Protein Translation Initiation Research Articles

Sphingosine-1-phosphate phosphohydrolase-1 regulates ER stress-induced autophagy

The sphingolipid metabolites ceramide and sphingosine-1-phosphate (S1P) have recently been implicated in autophagy. In this study, we report that depletion of sphingosine-1-phosphate phosphohydrolase-1 (SPP1), an endoplasmic reticulum (ER)-resident enzyme that specifically dephosphorylates S1P, induced autophagy. Although the mammalian target of rapamycin and class III phosphoinositide 3-kinase/Beclin-1 pathways were not involved and this autophagy was p53 independent, C/EBP homologous protein, BiP, and phospho-eucaryotic translation initiation factor-2α, and cleavage of procaspases 2 and 4, downstream targets of ER stress, were increased after SPP1 depletion. Autophagy was suppressed by depletion of protein kinase regulated by RNA-like ER kinase (PERK), inositol-requiring transmembrane kinase/endonuclease-1α, or activating transcription factor 6, three sensors of the unfolded protein response (UPR) to ER stress. Autophagy triggered by downregulation of SPP1 did not lead to apoptosis but rather stimulated, in a PERK dependent manner, the survival signal Akt, whose inhibition then sensitized cells to apoptosis. Although depletion of SPP1 increased intracellular levels of S1P and its secretion, activation of cell surface S1P receptors did not induce autophagy. Nevertheless, increases in intracellular pools of S1P, but not dihydro-S1P, induced autophagy and ER stress. Thus, SPP1, by regulating intracellular S1P homeostasis, can control the UPR and ER stress-induced autophagy.

Leucine and α-Ketoisocaproic Acid, but Not Norleucine, Stimulate Skeletal Muscle Protein Synthesis in Neonatal Pigs , ,

The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, α-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 μmol·kg−1·h−1 for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) and eIF4G, lower (P < 0.04) abundance of the inactive 4E-BP1·eIF4E complex, and greater (P < 0.05) active eIF4G·eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P < 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC.

Inhibition of Golgi Apparatus Glycosylation Causes Endoplasmic Reticulum Stress and Decreased Protein Synthesis

Nucleotide sugar transporters of the Golgi apparatus play an essential role in the glycosylation of proteins, lipids, and proteoglycans. Down-regulation of expression of the transporters for CMP-sialic acid, GDP-fucose, or both unexpectedly resulted in accumulation of glycoconjugates in the Golgi apparatus rather than in the plasma membrane. Pulse-chase experiments with radiolabeled sugars and amino acids showed decreased synthesis and secretion of both nonglycoproteins and glycoproteins. Further studies revealed that the above silencing induced endoplasmic reticulum stress and inhibited protein translation initiation. Together these results suggest that global inhibition of Golgi apparatus glycosylation may lead to important secondary metabolic changes, unrelated to glycosylation.

HSV Usurps Eukaryotic Initiation Factor 3 Subunit M for Viral Protein Translation: Novel Prevention Target

Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by ∼90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m). Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease.

Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Fibroblast growth factor (FGF) cooperates with the Wnt/beta-catenin pathway to promote mammary tumorigenesis. To investigate the mechanisms involved in FGF/Wnt cooperation, we genetically engineered a model of inducible FGF receptor (iFGFR) signaling in the context of the well-established mouse mammary tumor virus-Wnt-1 transgenic mouse. In the bigenic mice, iFGFR1 activation dramatically enhanced mammary tumorigenesis. Expression microarray analysis did not show transcriptional enhancement of Wnt/beta-catenin target genes but instead showed a translational gene signature that also correlated with elevated FGFR1 and FGFR2 in human breast cancer data sets. Additionally, iFGFR1 activation enhanced recruitment of RNA to polysomes, resulting in a marked increase in protein expression of several different Wnt/beta-catenin target genes. FGF pathway activation stimulated extracellular signal-regulated kinase and the phosphorylation of key translation regulators both in vivo in the mouse model and in vitro in a human breast cancer cell line. Our results suggest that cooperation of the FGF and Wnt pathways in mammary tumorigenesis is based on the activation of protein translational pathways that result in, but are not limited to, increased expression of Wnt/beta-catenin target genes (at the level of protein translation). Further, they reveal protein translation initiation factors as potential therapeutic targets for human breast cancers with alterations in FGF signaling.

Abstract 3790: Molecular mechanism of cell death by silibinin in colorectal cancer: Apoptosis versus autophagy

Abstract Recently, considerable research efforts have been directed toward the identification of naturally occurring dietary/non-dietary agents for both prevention/intervention of colorectal cancer (CRC). One such agent is silibinin, a flavonolignan isolated from the seeds of milk thistle (Silybum marianum), which has been used clinically and as dietary supplement against liver toxicity for over three decades. Several recent studies conducted by us and others have shown that silibinin effectively inhibits the growth of various cancers of epithelial origin. Regarding CRC, silibinin has been shown to inhibit CRC growth and progression in various pre-clinical models; however, the mechanistic basis for its efficacy against CRC remains largely unknown. Here, we demonstrate for the first time that silibinin rapidly induces oxidative stress in human CRC SW480 cells due to ROS generation with a concomitant dissipation of mitochondrial potential, cytochrome c release and apoptotic death. ROS generation preceded the changes in DiOC6 retention indicating that silibinin triggers oxidative stress prior to mitochondrial disruption and apoptosis. Longer-time exposure of cells with silibinin caused an intensified cytoplasmic vacuolization followed by sequestration of the organelles, which inhibited the release of cytochrome c. Interestingly, this decrease in apoptotic response co-related with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Further mechanistic studies revealed that silibinin strongly inhibits the activation of PI3K-Akt-mTOR pathway. Co-incident with this effect, energy restriction was induced by silibinin where an enhanced AMP/ATP ratio caused an increase in phospho AMPKTh172 resulting in increased phosphorylation and thereby decreased activity of acetyl-CoA carboxylase, an essential enzyme for fatty acid synthesis. Down regulation of mTOR kinase activity by silibinin also resulted in suppression of protein translation initiation complex leading to suppression of Cap-dependent translation. To further confirm that silibinin causes starvation-induced autophagic death, additional studies were performed to assess the metabolic profile of the cells employing investigative metabolomics study utilizing quantitative high-resolution proton nuclear magnetic resonance spectroscopy (1H-NMRS), which showed that silibinin significantly decreases glucose uptake in the cells eventually leading to cell death. Our completed studies show dual efficacy of silibinin where first it initiates an apoptotic response triggered by ROS generation and then imposes energy restriction within the cell activating autophagic machinery to induce autophagic cell death. Together, these might be major molecular mechanisms of silibinin in its chemopreventive efficacy against colon cancer. This work was supported by NCI RO1 grant CA112304. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3790.

Tyrosine phosphorylation regulates mechanical stretch‐induced activation of protein translation initiation in C2C12 myoblasts

It has been proposed that mechanically‐induced tension was the critical event for initiating muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, effect of mechanical stretch on the protein translation initiation was studied in C2C12 myoblasts. C2C12 cells were plated on fibronectin‐coated silicone elastomer chamber in DMEM containing 10% fetal bovine serum. Cells were grown in CO2 incubator and subjected to 30 min of cyclic stretch (15% uniaxial stretch at a frequency of 1 Hz). Western blot analysis revealed that 30 min of cyclic stretch increased the phosphorylation of p70 S6 kinase (p70S6K), which is a key regulator for translation initiation. Pre‐treatment of cells with broad range tyrosine kinase inhibitor, genistein, completely blocked the stretch‐induced phosphorylation of p70S6K. These results suggest that tyrosine phosphorylation plays a critical role in the activation of protein translation initiation process in response to cyclic stretch. This work is supported by Grant‐in‐Aid for Scientific Research C (20500577 to N.N.) from the Japan Society for the Promotion of Science (JSPS).

Differential Regulation of Protein Synthesis and mTOR Signaling in Skeletal Muscle and Visceral Tissues of Neonatal Pigs after a Meal

Neonates have a high rate of growth driven by elevated rates of skeletal muscle protein synthesis. Feeding stimulates protein synthesis but the time course of the changes in different tissues has not been determined. The aim of this study was to evaluate the time course of the changes in protein synthesis rates and translation initiation factor activation in skeletal muscle and visceral tissues after a meal. Neonatal pigs (n=36; 5–7 day old) received a meal providing one‐sixth of daily requirements and were sacrificed at 0, 30, 60, 90, 120, or 240 min after feeding. Protein synthesis increased 30 to 120 min in glycolytic muscles, 30 to 90 min in mixed fiber type muscle and did not increase in oxidative muscle post‐feeding. Protein synthesis increased in liver 30 to 120 min, jejunum 30 to 60 min and pancreas 30 min post‐feeding and did not increase in kidney and heart. Eukaryotic initiation factor (eIF) 4E binding protein (4EBP1) and rp S6 kinase 1 (S6K1) phosphorylation increased 30 to 90 min in all tissues. These results suggest that feeding stimulates mTOR signaling in skeletal muscle and visceral tissues. However, mTOR activation alone was not sufficient to stimulate protein synthesis in all tissues. (Supported by NIH grants AR‐44474 and AR‐51563 and USDA/ARS 6250510000‐33).Grant Funding Source: NIH grants AR‐44474 and AR‐51563 and USDA/ARS 6250510000‐33

The induction of G2/M cell-cycle arrest and apoptosis by cucurbitacin E is associated with increased phosphorylation of eIF2α in leukemia cells

The antiproliferative and apoptotic effects of cucurbitacin E, a natural product isolated from Cucurbitaceae, were determined in human leukemia HL-60 cells. Cucurbitacin E at low concentrations (3-50 nmol/l) inhibited the growth of HL-60 cells, which was associated with G2/M cell-cycle arrest, decrease in the levels of cyclin-dependent kinase1, and increase in the levels of p21. Cucurbitacin E at high concentrations (1-10 mol/l) induced apoptosis of HL-60 cells and activation of caspase-3, caspase-8, and caspase-9. Jurkat leukemia cells with or without caspase-8 expression were nearly equally sensitive to cucurbitacin E-induced apoptosis. Cucurbitacin E did not increase the levels of reactive oxygen species and antioxidants, N-acetylcysteine and catalase, did not block cucurbitacin E-induced apoptosis. Cucurbitacin E decreased the levels of the antiapoptotic proteins XIAP, survivin, and Mcl-1, but increased the level of the proapoptotic protein, Bax. The levels of phosphorylated eukaryotic translation initiation factor 2 subunit (eIF2) were induced in cells undergoing both apoptosis and cell-cycle arrest. As phosphorylated eIF2 is an inhibitor of protein translation initiation, our data suggest that cucurbitacin E induces cell growth arrest and apoptosis through the induction of eIF2 phosphorylation, which leads to the inhibition of cyclin-dependent kinase 1, Mcl-1, survivin, and/or XIAP protein synthesis and that cucurbitacin E induces apoptosis mainly through a mitochondrial-mediated pathway.

Maintaining Adequate Nutrition, Not Probiotic Administration, Prevents Growth Stunting and Maintains Skeletal Muscle Protein Synthesis Rates in a Piglet Model of Colitis

Malnutrition and cytokine-induced catabolism are pervasive in children with inflammatory bowel diseases (IBD), however, the benefits of aggressive nutrition support or of probiotics on nutrient and functional deficiencies and growth remain unclear. Piglets with dextran sulfate (DS)-induced colitis consuming a 50% macronutrient restricted diet (C-MR) were compared with those receiving probiotics (C-MRP) or adequate nutrition (C-WN) and with healthy well-nourished controls (REF). C-WN versus REF had reduced growth (-34% chest circumference and -22% snout-to-rump length gain) and a tendency toward lesser weight gain, but no differences in skeletal muscle protein fractional synthesis rates (FSR) or initiation of translation via the mTOR pathway were observed. Compared with C-WN, the C-MR and C-MRP piglets had lower weight gain, growth, and skeletal muscle FSR, and lower phosphorylated p70S6K1 with higher eIF4E*4E-BP1, indicative of reduced initiation of protein translation. Finally, plasma leucine concentrations were positively correlated with weight and phosphorylated p70S6K1, whereas negatively correlated with eIF4E*4E-BP1. In conclusion, reductions in weight gain, growth, protein turnover, skeletal muscle FSR, and initiation of protein translation with moderate macronutrient restriction in colitis are not ameliorated by probiotic supplementation. However, maintaining adequate nutrient intake during colitis preserves whole body protein metabolism, but growth remains compromised.

EIF2α Phosphorylation-Dependent Translation in CA1 Pyramidal Cells Impairs Hippocampal Memory Consolidation without Affecting General Translation

Protein synthesis inhibitor antibiotics are widely used to produce amnesia, and have been recognized to inhibit general or global mRNA translation in the basic translational machinery. For instance, anisomycin interferes with protein synthesis by inhibiting peptidyl transferase or the 80S ribosomal function. Therefore, de novo general or global protein synthesis has been thought to be necessary for long-term memory formation. However, it is unclear which mode of translation-gene-specific translation or general/global translation-is actually crucial for the memory consolidation process in mammalian brains. Here, we generated a conditional transgenic mouse strain in which double-strand RNA-dependent protein kinase (PKR)-mediated phosphorylation of eIF2alpha, a key translation initiation protein, was specifically increased in hippocampal CA1 pyramidal cells by the chemical inducer AP20187. Administration of AP20187 significantly increased activating transcription factor 4 (ATF4) translation and concomitantly suppressed CREB-dependent pathways in CA1 cells; this led to impaired hippocampal late-phase LTP and memory consolidation, with no obvious reduction in general translation. Conversely, inhibition of general translation by low-dose anisomycin failed to block hippocampal-dependent memory consolidation. Together, these results indicated that CA1-restricted genetic manipulation of particular mRNA translations is sufficient to impair the consolidation and that consolidation of memories in CA1 pyramidal cells through eIF2alpha dephosphorylation depends more on transcription/translation of particular genes than on overall levels of general translation. The present study sheds light on the critical importance of gene-specific translations for hippocampal memory consolidation.

Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E

One strategy to improve therapies in advanced prostate cancer (PC) involves targeting genes that are activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. Heat shock protein 27 (Hsp27) expression becomes highly upregulated in PC cells after androgen withdrawal or chemotherapy, in which it functions as a cytoprotective chaperone to confer broad-spectrum treatment resistance. The purpose of this study is to elucidate anti-apoptotic pathways regulated by Hsp27 that are activated during PC progression. Using two-hybrid experiment, we found that Hsp27 was having a major role in the protein translational initiation process. Furthermore, using complementary DNA (cDNA) microarray analysis, 4E binding protein 1 was identified as being proportionately and highly regulated by Hsp27. These data led us to analyze the protein synthesis initiation pathway, which is a prerequisite for cell growth and proliferation. Using northern and western blot analysis, we found that Hsp27 downregulation decreased eukaryotic translation initiation factor 4E (eIF4E) expression at the protein, but not mRNA, level. The cytoprotection afforded by Hsp27 overexpression was attenuated by eIF4E knockdown using specific eIF4E short interfering RNA (siRNA). Co-immunoprecipitation and co-immunofluorescence confirmed that Hsp27 colocalizes and interacts directly with eIF4E. Hsp27-eIF4E interaction decreases eIF4E ubiquitination and proteasomal degradation. By chaperoning eIF4E, Hsp27 seems to protect the protein synthesis initiation process to enhance cell survival during cell stress induced by castration or chemotherapy. Forced overexpression of eIF4E induces resistance to androgen-withdrawal and paclitaxel treatment in the prostate LNCaP cells in vitro. These findings identify Hsp27 as a modulator of eIF4E and establish a potential mechanism for the eIF4E-regulated apoptosis after androgen ablation and chemotherapy. Targeting Hsp27-eIF4E interaction may serve as a therapeutic target in advanced PC.

Influence of EIF4E and HER2 Protein Expression on the Response to Neoadjuvant Chemotherapy and Trastuzumab in Breast Cancer.

Abstract Background:Resistance to trastuzumab (TTZ) is still poorly understood. Many proteins implicated in signaling pathways of breast cancer cells have so far been studied as potential markers of TTZ resistance in preclinical models.Method:63 consecutive patients were treated for HER2 + breast cancer with a neoadjuvant chemotherapy regimen containing TTZ between December 2003 and July 2008. This regimen was mostly 4 cycles of FEC 100 (5FU, Epirubicin and Cyclophosphamide) followed by 4 cycles of TTZ – Docetaxel 100mg/m².Immunohistochemistry (IHC) was used to analyse, in the initial biopsy, the expression of HER family receptors (EGFR, HER3, HER4 and HER2), PTEN, cMyc and p27. We also analyzed the expression of two proteins (eIF4E and 4EBP1) involved in translational control of mRNAs. 4EBP1 is a small protein that represses the initiation of protein translation through its association with eIF4E, the mRNA cap-binding subunit of the eIF4F complex. mTOR-dependent phosphorylation of 4E-BP1 decreases the affinity of the protein for eIF4E, which facilitates the formation of eIF4F complex. Furthermore, the expression of eIF4E is elevated in many cancers.We analysed the pathologic response rate (pRR) after chemotherapy + TTZ, and we established correlations between the expression of these markers and pRR.Results:Among the 63 patients, 54 had a valuable pre-chemotherapy biopsy.For those 54 patients, median age was 48 years (range [21;80]), median tumor size was 50 mm clinically (range [20;120]), and 26 mm by ultrasound (range [10;80]). Respectively 63%, 57% and 52% were estrogen receptor (ER) negative, progesterone receptor (PR) negative and ER/PR negative. Histology type was ductal infiltrant carcinoma for 91% of patients. Respectively 28% and 72% were grade SBR (EE) II and III.A pathologic complete response (pCR) (Sataloff TA) was observed on 63% of patients, 28% had a partial response but superior to 50% (Sataloff TB) and 9% had a poor or absent pathologic response (Sataloff TC and TD).This pCR was not statistically linked to any initial clinical characteristics (age, grade, hormonal status and tumoral size).pCR was strongly correlated with weak eIf4E and with percentage of HER2 strong-stained cells (respectively p=0,0114 et 0,0056). 93% of tumors with an eIF4E IRS &amp;lt;7 (n=13/14) had a pCR, and only 5% (1/19) with an incomplete pRR had an eIF4E IRS &amp;lt;7. Moreover, 78% (25/32) of tumors with 100% of tumoral cells harboring a strong intensity of HER2 had a pCR versus 41% (9/22) of tumors with less than 100% of cells expressing HER2 with a strong staining.Conclusion:Our study shows a strong correlation between eIF4E expression or HER2 expression and pRR in patients with HER2 + breast treated with a neoadjuvant TTZ-containing regimen. IHC of eIF4E might be a powerful test to predict sensitivity to TTZ. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2043.

Mammalian Target of Rapamycin Is a Critical Regulator of Cardiac Hypertrophy in Spontaneously Hypertensive Rats

Evidence exists that protein kinase C and the mammalian target of rapamycin are important regulators of cardiac hypertrophy. We examined the contribution of these signaling kinases to cardiac growth in spontaneously hypertensive rats (SHRs). Systolic blood pressure was increased (P<0.001) at 10 weeks in SHRs versus Wistar-Kyoto controls (162+/-3 versus 128+/-1 mm Hg) and was further elevated (P<0.001) at 17 weeks in SHRs (184+/-7 mm Hg). Heart:body weight ratio was not different between groups at 10 weeks but was 22% greater (P<0.01) in SHRs versus Wistar-Kyoto controls at 17 weeks. At 10 weeks, activation of Akt and S6 ribosomal protein was greater (P<0.01) in SHRs but returned to normal by 17 weeks. In contrast, SHRs had protein kinase C activation only at 17 weeks. To determine whether mammalian target of rapamycin regulates the initial development of hypertrophy, rats were treated with rapamycin (2 mg/kg per day IP) or saline vehicle from 13 to 16 weeks of age. Rapamycin inhibited cardiac mammalian target of rapamycin in SHRs, as evidenced by reductions (P<0.001) in phosphorylation of S6 ribosomal protein and eukaryotic translation initiation factor-4E binding protein 1. Rapamycin treatment also reduced (P<0.001) heart weight and hypertrophy by 47% and 53%, respectively, in SHRs in spite of increased (P<0.001) systolic blood pressure versus untreated SHRs (213+/-8 versus 189+/-6 mm Hg). Atrial natriuretic peptide, brain natriuretic peptide, and cardiac function were unchanged between SHRs treated with rapamycin or vehicle. These data show that mammalian target of rapamycin is required for the development of cardiac hypertrophy evoked by rising blood pressure in SHRs.

Arabidopsis OBG-Like GTPase (AtOBGL) Is Localized in Chloroplasts and Has an Essential Function in Embryo Development

OBG-like GTPases, a subfamily of P-loop GTPases, have divers and important functions in bacteria, including initiation of sporulation, DNA replication, and protein translation. Homologs of the Bacillus subtilis spo0B GTP-binding protein (OBG) can be found in plants and algae but their specific function in these organisms has not yet been elucidated. Here, it is shown that AT5G18570 encodes an Arabidopsis thaliana OBG-like protein (AtOBGL) that is localized in chloroplasts. In contrast to the bacterial members of this protein family, AtOBGL and other OBG-like proteins from green algae and plants possess an additional N-terminal domain, indicating functional adaptation. Disruption of the gene locus of ATOBGL by TDNA insertion resulted in an embryo-lethal phenotype and light microscopy using Normarski optics revealed that embryo maturation in the atobgl mutant is arrested at the late globular stage before development of a green embryo. Expression of 35S::ATOBGL within the atobgl mutant background could rescue the mutant phenotype, confirming that embryo-lethality is caused by the loss of AtOBGL. Together, the data show that the bacterial-derived OBG-like GTPases have retained an essential role in chloroplasts of plants and algae. They furthermore corroborate the significance of chloroplast functions for embryo development - an important stage within the Arabidopsis lifecycle.

A Host RNA Helicase-Like Protein, AtRH8, Interacts with the Potyviral Genome-Linked Protein, VPg, Associates with the Virus Accumulation Complex, and Is Essential for Infection

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.

Regulation of REDD1 by insulin‐like growth factor‐I in skeletal muscle and myotubes

Insulin-like growth factor-I (IGF-I) is a major anabolic hormone for skeletal muscle and a potent stimulus for protein synthesis and translation initiation. Recent studies suggest that translation can be inhibited by over expression of the mammalian target of rapamycin (mTOR) repressor REDD1. The purpose of the present study was to determine whether IGF-I alters the expression of REDD1 and whether this is associated with a concomitant change in protein synthesis in vitro. Subcutaneous injection of IGF-I or intravenous delivery of insulin for 3-4 h increased REDD1 mRNA in skeletal muscle 7-10-fold. A threefold increase in REDD1 was observed when C2C12 myotubes were treated with IGF-I. REDD1 protein continued to be expressed for up to 24 h after addition of IGF-I to cells. Withdrawal of IGF-I from myotubes lead to a rapid loss of REDD1 protein content. IGF-I-induced REDD1 mRNA and protein expression were prevented by inhibitors of transcription and translation. IGF-I had an additive effect with dexamethasone (Dex) on REDD1 protein content in myotubes. The PI3K inhibitor LY294002 blocked IGF-I but not Dex induced REDD1. IGF-I also stimulated REDD1 promoter activity. Although REDD1 protein was elevated 5-6 h after addition of IGF-I to myotubes, protein synthesis measured during this 1 h window was paradoxically greater in myotubes expressing more REDD1. In contrast to the IGF-I induced increase in REDD1 mRNA, REDD2 mRNA was decreased by IGF-I. We conclude that IGF-I stimulates REDD1 expression in skeletal muscle and myotubes but under these conditions the REDD1 response is not sufficient to repress protein synthesis.

Differential Regulation of Type I Interferon and Epidermal Growth Factor Pathways by a Human Respirovirus Virulence Factor

A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.

ABC50 Promotes Translation Initiation in Mammalian Cells

ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.

Enhanced IFNγ production in adenosine‐treated CHOCells: A mechanistic study

Adenosine causes growth arrest in recombinant mammalian cell cultures, which results in enhanced productivity of the recombinant protein. Adenosine is also known to increase intracellular ATP level when added to mammalian cells. As a cell's energy level affects its protein expression capacity, we investigated the factors that contribute to the increase in recombinant protein productivity. Chinese hamster ovary (CHO) cells expressing human interferon-gamma (IFNgamma) were treated with 1 mM adenosine on Day 2 of culture. The growth arrest resulted in 60% reduction in integral viable cell density when compared with control. However, IFNgamma titer improved 1.4-fold alongside a 2.5-fold increase in average specific productivity. The adenosine-treated cells also experienced a two-fold increase in ATP level that sustained for 3 days. Western blot studies revealed a relatively short-lived but strong activation of the energy sensor AMP-activated protein kinase (AMPK) in adenosine-treated cells. Activation of AMPK was probably due to adenosine being temporarily converted to AMP. Activated AMPK should have down-regulated protein translation by preventing mammalian target of rapamycin (mTOR) from phosphorylating and inactivating 4E-binding protein 1 (4E-BP1), a key repressor of protein translation initiation. However, Western blots showed increased phosphorylation of 4E-BP1 on Day 2 that lasted 3 days. This implied that a high concentration of ATP could keep 4E-BP1 inhibited, probably by directly modulating mTOR. This corroborated with an earlier in vitro observation (Dennis et al., Science. 2001;294:1102-1105). Inhibition of translation initiation repression is thus likely to contribute in part to the improvement in IFNgamma-specific productivity and titer.