Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.     

Identification of caspase 3-mediated cleavage and functional alteration of eukaryotic initiation factor 2alpha in apoptosis

Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis.     

Initiation of endogenous messenger RNA translation on hen oviduct polysomes.

The eIF-2A fraction of reticulocyte ribosomal salt wash is capable of maximally stimulating the translation of endogenous messenger RNA by hen oviduct polysomes. The factor increases the initiation of protein synthesis 2--3-fold when measured by the factor-dependent synthesis of NH2-terminal peptides. The addition to these polysomes of elongation factor, EF-1, also increases protein synthesis but at a distinctly different rate and Mg2+ concentration optimum than the eIF-2A fraction. Moreover, there is no stimulation of NH2-terminal peptide synthesis with EF-1 alone. In contrast, all the known initiation factors are required for the translation of exogenous globulin mRNA on oviduct polysomes. Reticulocyte polysomes isolated by an identical procedure to that used for oviduct polysomes or by standard methods also require all the initiation factors for the translation of either endogenous mRNA or exogenous ovalbumin mRNA. Addition of 7-methylguanosine 5'-monophosphate does not inhibit the factor-dependent stimulation of oviduct polysomes except at high concentrations (1.0 mM) indicating that the sites with which 7-methylguanosine 5'-monophosphate normally competes are already occupied. These findings suggest that the messenger RNA remains bound to the oviduct polysomes or initiation factors. Hence the addition of exogenous factors which are involved with mRNA recognition and binding to the ribosome are not required. It has been previously shown that eIF-2A is capable of binding in vitro the initiatior tRNA to an existing Ado-Urd-Gua-40 S complex and initiating protein synthesis when such a complex is present. These present studies indicate that such an initiation complex may exist within the oviduct cell on membrane-associated polysomes. Under these circumstances eIF-2A mediates binding of the initiator tRNA and initiates protein synthesis.     

MAGE-3原核表达载体的构建和表达

通过RT-PCR扩增957bp的MAGE-3全长编码序列,将该片段克隆至Pgex-4T-2原核表达载体,转化大肠杆菌BL-21,经IPTG诱导表达,并经12%SDSPAGE凝胶电泳,考马斯亮蓝染色及Western blot鉴定,证明了目的基因的有效表达,目的蛋白高达细菌总蛋白的32%。表达产物经Glutathione Sepharose 4B 纯化后,每100mL菌液最终可获得3mg的目的蛋白,蛋白纯度在90%以上。纯化的GST-MAGE-3蛋白在体外冲击树突状细胞,能诱导特异性CTL杀伤肿瘤细胞活性。     

Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus.

Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3Dpol. There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3Dpol in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3Dpol was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 h and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 microliters) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 h. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.     

Purification and some properties of a protein binding and deacylating initiator transfer ribonucleic acid

1. A protein factor promoting the binding of initiator tRNA to the 40S ribosomal subunit was purified to homogeneity (more than 2500-fold) from rat liver cytosol. It has a mol.wt. of 265000 and is composed of four subunits of identical molecular weight. 2. This factor directs the binding of methionyl-tRNA(fMet) and to a lesser extent also of N-acetylphenylalanyl-tRNA, but not of methionyl-tRNA(Met) or phenylalanyl-tRNA, to the smaller ribosomal subunit at high concentrations of GTP (8-10mm) with an optimum at pH4.0. As evidenced by sucrose-density-gradient centrifugation, initiator tRNA becomes bound to the 40S subunit or to 80S ribosomes. 3. A deacylase activity specific for methionyl-tRNA(fMet) is associated with the pure factor. The factor significantly stimulates the translation of natural message in systems containing polyribosomes and both purified peptide-elongation factors. 4. The factor binds initiator tRNA or GTP to form unstable binary complexes and forms a ternary complex with methionyl-tRNA(fMet) and GTP. This complex is relatively stable. 5. In the absence of any cofactors the factor forms a stable complex with 40S and 80S ribosomes. This preformed ribosomal complex binds efficiently initiator tRNA at pH7.5 and low concentrations of GTP (1-2mm). The ternary complex of the factor with methionyl-tRNA(fMet) and GTP may be liberated from this ribosomal complex. 6. A protein factor capable of promoting the binding and simultaneously the deacylation of initiator tRNA may apparently have a regulatory function in physiological gene translation by removing an excess of methionyl-tRNA(fMet) not required for translation.     

Ribosomal production and in vitro selection of natural product-like peptidomimetics: the FIT and RaPID systems

Bioactive natural product peptides have diverse architectures such as non-standard sidechains and a macrocyclic backbone bearing modifications. In vitro translation of peptides bearing these features would provide the research community with a diverse collection of natural product peptide-like molecules with a potential for drug development. The ordinary in vitro translation system, however, is not amenable to the incorporation of non-proteinogenic amino acids or genetic encoding of macrocyclic backbones. To circumvent this problem, flexible tRNA-acylation ribozymes (flexizymes) were combined with a custom-made reconstituted translation system to produce the flexible in vitro translation (FIT) system. The FIT system was integrated with mRNA display to devise an in vitro selection technique, referred to as the random non-standard peptide integrated discovery (RaPID) system. It has recently yielded an N-methylated macrocyclic peptide having high affinity (Kd=0.60 nM) for its target protein, E6AP.     

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.     

Combinatorial peptidomics: a generic approach for protein expression profiling

Traditional approaches to protein profiling were built around the concept of investigating one protein at a time and have long since reached their limits of throughput. Here we present a completely new approach for comprehensive compositional analysis of complex protein mixtures, capable of overcoming the deficiencies of current proteomics techniques. The Combinatorial methodology utilises the peptidomics approach, in which protein samples are proteolytically digested using one or a combination of proteases prior to any assay being carried out. The second fundamental principle is the combinatorial depletion of the crude protein digest (i.e. of the peptide pool) by chemical crosslinking through amino acid side chains. Our approach relies on the chemical reactivities of the amino acids and therefore the amino acid content of the peptides (i.e. their information content) rather than their physical properties. Combinatorial peptidomics does not use affinity reagents and relies on neither chromatography nor electrophoretic separation techniques. It is the first generic methodology applicable to protein expression profiling, that is independent of the physical properties of proteins and does not require any prior knowledge of the proteins. Alternatively, a specific combinatorial strategy may be designed to analyse a particular known protein on the basis of that protein sequence alone or, in the absence of reliable protein sequence, even the predicted amino acid translation of an EST sequence. Combinatorial peptidomics is especially suitable for use with high throughput micro- and nano-fluidic platforms capable of running multiple depletion reactions in a single disposable chip.     

The effect of elevated temperature on protein synthesis in cell-free extracts of cultured Chinese hamster ovary cells

The effect of elevated temperature on the activity of various components involved in protein synthesis was investigated in extracts from cultured Chinese hamster ovary cells. The translation of exogenous mRNA was markedly inhibited by preincubation of the extract for 15 to 20 minutes at 42°C. However, the following intermediary reactions were not affected, or only slightly inhibited, at 42°C: 1) the incorporation of Met-tRNA_f into eIF-2·Met-tRNA_f·GTP ternary complex; 2) the interaction of the ternary complex with 40S ribosomal subunits to form the 40S preinitiation intermediate; 3) the binding of mRNA and 60S subunits to form the 80S initiation complex; and 4) the reactions catalyzed by elongation factors EF-1 and EF-2. The activity of Met-tRNA synthetase was markedly inhibited, affecting the formation of initiator Met-tRNA_f required for the initiation of protein synthesis and the translation of natural mRNA. Other aminoacyl-tRNA synthetases were not significantly affected by the elevated temperature.     

Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

The 5′-untranslated region (5′-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5′-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5′-UTRs with high translation efficiency using a ribosome display technique. A 5′-UTR random library, comprised of 5′-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5′-UTR with high translation efficiency was obtained from random 5′-UTR library.     

The p75 neurotrophin receptor interacts with multiple MAGE proteins

The p75 neurotrophin receptor has been implicated in diverse aspects of neurotrophin signaling, but the mechanisms by which its effects are mediated are not well understood. Here we identify two MAGE proteins, necdin and MAGE-H1, as interactors for the intracellular domain of p75 and show that the interaction is enhanced by ligand stimulation. PC12 cells transfected with necdin or MAGE-H1 exhibit accelerated differentiation in response to nerve growth factor. Expression of these two MAGE proteins is predominantly cytoplasmic in PC12 cells, and necdin was found to be capable of homodimerization, suggesting that it may act as a cytoplasmic adaptor to recruit a signaling complex to p75. These findings indicate that diverse MAGE family members can interact with the p75 receptor and highlight type II MAGE proteins as a potential family of interactors for signaling proteins containing type II death domains.     

Expression of Drosophila MAGE gene encoding a necdin homologous protein in postembryonic neurogenesis

The MAGE (melanoma antigen) family is characterized by a large conserved domain termed MAGE homology domain. Originally identified MAGE genes encoding tumor rejection antigens are expressed only in cancers and male germ cells. Necdin, which contains the MAGE homology domain, is highly expressed in postmitotic cells such as neurons and skeletal muscle cells. The human necdin gene NDN is transcribed only from the paternal allele through genomic imprinting, and its deficiency is implicated in the pathogenesis of the neurodevelopmental disorder Prader-Willi syndrome. Although over 30 MAGE genes have been identified in humans, fruit fly (Drosophila melanogaster) has only a single MAGE gene that encodes a protein similar to necdin homologous MAGE proteins. In this study, we analyzed the spatiotemporal expression patterns of MAGE mRNA and the encoded protein during fly development. Whole-mount embryo in situ hybridization analysis revealed that MAGE mRNA was highly expressed at the syncytial blastoderm stage and in the ventral and procephalic neurogenic regions of the ectoderm during gastrulation. In contrast, MAGE expression was nearly undetectable in postmitotic neurons of the central nervous system at late embryonic stages. During postembryonic neurogenesis, MAGE was highly expressed in neural stem cells (neuroblasts) and their progeny (ganglion mother cells and postmitotic neurons) at larval and pupal stages. MAGE was also expressed in postmitotic neurons including mushroom body neurons and retinal photoreceptors in adulthood. These results indicate that MAGE expression lasts throughout the postembryonic neurogenesis in Drosophila.     

The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7-45

The effects of incubation of yeast spheroplasts at elevated temperature (40 degrees C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells (wt A364A ) and a temperature-sensitive mutant (ts 7-45) derived from it. With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: the ribosomal subunit-polysome pattern; the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; the translation of poly(U) in postpolysomal extracts; the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and the formation of eIF-2 X GTP X Met-tRNAf ternary complex in the cytosol. With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations. Preincubation of temperature-sensitive spheroplasts at 40 degrees C resulted in: a further decrease in the level of 40 S subunits; disaggregation of polysomes; loss of ability to translate natural mRNA but not poly(U); decreased ability to form 40 S preinitiation intermediates; and production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly. The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.     

Phosphomimetic substitution of heterogeneous nuclear ribonucleoprotein A1 at serine 199 abolishes AKT-dependent internal ribosome entry site-transacting factor (ITAF) function via effects on strand annealing and results in mammalian target of rapamycin complex 1 (mTORC1) inhibitor sensitivity

The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G(1) arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P)(199)-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.