Reticulocytes protein synthesis

Farrell, P. J., Balkow, K., Hunt, T., Jackson, R. J., andTrachsel, H. (1977). Phosphorylation of initiation factor eIF-2 and the control of reticulocyte protein synthesis. Cell 11, 187-200. 

Kemper WM, Berry KW, Merrick WC (1976) Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Balpha and M2Bbeta. J Biol Chem 251 5551-5557 Kim SC, Sprung R, Chen Y et al (2006a) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23 607-618 Kim YS, Kang KR, Wolff EC d al (2006b) Deoxyhypusine hydroxylase is a Fe(ll)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected]. J Biol Chem 281 13217—13225... 

The mature red blood cell cannot synthesize protein. Reticulocytes are active in protein synthesis. Once reticulocytes enter the circulation, they lose their intracellular organelles (ribosomes, mitochondria, etc) within about 24 hours, becoming young red blood cells and concomitandy losing their ability to synthesize protein. Extracts of rabbit reticulocytes (obtained by injecting rabbits with a chemical—phenylhydrazine—that causes a severe hemolytic anemia, so that the red cells are almost completely replaced by reticulocytes) are widely used as an in vitro system for synthesizing proteins. Endogenous mRNAs present in these reticulocytes are destroyed by use of a nuclease, whose activity can be inhibited by addition of Ca +. The system is then pro-... 

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

Protein synthesis inhibition. Chromatographic fraction of the dried seed, in cell culture, was active on reticulocyte lysate of rabbits, inhibitory concentrationjf, 15.25 ng/ mL vo84. 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

Similar evaluations employing an in vitro reticulocyte assay system failed to provide evidence of diminished polypeptide formation (unpub. obser.). Thus, a consistent pattern emerges in these three systems canavanine does not impede protein synthesis, including aberrant, canavanyl proteins. 

Regulation of protein synthesis in the rabbit reticulocyte. The vast majority of the protein synthesized in the rabbit reticulocyte is hemoglobin. The gross rate of protein synthesis in the reticulocyte is controlled indirectly by the concentration of heme. Heme inactivates a kinase that would otherwise inactivate the initiation complex involving eIF-2 and eIF-2B. The kinase phosphorylates the eIF-2 factor, making it impossible for the eIF-2-eIF-2B complex to exchange GDP for GTP. 

Globin is synthesized in reticulocytes (see Chap. 1, Prob. 1.1). which have no nucleus and therefore cannot utilize transcriptional and other potential modes of control. Control of globin synthesis from the pool of globin-enriched mRNA is geared to the concentration of hemin (Fe(III)-protoporphyrin]. which has the ability to inactivate a translational inhibitor of protein synthesis. The inhibitor is a protein kinase that phosphorylates and inactivates one of the initiation factors involved in initiation of translation. When the concentration of hemin is high, it binds to a regulatory subunit of the kinase and. as a result, initiation of globin synthesis can proceed. 

Incubate the reticulocytes with [3H]leucine, which will be incorporated into proteins. Prepare electron microscope autoradiographs and count silver grains per cell and the number of polysomes. The latter appear as rosettes of five ribosomes in these cells. A statistical comparison between the number of polysomes and the amount of protein synthesized during the incubation time (proportional to the number of silver grains) indicates whether there are nonactive polysomes. In fact, many of the polysomes are inactive i.e., they are switched off (see Chap. 17 for the control of protein synthesis). 

Some members of the human RNase A superfamily of proteins are known to have host defense activities (reviewed in ref. 9). These include, for example, two of the eosinophil cytotoxic granular proteins, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) (10). Angiogenin, a protein 65 % homologous to pancreatic RNase (11,12) that was originally isolated on the basis of its angiogenic activity (13), is a potent inhibitor of protein synthesis in the rabbit reticulocyte lysate (14) and when injected into Xenopus oocytes (15). We have therefore sought to fuse RNases to MAbs to evaluate their usefulness as immunotoxins (16,17). 

Breitbart, H., Atlan, H., Eltes, F., Herzberg, M. Mol. Biol. Rep. 2 (2), 167 (1975). Interaction between membrane properties and proteins synthesis in reticulocytes - a two step inhibition of protein synthesis by valinomycin... 

Herzberg, M., Breitbart, H., Atlan, H. Eur. J. Biochem. 45 (1), 161 (1974). Interactions between membrane functions and protein synthesis in reticulocytes. Effects of valinomycin and dicyclohexyl-18-crown-6... 

In reticulocytes, elF-2 is subject to phosphorylation by the heme-regulated elF-2-kinase (HRI) which is regulated via the heme concentration. Another protein kinase that can phosphorylate and regulate elF-2 is the RNA-dependent elF2a-kinase (PKR). The latter is induced by interferons and activated by double stranded RNA. Stress influences activate the protein kinases PRPK and GCN2 allowing for inhibition of protein synthesis via elF-2 phosphorylation too. 

TRANSLATIONAL CONTROL Eukaryotic cells can respond to various stimuli (e.g., heat shock, viral infections, and cell cycle phase changes) by selectively altering protein synthesis. The covalent modification of several translation factors (nonribosomal proteins that assist in the translation process) has been observed to alter the overall protein synthesis rate and/or enhance the translation of specific mRNAs. For example, the phosphorylation of the protein eIF-2 affects the rate of hemoglobin synthesis in rabbit reticulocytes (immature red blood cells). 

Thus, 3 -amino-3 -deoxy-2, 3 -ieco-adenosine and 3 -deoxy-2, 3 -5eco-inosine were prepared, and the former was used for the synthesis of a seco-puromycin analogue, which was not capable of inhibiting protein synthesis in reticulocyte systems using globin mRNA (88T6419). 

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