Brian Safer

The Regulation of Initiation of Mammalian Protein Synthesis

Publisher Summary This chapter describes the regulation of initiation of mammalian protein synthesis. It discusses the current knowledge of the initiation of protein synthesis. The regulation of the rate of initiation has been shown to be of great importance in many cells and tissues during rapid transitions from one metabolic state to another. Although mechanisms by which initiation is regulated in reticulocyte lysate and in extracts from interferon-treated cells have been determined, it is not known to what extent these mechanisms represent general regulatory mechanisms that may be found in a wide variety of cell types and physiological conditions, such as those discussed. The accumulation of information on both the sequence of events and the regulation of initiation derived from reticulocyte lysate has provided a solid foundation from which to ask questions of a wide range of cells and tissues. However, it is not yet known if a ubiquitous regulatory mechanism exists for the control of initiation, or if the regulation of eIF-2 activity is the prevalent point of control. Although data are accumulating to suggest that the control of eIF-2 activity may represent a general regulatory mechanism, it is becoming increasingly apparent that more than one step in the initiation pathway is subject to control. It is expected that the development of cell-free protein synthesizing systems from nucleated cells will allow the pursuit of these questions.

Evidence for role of m7G5'-phosphate group in recognition of eukaryotic mRNA by initiation factor IF-M3.

7-methylguanosine 5′-monophosphate inhibits protein synthesis in a fractionated, messenger-dependent, reticulocyte cell-free system. This compound also inhibits binding of histone mRNA to reticulocyte ribosomes as well as interaction of VSV mRNA and histone mRNA but not EMC virus RNA with purified initiation factor IF-M3. These studies provide evidence that the role of 7-methylguanosine in the mechanism for initiation of eukaryotic mRNA translation may be related to specific recognition of mRNA by initiation factor IF-M3.

The Roles of AAV Rep Proteins in Gene Expression and Targeted Integration

Adeno-associated virus 2 (AAV), a nonpathogenic human virus, can either integrate into host chromatin and remain latent or replicate following infection. The outcome depends on the cellular conditions. Under conditions permissive for AAV DNA replication (e.g., during adenovirus coinfection), AAV gene expression is induced. The single-stranded AAV genome of 4680 nucleotides is organized into two open reading frames (ORFs) that encode structural capsid proteins (Cap) and nonstructural proteins (Rep) (Fig. 1). Two promoters at AAV map units 5 and 19, p5 and p19, direct expression of the rep gene. The cap gene is regulated by the p40 promoter. A common intron results in the production of four Rep proteins: p5 initiated Rep78, Rep68 and p19 initiated Rep52, Rep40. The inverted terminal repeats (ITRs) function as viral origins of replication required for encapsidation of the of AAV DNA. The production of AAV Rep proteins enables viral DNA to replicate, resulting in a geometric increase in the number of viral genomes. The AAV p5 initiated regulatory proteins Rep78 and Rep68 interact with the viral promoters to establish a feedback loop. These Rep proteins are involved directly in viral DNA replication as well.

Signal transduction pathways that contribute to increased protein synthesis during T-cell activation

Protein synthesis rates were maximally stimulated in human lymphocytes by ionomycin and the phorbol ester PMA (I+P), which promotes proliferation, whereas PMA alone, which does not promote proliferation, stimulated protein synthesis to a lesser degree. Three translation-associated activities, eIF4E phosphorylation, eIF2B activity and 4E-BP1 phosphorylation also increased with stimulation by I+P and PMA, but only 4E-BP1 phosphorylation was differentially stimulated by these conditions. Correspondingly, signaling pathways activated in T cells were probed for their connection to these activities. Immunosuppressants FK506 and rapamycin partially blocked the protein synthesis rate increases by I+P stimulation. FK506 had less of an inhibitory effect with PMA stimulation suggesting that its mechanism mostly affected ionomycin-activated signals. I+P and PMA equally stimulated phosphorylation of ERK1/2, but I+P more strongly stimulated Akt, and p70(S6K) phosphorylation. An inhibitor that blocks ERK1/2 phosphorylation only slightly reduced protein synthesis rates stimulated by I+P or PMA, but greatly reduced eIF4E phosphorylation and eIF2B activity. In contrast, inhibitors of the PI-3 kinase and mTOR pathways strongly blocked early protein synthesis rate stimulated by I+P and PMA and also blocked 4E-BP1 phosphorylation and release of eIF4E suggesting that these pathways regulate protein synthesis activities, which are important for proliferation in T cells.

Transcriptional regulation of E2F-1 and eIF-2 genes by α-Pal: a potential mechanism for coordinated regulation of protein synthesis, growth, and the cell cycle

alpha-Pal regulates the basal transcription of the alpha and beta subunits of eukaryotic initiation factor two (eIF-2), a rate-limiting enzyme for the initiation of protein biosynthesis. We recently showed that its global function may be to modulate the expression of key metabolic genes in response to cellular proliferation. In this paper, we examined a potential molecular mechanism by which alpha-Pal may achieve this function. When overexpressed, alpha-Pal upregulated protein synthesis and growth, but downregulated the cell cycle. The mechanism for the increased protein synthesis and growth appeared to be a transcriptional upregulation of the eIF-2alpha and eIF-2beta genes. The mechanism for the cell cycle downregulation appeared to be a transcriptional downregulation of E2F-1, a transcription factor that regulates genes required for cell cycle progression beyond the G1/S interphase. Specifically, an apparently modified species of alpha-Pal bound to the eIF-2 promoters and induced transcriptional upregulation, whereas, an apparently unmodified species of the alpha-Pal bound to the E2F-1 promoter and induced transcriptional downregulation. By this mechanism, alpha-Pal may participate in coordinating the regulation of global protein synthesis, growth and the cell cycle; a regulation that is essential to cellular differentiation.

The role of phosphorylation in the regulation of eukaryotic initiation factor 2 activity.

Publisher Summary The regulation of protein synthesis by changes in the rate of translational initiation is a rapid, short-term mechanism for modifying gene expression in animal cells. Prominent examples include viral inhibition of host protein synthesis, the expression of latent mRNA during embryonic development, the coordination of ribosome and ribosomal protein synthesis, the rapid adjustment of protein synthesis with the energy and nutritional status of the cell, and the coordination of heme and globin biosynthesis during reticulocyte maturation. The regulation of the initiation of protein synthesis in reticulocyte lysate is seen as involving a series of events leading to a progressive reduction of the active eukaryotic initiation factor (eIF)-2 pool. This is initiated by a small increase in the steady-state level of phosphorylation of the subunit of eIF-2 that, by subsequent modifications, leads to conformational changes in the β subunit and a reduction in the capacity of eIF-2 to be recycled. Further understanding of this regulation requires an appreciation of the mechanisms by which eIF-2 is reutilized and how the reversal factors and other eIF-2 cofactors participate in the eIF-2 activity cycle.

Immunosuppressants FK506 and rapamycin have different effects on the biosynthesis of cytoplasmic actin during the early period of T cell activation.

FK506 and rapamycin are immunosuppressants that interfere with T cell activation. FK506 inhibits early events of T cell activation such as the induction of cytokine transcription, whereas rapamycin inhibits later interleukin 2 signalling events. However, both reagents either directly or indirectly reduce protein synthesis. Therefore a kinetic study was conducted in human primary T lymphocytes examining increased synthesis of proteins stimulated by either ionomycin+phorbol myristate acetate (PMA) or PMA alone. Three patterns of protein expression were observed. Synthesis of one group of proteins had enhanced synthesis with FK506, but reduced synthesis with rapamycin. A second group had reduced synthesis with rapamycin and either no change or a slight reduction with FK506 and a third group had reduction with both FK506 and rapamycin. One major protein of the first group, p42, had a rapid increase in synthesis that decreased by 8 h. Its synthesis was strongly enhanced by FK506, but reduced by rapamycin after ionomycin+PMA stimulation. In contrast, this protein was strongly induced by PMA alone in these cells and not affected by FK506 treatment, but still reduced by rapamycin. p42 was identified as cytoplasmic actin. mRNA levels of both gamma- and beta-actin were found to be enhanced with FK506 treatment suggesting that regulation of actin was at a transcriptional or post-transcriptional level. Results with actinomycin D indicated that FK506 is regulating actin biosynthesis at the post-transcriptional level. Rapamycin, however, appeared to be operating at the level of translation.

Classification of an eIF-2 phosphatase as a type-2 protein phosphatase

As more and more proteins are reported to undergo regulation by phosphorylation-dephosphorylation, the number of protein phosphatases described in the literature has increased exponentially. However most of these enzymes have been reported to dephosphorylate several substrates, and this has raised the possibility that there are relatively few protein phosphatase catalytic subunits in mammalian tissues. This idea is supported by the observation that several high h4, protein phosphatases each contain a catalytic subunit of M, 30 000-35 000 [l-5], which can be generated by the addition of ethanol at room temperature [ 1,2,5], freezing and thawing in 0.2 M 2-mercaptoethanol [3,4] or treatment with urea [6]. This subunit has been termed protein phosphatase C [ 11, and found to possess a broad substrate specificity [ 1,7]. However, this preparation is a mixture of two distinct enzymes with very similar physical properties [S]. Furthermore, these activities are closely related to two enzymes termed protein phosphatase-I and protein phosphatase-2, which have been implicated in the regulation of glycogen metabolism in mammalian muscle [9-l 11. The two enzymes termed type-l and type-2 can be distinguished readily by the use of two criteria:

Detection of early gene expression changes during activation of human primary lymphocytes by in vitro synthesis of proteins from polysome-associated mRNAs

The rapid increase in protein synthesis during the mitogenic stimulation of human peripheral blood lymphocyte is the result of global and specific translational control mechanisms. To study some of these mechanisms, we examined the in vitro translatability of mRNAs associated with the polyribosome fraction. Polyribosome fractions were isolated from lymphocytes after activation with ionomycin and the phorbol ester PMA. The associated PAmRNAs were translated in the presence of mRNA‐depleted rabbit reticulocyte lysate and [35S]Met, and the protein products were analyzed by SDS–PAGE and autoradiography. There was little synthesis of protein from the PAmRNAs isolated from unactivated T cells, but the PAmRNAs isolated from activated T cells showed a rapid increase in translatability. Translation of the PAmRNAs was sensitive to edeine and m7GTP, suggesting their cap‐dependent translation. With activation, the majority of proteins showed increasing in vitro translation, but two proteins, p72 and p33, were found to have increased synthesis within 30 min, which decreased in 1 h. Transcription inhibitors were used to ascertain if regulation of their expression was transcriptional or translational. To identify these proteins, we used biotinylated lysine during the in vitro translation reaction, and we extracted the biotinylated protein by using streptavidin magnetic beads. The protein product was analyzed by mass spectrometry. p33 was identified as a prohibitin‐like protein (BAP37), but the identification of p72 was not found in the databases. The distinct up‐regulation and down‐regulation of their protein expression suggest their tightly controlled regulation during early T cell activation.

The purification and characterization of subunits α, β, and γ from the rabbit reticulocyte eukaryotic initiation factor 2

Abstract Eukaryotic initiation factor 2 (eIF-2) contains three nonidentical subunits, α, β, and γ. The simultaneous purification of all three subunits was achieved by reverse-phase HPLC using a 0.1% trifluoroacetic acid-acetonitrile binary solvent system. The order of the eluted subunits, β, α, and γ, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After hydrolysis in 6 n HCl, picomole level amino acid composition analysis was achieved by the ninhydrin reaction on a Beckman 6300 system. Using second-derivative spectroscopic analysis, Trp was detected in all three subunits. All three subunits were subjected to amino-terminal sequence analysis. The aminoterminal of eIF-2α from amino acid positions 1 to 23 inclusive was determined. The order of eight amino acids from the amino-terminal of eIF-2γ was also determined. This characterization and partial determination of the primary sequence of these subunits permit the utilization of molecular biology techniques in order to elucidate the complete primary structure. Additionally, the partial amino acid sequence data permitted the designation of synthetic gene probes as well as the identification of eIF-2α and γ cDNA and/or genomic clones.