Protein Synthesis In HeLa Cells Research Articles

MTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.

Cytotoxic activity of Pinus cembra L. needle extract: A preliminary study on HeLa cell line

The aim of this study was to investigate the cytotoxic effects of a hydromethanolic extract obtained from cembran pine needles in HeLa cell line. In this respect, the effects of needle extract on protein synthesis, viability, proliferation and cell cycle in HeLa cells were evaluated after 48 h treatment. Cembran pine needle extract dose-dependently decreased protein synthesis in HeLa cells causing 44.26% reduction in protein synthesis at 100µg/ml. At 25, 50 and 100µg/ml, it increased cell death in comparison with the control (20.99%, 21.49% and 23.63%, respectively vs. 9.83%). In addition, at 100µg/ml, cembran pine needle extract showed a remarkable antiproliferative effect whereas at 25 and 50µg/ml, it induced sub-G1 phase cells accumulation (11.68 ± 0.81% and 14.69 ± 0.56%, respectively in comparison with control, 6.03 ± 0.55%), an indicator of proapoptotic effects. Taken together, these results indicate that cembran pine needles are a source of compounds with antitumor potential which needs to be further investigated and exploited.

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

Assessment of the Inhibition of Ricin Toxicity by Lactose in Milk

The effect of lactose at the concentration typically found in milk (134 mM) on the ability of ricin to inhibit protein synthesis in HeLa cells was studied. Ricin (0.001 to 300 μg/ml) that was either not treated or treated with 134 mM lactose was added to test tubes containing 1 ml of HeLa cells (approximately 3 × 10(5) cells in a low-leucine medium). After 2 h of incubation at 37°C, 0.5 μCi of L-[U-(14)C]-leucine was added to each tube and incubated for another 60 min. The cells were harvested by centrifugation and lysed, and cellular proteins were separated. The amount of radioactivity incorporated into the proteins was determined by liquid scintillation. The biological activity of ricin, i. e., the amount of radioactivity in a sample relative to that of the control (cells not treated with ricin), was calculated for each treatment. The inhibitory effect of 134 mM lactose on the biological activity of ricin was only significant at concentrations of ricin below 1 μg/ml. At higher ricin concentrations, the effect of 134 mM lactose decreased as the concentration of ricin increased, resulting in an increase in the inhibition of proteins synthesis. Our results also indicated that bovine milk, when used in place of 134 mM lactose, was more effective for reducing the activity of ricin at concentrations below 1 μg/ml but was ineffective against ricin concentrations greater than 1 μg/ml. These results suggest that milk may not protect against ricin intoxication at the concentration (0.89 μg/ml) equivalent to the lowest limit of its 50 % lethal dose for a 20-kg child consuming 225 ml (8 oz) of milk.

UAP56 is an important regulator of protein synthesis and growth in cardiomyocytes

UAP56, an ATP dependent RNA helicase that also has ATPase activity, is a DExD/H box protein that is phylogenetically grouped with the eukaryotic initiation factor eIF4A, the prototypical member of the DExD/H box family of helicases. UAP56, also known as BAT1, is an essential RNA splicing factor required for spliceosome assembly and mRNA export but its role in protein synthesis is not known. Here we demonstrate that UAP56 regulates protein synthesis and growth in cardiomyocytes. We found that wild-type (WT) UAP56 increased serum induced protein synthesis in HeLa cells. UAP56 mutants lacking ATPase and/or helicase activity inhibited protein synthesis compared with WT UAP56, suggesting that the ATPase and RNA helicase activity of UAP56 is important for protein synthesis. UAP56 siRNA inhibited phenylephrine (PE) induced protein synthesis in cardiomyocytes and inhibited PE induced cardiomyocyte hypertrophy. Our data demonstrate that UAP56 is an important regulator of protein synthesis and plays an important role in the regulation of cardiomyocyte growth.

Ubiquitination Is Required for Effective Replication of Coxsackievirus B3

BackgroundProtein ubiquitination and/or degradation by the ubiquitin/proteasome system (UPS) have been recognized as critical mechanisms in the regulation of numerous essential cellular functions. The importance of the UPS in viral pathogenesis has become increasingly apparent. Using murine cardiomyocytes, we have previously demonstrated that the UPS plays a key role in the replication of coxsackievirus B3 (CVB3), an important human pathogen associated with various diseases. To further elucidate the underlying mechanisms, we examined the interplay between the UPS and CVB3, focusing on the role of ubiquitination in viral lifecycle.Methodology/Principal FindingsAs assessed by in situ hybridization, Western blot, and plaque assay, we showed that proteasome inhibition decreased CVB3 RNA replication, protein synthesis, and viral titers in HeLa cells. There were no apparent changes in 20S proteasome activities following CVB3 infection. However, we found viral infection led to an accumulation of protein-ubiquitin conjugates, accompanied by a decreased protein expression of free ubiquitin, implicating an important role of ubiquitination in the UPS-mediated viral replication. Using small-interfering RNA, we demonstrated that gene-silencing of ubiquitin significantly reduced viral titers, possibly through downregulation of protein ubiquitination and subsequent alteration of protein function and/or degradation. Inhibition of deubiquitinating enzymes apparently enhances the inhibitory effects of proteasome inhibitors on CVB3 replication. Finally, by immunoprecipitation, we showed that coxsackieviral polymerase 3D was post-translationally modified by ubiquitination and such modification might be a prerequisite for its function in transcriptional regulation of viral genome.ConclusionCoxsackievirus infection promotes protein ubiquitination, contributing to effective viral replication, probably through ubiquitin modification of viral polymerase.

Paramyxovirus-Induced Shutoff of Host and Viral Protein Synthesis: Role of the P and V Proteins in Limiting PKR Activation

The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI(-) mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI(-) mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI(-) virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2alpha. WT SV5 was a poor activator of the eIF-2alpha kinase protein kinase R (PKR). By contrast, the P/V-CPI(-) mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI(-) mutant was restored to approximately 50% that of control HeLa cells. By contrast, the rates of P/V-CPI(-) viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI(-) mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI(-) mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

Enantioselective Synthesis of the Complex Rocaglate (−)‐Silvestrol

The total synthesis of the natural product (−)-silvestrol (1) has been accomplished and features enantioselective [3+2] photocycloaddition of a substituted 3-hydroxyflavone and methyl cinnamate promoted by a chiral Brønsted acid. Initial biological studies indicate a 5–10-fold greater activity of silvestrol as an inhibitor of protein synthesis in HeLa cells than its 1′′′′ diastereomer.

Molecular basis of the permeabilization of mammalian cells by ionophores.

Mammalian cells become permeable to low-molecular-weight compounds and proteins when they are treated with nigericin. Concentrations of this ionophore that have no effect on protein synthesis induce the entry of the translation inhibitors hygromycin B (Mr 550) and kappa-sarcin (Mr 16800). Treatment of HeLa cells with low concentrations of nigericin stimulate the (Na+/K+)ATPase and, as a consequence, increase the plasma membrane potential. A correlation is observed between the increase in membrane potential and the entry of antibiotics and protein toxins into cells. These results suggest a relationship between the two phenomena and help an understanding of the molecular mechanism for the passage of charged molecules and proteins into mammalian cells. This permeabilization is specific for certain ionophores, such as nigericin and monensin, and does not occur on treatment with amphotericin B, valinomycin or A23187. These compounds depolarize the plasma membrane and drastically block protein synthesis in HeLa cells under these conditions.

Fast control of DNA replication in response to hypoxia and to inhibited protein synthesis in CCRF-CEM and HeLa cells.

In order to elucidate whether data about the fast regulation of DNA replication in dependence on oxygen supply and on a functioning protein synthesis, previously elaborated with Ehrlich ascites cells, are valid for human cells too, we repeated key experiments with CCRF-CEM and HeLa cells. The most important techniques employed were DNA fibre autoradiography and alkaline sedimentation analyses of growing (pulse-labeled) daughter strand DNA. It was found that CCRF-CEM and HeLa cells responded to transient hypoxia and to transient inhibition of protein synthesis in an almost identical fashion. Scheduled replicon initiations were reversibly suppressed and the progress rates of replication forks, which were already active before the respective inhibitory conditions were established, were reversibly slowed down. The inclusion of the fork progress rate in the response differs from Ehrlich ascites cells, which respond only by suppressing initiation. Further circumstances of the fast oxygen dependent response, concerning the behaviour of ribonucleotide reductase and of the dNTP pools, revealed no significant differences among the three cell lines. The striking identity of the response of each of the cell lines to hypoxia and to inhibited protein synthesis prompts the suspicion that converging fast regulatory pathways act on the cellular replication machinery. The phenomena as such seem to be rather widespread among mammalian cells.

Morphological transformation caused by a partial sequence of U5 small nuclear RNA

My colleagues and I have reported that a small nuclear RNA, U5, can cause transformation of and chromosomal aberrations in rat fibroblast 3Y1 cells. In the study described here, I found that a partial U5 sequence, the 3′ half of the first stem structure (designated RNA3S), transformed the cells morphologically at a high frequency when expressed with a poly(A) tail (RNA3S+). The transformation critically depended upon a polypurine sequence in RNA3S+. The expressed RNA3S+ was associated with polysomes. The transformed cells did not exhibit significant frequencies of chromosome aberrations when compared with control cells, suggesting that the transformation occurs without significant induction of chromosome damage. Heterogeneous subcolonies emerged in monolayers when the morphologically transformed cells were subcultured and maintained at postconfluence. Cells from subcolonies acquired the ability to form small colonies in soft agar and tumors in nude mice, suggesting that RNA3S+ can drive the cells into the neoplastic stage. RNA3S+ also conferred morphological alterations and a growth advantage and decreased levels of fibronectin protein synthesis in human HeLa cells, confirming the transforming potential of the RNA. RNA3S+ transformation may therefore be a useful model system for studying a transformation process. Mol. Carcinog. 20:175–188, 1997. © 1997 Wiley-Liss, Inc.

Morphological transformation caused by a partial sequence of U5 small nuclear RNA

My colleagues and I have reported that a small nuclear RNA, U5, can cause transformation of and chromosomal aberrations in rat fibroblast 3Y1 cells. In the study described here, I found that a partial U5 sequence, the 3' half of the first stem structure (designated RNA3S), transformed the cells morphologically at a high frequency when expressed with a poly(A) tail (RNA3S+). The transformation critically depended upon a polypurine sequence in RNA3S+. The expressed RNA3S+ was associated with polysomes. The transformed cells did not exhibit significant frequencies of chromosome aberrations when compared with control cells, suggesting that the transformation occurs without significant induction of chromosome damage. Heterogeneous subcolonies emerged in monolayers when the morphologically transformed cells were subcultured and maintained at postconfluence. Cells from subcolonies acquired the ability to form small colonies in soft agar and tumors in nude mice, suggesting that RNA3S+ can drive the cells into the neoplastic stage. RNA3S+ also conferred morphological alterations and a growth advantage and decreased levels of fibronectin protein synthesis in human HeLa cells, confirming the transforming potential of the RNA. RNA3S+ transformation may therefore be a useful model system for studying a transformation process.

An intracellular calcium store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store

There is considerable evidence, reviewed by Brostrom and Brostrom [1], that Ca 2+ stores are involved in the regulation of protein synthesis. We provide evidence in HeLa cells that is consistent with their findings that depletion of Ca 2+ stores and not changes in cytosolic free Ca 2+ ([Ca 2+] i) inhibit protein synthesis, but we also show that the mechanism leading to depletion is critical. Specifically, depletion of stores by the Ca 2+-mobilizing hormone histamine does not inhibit protein synthesis. In assessing the role of Ca 2+ stores in protein synthesis, experiments in certain cell types have been complicated by the use of Ca 2+ ionophores, which simultaneously elevate [Ca 2+] i and deplete Ca 2+ stores. We have measured total cell Ca 2+, [Ca 2+] i and protein synthesis in HeLa cells under conditions that allowed evaluation of the separate contributions of stores and [Ca 2+] i. Using 1,2-bis(2-aminophenoxyethane)-N,N,N′N′-tetraacetic acid (BAPTA) as an intracellular Ca 2+, chelator and thapsigargin, which inhibits the membrane Ca 2+-ATPase of storage vesicles, total cell Ca 2+ can be depleted and this depletion is enhanced by extracellular EGTA which blocks Ca 2+ influx; [Ca 2+] i is actually lowered by BAPTA under these conditions. Protein synthesis is inhibited by BAPTA in the presence of EGTA and by thapsigargin with or without EGTA. However, histamine which with EGTA, affects an equal degree of Ca 2+ depletion does not inhibit protein synthesis. Thus, it is suggested that Ca 2+ stores are not homogeneous, and that the hormone-sensitive store specifically does not play a role in the regulation of protein synthesis. In this respect, the hormone-sensitive and insensitive stores do not functionally communicate and may be separately regulated.

Polyadenylated RNA sequences produced in vaccinia virus-infected cells under aberrant conditions inhibit protein synthesis in vitro

We have previously demonstrated that small nontranslated polyadenylated RNAs (POLADS) produced in vaccinia virus (VV)-infected cells inhibit the translation of cellular mRNAs, but minimally affect the translation of VV mRNAs in a cell-free protein synthesizing system. Infection of HeLa cells with ultraviolet-irradiated vaccinia virus or infection in the presence of actinomycin D (ACD) amplifies the synthesis of POLADS compared to the amount produced in cells infected under normal conditions. The effect of these POLADS on translation was studied in the reticulocyte lysate system. Polyadenylated RNAs isolated from cells infected with wild-type virus (V-POLADS) had a greater inhibitory effect on HeLa cell protein synthesis than on VV protein synthesis. Polyadenylated sequences obtained from cells infected with ultraviolet-irradiated virus (UV-POLADS) or from cells infected in the presence of ACD (ACD-POLADS), however, inhibited translation of both HeLa and viral mRNAs. Ultraviolet-POLADS and ACD-POLADS were found to possess, on average, longer poly(A) tails than V-POLADS. The inhibition of translation of both host and viral mRNAs effected by V-POLADS, UV-POLADS, and ACD-POLADS was reversed by poly(A) binding protein.

Isolation and partial characterization of three protein-synthesis inhibitory proteins from the seeds of Luffa cylindrica.

Three new proteins which inhibit protein synthesis in rabbit reticulocyte lysates were isolated from an extract of sponge gourd (Luffa cylindrica) seeds by chromatography on a AF-Blue Toyopearl column followed by FPLC with a Mono S column. These three protein-synthesis inhibitory proteins (PSIs) have molecular masses of 19 kDa, 15 kDa, and 9 kDa, and were designated 19K-PSI, 15K-PSI, and 9K-PSI, respectively. Although the 19K-PSI had no effect on protein synthesis in HeLa cells, its inhibitory activity on the cell-free protein synthesis was 340- and 83-fold stronger than those of ricin A-chain and luffin-a, respectively, probably due to hydrolyzing mRNA. The inhibitory activities of 15K- and 9K-PSIs on the cell-free protein synthesis were weaker than those of ricin A-chain and luffin-a. The 19K-PSI was a glycoprotein having an ordinary amino acid composition, three intramolecular disulfide bonds and a blocked N-terminal residue, while the 15K-PSI was extraordinarily rich in glycine and the 9K-PSI in arginine and glutamic acid (and/or glutamine). The amino acid composition of 19K-PSI was: Ser27Glx3Gly164Tyr7Lys9His6, and that of 9K-PSI was: Asx3Glx25Pro2Gly5Lys2His2Arg25Trp3.

Inhibition of Translational Initiation in Eukaryotic Cells by Calcium Ionophore

Ca2+ has been recently reported to be required for high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). In the present investigation low concentrations of the Ca2+ ionophores, A23187 and ionomycin, were found to rapidly suppress the Ca2+-dependent component of protein synthesis in GH3 cells. More ionophore was required to inhibit amino acid incorporation into protein as extracellular Ca2+ was increased. Pre-existing inhibitions of protein synthesis produced by low concentrations of ionophore at low extracellular Ca2+ concentrations were reversed by adjustment to high extracellular Ca2+. Treatment with ionophore reduced the cellular contents of polysomes and 43 S preinitiation complex to values equivalent to those found for Ca2+-depleted cells. Average ribosomal transit times were unaffected by ionophore, and treated cells retained the ability to accumulate polysomes when incubated with cycloheximide. Cell types, such as HeLa and Chinese hamster ovary, that normally display only a modest Ca2+-dependent component of protein synthesis, manifested a strong underlying Ca2+ dependence in amino acid incorporation and polysome formation following treatment with low concentrations of ionophore. Protein synthesis in GH3 or HeLa cells during recovery from heat shock and arsenite treatment was not affected by cellular Ca2+ depletion or ionophore treatment. On the basis of these results, Ca2+ ionophore is proposed to inhibit Ca2+-dependent translational initiation through facilitating the mobilization of sequestered intracellular Ca2+.

Characterization of the double-stranded RNA implicated in the inhibition of protein synthesis in cells infected with a mutant adenovirus defective for VA RNA

In the absence of VA RNA,, protein synthesis in adenovirus-infected HeLa cells fails at late times of infection because of defective initiation. The defect is due to the activation of a protein kinase that phosphorylates the α-subunit of initiation factor eIF-2. The kinase responsible for the translational defect is DAI, the double-stranded RNA (dsRNA)-activated inhibitor of protein synthesis, which is present in uninfected HeLa cells at a basal level and in a largely inactive, latent state. In vitro it can be activated by incubation with ATP and a low concentration of dsRNA. Previous studies suggested that RNA generated during the course of infection can activate DAL We show here that the activator RNA has the properties of dsRNA: it chromatographs with dsRNA, can be denatured and reannealed, and is destroyed by a dsRNA-specific nuclease. At least some of the dsRNA is viral. It hybridizes to DNA sequences in the center of the viral genome, principally between map units 47 and 51 and 73 and 76, consistent with an origin in the symmetrical transcription of both viral DNA strands.

Hyperthermic toxicity and the modulation of heat damage to cell protein synthesis in HeLa cells.

That some of the effects of hyperthermia on HeLa cell protein synthesis may involve free radical activity is suggested by experiments with free radical scavengers. A possible source of damaging free radicals induced by heat may lie in peroxidative events within lipid-containing membranes. These can be blocked by EGTA. Other experiments with t-butylhydroperoxide indicate that cycloheximide can also neutralise peroxidative effects. These effects are discussed in the light of observations that calcium depletion (or EGTA treatment) can induce thermotolerance and that cycloheximide does not prevent the development of thermotolerance.

Attachment, uncoating and blockage of cell protein synthesis by Sabin derivatives of poliovirus in HeLa and Vero cells.

Virulent Mah type 1 poliovirus and the attenuated Sab types 1, 2 and 3 poliovirus do not efficiently produce plaques or grow as efficiently with Vero cells as with HeLa cells. All viruses attach poorly to simian cells on monolayer and, except for Mah and Sab1, eclipse poorly. Additionally, all three attenuated viruses block initiation of Vero cell protein synthesis less effectively than HeLa cell protein synthesis. The lower rates of attachment and eclipse of these viruses with Vero cells and their limited capacities to block cellular protein synthesis and thereby provide ribosomes for translation of viral RNA probably contribute to their more restricted growth in Vero cells.

Baciphelacin: a new eukaryotic translation inhibitor

Baciphelacin an antibiotic produced by Bacillus thiaminolyticus was a potent inhibitor of protein synthesis in HeLa cells and other mammalian cell lines. It had no effect on DNA or RNA synthesis. Concentrations of baciphelacin around 10 −7 M inhibited protein synthesis by 50% in intact cells. The antibiotic had no effect on protein synthesis in Saccharomyces cerevisiae or Escherichia coli, but inhibited the protozoan Trypanosoma brucei. In vitro protein synthesis in a rabbit reticulocyte cell-free system was blocked by baciphelacin. However, translation of globin mRNA in a wheat cell-free system was not affected by this antibiotic. Baciphelacin had no activity against a number of cell-free systems used to measure different steps of translation, including binding of substrates to the ribosome, peptide bond formation and polyphenylalanine synthesis. Therefore, it is assumed that it affects the initiation of translation or the charging of tRNA. Finally, the inhibition of protein synthesis by compounds structurally related to baciphelacin was tested and their effects compared to baciphelacin.