dose-dependently stimulated calcineurin activation, specific calcineurin inhibitor cyclosporine A (CsA), abolished ET-1-induced HIF1α elevation, and reversed ET-1-induced RACK1 (receptor of activated protein kinase C 1) de-phosphorylation. Endothelin receptor A was found to specifically mediate the effects of ET-1. To examine whether RACK1 is particularly involved in proteasome-dependent HIF1α degradation, RACK1 was silenced by siRNA transfection. Cells lacking RACK1 exhibited significant elevation of HIF1α protein level. Taken together, our study suggests that ET-1 suppressed proteasome-dependent HIF1α degradation by calcineurin-dependent RACK1 de-phosphorylation., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The prion protein (PrP(C)) is a cell surface protein expressed mainly in the nervous system. In addition to the role of its abnormal conformer in transmissible spongiform encephalopathies, normal PrP(C) may be implicated in other degenerative conditions often associated with inflammation. PrP(C) is also present in cells of hematopoietic origin, including T cells, dendritic cells, and macrophages, and it has been shown to modulate their functions. Here, we investigated the impact of inflammation and stress on the expression and function of PrP(C) in neutrophils, a cell type critically involved in both acute and chronic inflammation. We found that systemic injection of LPS induced transcription and translation of PrP(C) in mouse neutrophils. Up-regulation of PrP(C) was dependent on the serum content of TGF-β and glucocorticoids (GC), which, in turn, are contingent on the activation of the hypothalamic-pituitary-adrenal axis in response to systemic inflammation. GC and TGF-β, either alone or in combination, directly up-regulated PrP(C) in neutrophils, and accordingly, the blockade of GC receptors in vivo curtailed the LPS-induced increase in the content of PrP(C). Moreover, GC also mediated up-regulation of PrP(C) in neutrophils following noninflammatory restraint stress. Finally, neutrophils with up-regulated PrP(C) presented enhanced peroxide-dependent cytotoxicity to endothelial cells. The data demonstrate a novel interplay of the nervous, endocrine, and immune systems upon both the expression and function of PrP(C) in neutrophils, which may have a broad impact upon the physiology and pathology of various organs and systems.

In this study, the effects of L-arginine-nitric-oxide ((∙)NO)-producing pathway on protein content of ubiquitin, as an important component of ubiquitin-proteasome system for protein removal, were investigated. We showed that L-arginine markedly decreased ubiquitin protein content in interscapular brown adipose tissue, both in thermogenic inactive (at room temperature) and thermogenic active (on cold) states; while in L-NAME-treated groups this effect was abolished. This result suggests that nitric oxide ((∙)NO), besides well established roles, is involved in this aspect of structure remodeling, as well.

The mTOR complex 1 (mTORC1) kinase nucleates a pathway that promotes cell growth and proliferation and is the target of rapamycin, a drug with many clinical uses. mTORC1 regulates messenger RNA translation, but the overall translational program is poorly defined and no unifying model exists to explain how mTORC1 differentially controls the translation of specific mRNAs. Here we use high-resolution transcriptome-scale ribosome profiling to monitor translation in mouse cells acutely treated with the mTOR inhibitor Torin 1, which, unlike rapamycin, fully inhibits mTORC1 (ref. 2). Our data reveal a surprisingly simple model of the mRNA features and mechanisms that confer mTORC1-dependent translation control. The subset of mRNAs that are specifically regulated by mTORC1 consists almost entirely of transcripts with established 5' terminal oligopyrimidine (TOP) motifs, or, like Hsp90ab1 and Ybx1, with previously unrecognized TOP or related TOP-like motifs that we identified. We find no evidence to support proposals that mTORC1 preferentially regulates mRNAs with increased 5' untranslated region length or complexity. mTORC1 phosphorylates a myriad of translational regulators, but how it controls TOP mRNA translation is unknown. Remarkably, loss of just the 4E-BP family of translational repressors, arguably the best characterized mTORC1 substrates, is sufficient to render TOP and TOP-like mRNA translation resistant to Torin 1. The 4E-BPs inhibit translation initiation by interfering with the interaction between the cap-binding protein eIF4E and eIF4G1. Loss of this interaction diminishes the capacity of eIF4E to bind TOP and TOP-like mRNAs much more than other mRNAs, explaining why mTOR inhibition selectively suppresses their translation. Our results clarify the translational program controlled by mTORC1 and identify 4E-BPs and eIF4G1 as its master effectors.

Histone deacetylase 6 (HDAC6) is known as a cytoplasmic enzyme that regulates cell migration, cell adhesion, and degradation of misfolded proteins by deacetylating substrates such as α-tubulin and Hsp90. When HaCaT keratinocytes were exposed to 1-200μM sodium arsenite, we observed perinuclear localization of HDAC6 within 30 min. Although the overall level of HDAC6 protein did not change, sodium arsenite caused an increase of HDAC6 in ribosomal fractions. Separation of ribosomal subunits versus intact ribosomes or polysomes indicated that HDAC6 was mainly detected in 40/43S fractions containing the small ribosomal subunit in untreated cells but was associated with 40/43S and 60/80S ribosomal fractions in arsenite-treated cells. Immunocytochemistry studies revealed that arsenite caused colocalization of HDAC6 with the ribosomal large and small subunit protein L36a and S6. Both L36a and S6 were detected in the immunocomplex of HDAC6 isolated from arsenite-treated cells. The observed physical interaction of HDAC6 with ribosomes pointed to a role of HDAC6 in stress-induced protein translation. Among arsenite stress-induced proteins, de novo Nrf2 protein translation was inhibited by Tubastatin A. These data demonstrate that HDAC6 was recruited to ribosomes, physically interacted with ribosomal proteins, and regulated de novo protein translation in keratinocytes responding to arsenite stress.

In a glucose-salt solution (Earle's balanced salt solution), asparagine (Asn) stimulates ornithine decarboxylase (ODC) activity in a dose-dependent manner, and the addition of epidermal growth factor (EGF) potentiates the effect of Asn. However, EGF alone fails to activate ODC. Thus, the mechanism by which Asn activates ODC is important for understanding the regulation of ODC activity. Asn reduced antizyme-1 (AZ1) mRNA and protein. Among the amino acids tested, Asn and glutamine (Gln) effectively inhibited AZ1 expression, suggesting a differential role for amino acids in the regulation of ODC activity. Asn decreased the putrescine-induced AZ1 translation. The absence of amino acids increased the binding of eukaryotic initiation factor 4E-binding protein (4EBP1) to 5'-mRNA cap and thereby inhibited global protein synthesis. Asn failed to prevent the binding of 4EBP1 to mRNA, and the bound 4EBP1 was unphosphorylated, suggesting the involvement of the mammalian target of rapamycin (mTOR) in the regulation of AZ1 synthesis. Rapamycin treatment (4 h) failed to alter the expression of AZ1. However, extending the treatment (24 h) allowed expression in the presence of amino acids, indicating that AZ1 is expressed when TORC1 signaling is decreased. This suggests the involvement of cap-independent translation. However, transient inhibition of mTORC2 by PP242 completely abolished the phosphorylation of 4EBP1 and decreased basal as well as putrescine-induced AZ1 expression. Asn decreased the phosphorylation of mTOR-Ser(2448) and AKT-Ser(473), suggesting the inhibition of mTORC2. In the absence of amino acids, mTORC1 is inhibited, whereas mTORC2 is activated, leading to the inhibition of global protein synthesis and increased AZ1 synthesis via a cap-independent mechanism.

To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK-dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1(-/-); ERK2(flox/flox); Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1(-/-); ERK2 (flox/flox); Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation.

Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97 ± 0.02 and 0.99 ± 0.05%/hr calculated from 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68 ± 0.03 and 0.60 ± 0.06%/hr in the TG treatment group (p<0.05, vs. control). TG-induced ER stress inhibited hepatic protein synthesis. The stable isotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis.

Cardiomyocyte stretching has been reported to be a major trigger for brain natriuretic peptide (BNP) release; however, an increase in circulating BNP is observed in patients with acute myocardial ischemia in the absence of increased left ventricular wall stress or cardiomyocyte stretching. In the present study, to investigate the direct and independent effects of acute myocardial ischemia on BNP expression and its mechanism, we established an in vitro glucose-free ischemia and hypoxia injured model of cultured rat cardiomyotes and proved hypoxia upregulated expressions of interleukin-6(il-6) and BNP. Further treatment with il-6 elicited dose- and time-dependent increases in BNP mRNA and protein expression as well as an upregulation in transforming growth factor-β1 (TGF-β1)/Smad2 expression, which was partially suppressed by a neutralizing antibody. In conclusion, our study showed that acute myocardial ischemia can directly upregulate BNP expression at the translational and transcriptional levels through the action of il-6, and this process is associated with the upregulation of TGF-β1/Smad2 signal path., (Copyright © 2011 Elsevier B.V. All rights reserved.)