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Changes in activity and amount of active elongation factor 1α in aging and immortal human fibroblast cultures
Abstract
Stoichiometrically estimated amounts of active elongation factor, EF-1 alpha, remain constant in serially passaged Phase II cultures of human fibroblasts, MRC-5, but decrease by 45% towards the end (Phase III) of their lifespan. Catalytic activity of EF-1 alpha is also reduced by 35% in Phase III old cells. The SV40 transformed immortal cell line MRC-5V2 has 30% higher levels of active EF-1 alpha without significant increase in its catalytic activity. Low-serum-associated G1 arrest of normal and transformed cells reduces amounts of active EF-1 alpha by 35% and 20%, respectively. Catalytic activity, however, is reduced rapidly only in G1 arrested normal cells and not in transformed cells. Even though the cell cycle-related changes are reversible both in normal and transformed cells, the age-related decline in amounts of active EF-1 alpha and its activity are irreversible and, most probably, crucial.
... 7). Die Menge an EF1-alpha im Zytoplasma korreliert außerdem direkt mit der Zellzyklus-Rate (Cavallius et al. 1986;Duttaroy et al. 1998;Grassi et al. 2007), was erklären könnte, dass es auch in stark metastasierenden TIC höher exprimiert wird (Tab. 7). ...
... Des Weiteren schützt es vor Apoptose nach Interleukin-3-Entzug (Talapatra et al. 2002) und die Überexpression führt zu einer Resistenz gegenüber Cisplatin in humanen Zelllinien von Kopf-Hals-Karzinomen (Johnsson et al. 2000). Die Menge an EF1-alpha im Zytoplasma korreliert direkt mit der Zellzyklus-Rate (Cavallius et al. 1986;Duttaroy et al. 1998;Grassi et al. 2007). Durch diese Eigenschaften gilt es als Onkogen beim Ovarialkarzinom (Anand et al. 2002). ...
T cell responses in cancer patients correlate with improved prognosis. This effect can partially be attributed to cytotoxic T cells that recognize tumor antigens on cancer cells. Hence, the induction or improvement of such therapeutic tumor antigen-specific T cell responses is a major goal of tumor immunotherapy. So far the target-antigens of relevant prognostic T cell responses are unknown.
In this thesis a systematic analysis was performed to identify those antigens that are able to elicit spontaneous T cell responses in patients with colorectal carcinoma. Those responses against the newly identified antigens were then compared, in a larger patient cohort, to T cell responses against established “canonical” tumor antigens that are commonly used for immunotherapy.
The establishment of the proteome-based technology PF2D (Protein fractionation in two dimensions) in our group allowed the identification of protein fractions out of lysates from primary and metastatic tumor tissue that were capable to elicit strong T cell responses in ELISpot assays. With mass spectrometry and further ELISpot tests we could identify 21 so far unknown tumor-associated antigens. We could show that the T cell response was directed against different antigens in primary tumors or liver metastasis. Some of the identified antigens were selectively overexpressed on tumor stem cells. Additionally in the blood of these patients there was a higher frequency of T cells specific for the newly identified TAA in comparison with canonical TAA, that have been described in the past.
In part two of this thesis we could show that patients suffering from colorectal cancer are able to induce spontaneous T cell responses against mutated forms of tumor-associated antigens. These patients harbor higher frequencies of T cells in the blood that are specific for the mutated forms compared to the corresponding wildtyp antigens and they show an increased immune response.
This work shows that classical tumor-associated antigens play a minor role in the immune response against tumors. Patient-individual antigens, a result of the unique tumor development in every patient, are highly immunogenic and serve as better targets for immune monitoring and directed T cell therapies.
... The translation process includes three phases: initiation, elongation, and termination. Many studies indicated that the regulation of the elongation phase can be important during the cell cycle (Moldave, 1985), aging (Cavallius et al., 1986), and oxidative stress (Ayala et al., 1996). The eukaryotic translation elongation factor 1A (eEF1A), whose primary function is to shuttle aminoacyl-tRNA during protein translation, plays a key role in the elongation process (Kaziro et al., 1991). ...
Translation elongation factor 1A (eEF1A) is a factor critically involved in the process of protein synthesis. The activity of eEF1A has been shown by several studies to be regulated by post-translational modifi cations such as phosphorylation and dephosphorylation. However, until now less research has focused on other post-translational modifi cations of eEF1A, especially acetylation. In this report, we provide new evidence for the existence of eEF1A acetylation in PLC5 cells by immunoprecipitation and Western blotting. Using the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), we found that the deacetylation of eEF1A is mainly attributable to classes I and II HDAC rather than class III HDAC, and, furthermore, that the antitumour agent etoposide phosphate (VP 16) enhances the acetylation of eEF1A in a synergistic way with TSA. Our data suggest the possibility that the increased acetylation of eEF1A could be a new mechanism for the antitumour effect of etoposide
... (eEFIA) — высокоэкспрессирующийся белок, игра ющий главную роль в цикле элонгации трансляции эукариот. Важно, также что eEFIA, кроме белко вого синтеза, выполняет немало других неканони ческих функций, участвуя в таких клеточных про цессах, как старение[1], апоптоз[2], репликация ДНК[3 ], стимуляция злокачественной трансфор мации[4 ]и транскрипции TAR РНК[5 ]. Показа но взаимодействие eEFIA с актином, микротрубоч ками и кальмодулином в цитоскелете[6 ], а также участие белка в активации фосфатидилинозитол-4- киназы[7 ]. ...
Eukaryotic translation elongation factor lA (eEF1A) is one of the main components of the translation machinery in cells. It performs also a number of non-canonical functions which are not related to the protein synthesis. In this work eEF1A was subjected to limited trypsinolysis to obtain the truncated by 67 amino acids polypeptide eEF1A similar to a potential product of the oncogene PTI-1. eEF1A was used as a model to study possible translation functions of the PTI-1 oncogene product. Comparison of eEF1A and eEF1A in four different functional tests suggests a possibility of the participation of the PTI-1 protein in translation.
... The previous study showed the EEF1A gene was detected down-regulation expression from 75 day-fetal to 1 week-postnatal muscle in pig using cDNA macroarray method . Another study showed EEF1A1 expression level was decreased with aging in human fibroblasts (Cavallius et al., 1986). Therefore, our results were consistent with the previous studies. ...
The eukaryotic elongation factor 1 A (EEF1A) participates in protein synthesis by forming the eEF1A·GTP·tRNA complex to deliver aminoacyl-tRNA to the A site of ribosomes. This study described cDNA sequences and partial genomic structure of porcine EEF1A1. The porcine EEF1A1 gene encoded a protein with 462 amino acids, which shared complete homology with human, chimpanzee and dog. The temporal expression pattern showed the diversity of EEF1A1 level in mRNA was relatively minor in prenatal embryo skeletal muscle, however, the expression decreased during aging after birth in skeletal muscle of the Chinese Tongcheng pig. The spatial expression patterns indicated that the gene expressed in skeletal muscle, heart, lung, liver, kidney, fat and spleen. In addition, we assigned the gene to porcine chromosome 1 using a radiation hybrid panel.
... EF-1 is a cellular protein complex that plays a role in protein synthesis by mediating the transfer of aminoacyl-tRNA to 80 S ribosomes [102]. Expression of the Ef-1-alpha gene decreases with age in mice and human fibroblasts [103] and forced expression of EF-1-alpha prolongs the lifespan of Drosophila melanogaster [104]. These reports suggest that the EF-1 complex has trophic effects on cellular function. ...
After the onset of brain ischemia, a series of events leads ultimately to the death of neurons. Many molecules can be pharmacologically targeted to protect neurons during these events, which include glutamate release, glutamate receptor activation, excitotoxicity, Ca2+ influx into cells, mitochondrial dysfunction, activation of intracellular enzymes, free radical production, nitric oxide production, and inflammation. There have been a number of attempts to develop neuroprotectants for brain ischemia, but many of these attempts have failed. It was reported that cyclosporin A (CsA) dramatically ameliorates neuronal cell damage during ischemia. Some researchers consider ischemic cell death as a unique process that is distinct from both apoptosis and necrosis, and suggested that mitochondrial dysfunction and Δψ collapse are key steps for ischemic cell death. It was also suggested that CsA has a unique neuroprotective effect that is related to mitochondrial dysfunction. Here, I will exhibit examples of neuroprotectants that are now being developed or in clinical trials, and will discuss previous researches about the mechanism underlying the unique CsA action. I will then introduce the results of our cDNA subtraction experiment with or without CsA administration in the rat brain, along with our hypothesis about the mechanism underlying CsA's effect on transcriptional regulation.
... blasts (Cavallius et al., 1986). The expression levels of EFla vary among tissues and several-fold higher expression is found in cultured cells compared to tissues (Sanders et al., 1992). ...
A novel isoform of human elongation factor-1α (EF-1α2) has been characterised. It shows a high similarity to other EF-1α proteins, especially to a rat EF-1α variant and it has all the characteristics of a functional EF-1α protein. The pattern of expression of both EF-1α2 and EF-1α was analysed in different human tissues. This showed that the two proteins were differentially expressed, EF-1α2 was expressed in brain, heart, skeletal muscle and in the transformed cell lines AMA and K14, but was undetectable in other tissues and in both primary and transformed human fibroblasts. EF-1α was expressed in brain, placenta, lung, liver, kidney, pancreas and in all the cell lines that we have analysed but barely detectable in heart and skeletal muscle.
... Besides its essential role in protein synthesis, there are several reports showing the involvement of EF-1α in a number of other functions: complex formation with mitotic apparatus (Kuriyama et al., 1990;Ohta et al., 1990), interaction with cytoskeletal elements (Yang et al., 1990;Shiina et al., 1994), with the endoplasmic reticulum (Hayashi et al., 1989), and with ribosomal subunits (Berchtold et al., 1993). Furthermore, a possible implication has been inferred in cellular senescence and aging (Cavallius et al., 1986;Shepherd et al., 1989). All these facts suggest the coupling of protein synthesis and other cellular functions. ...
The cDNA for a protein that is related to translation elongation factor 1α (EF-1α) has been cloned from the sea urchin Anthocidaris crassispina. The sea urchin EF-1α-related protein (AcEFP) seems somewhat unique in structure compared to EF-1α of other organisms so that it is uncertain if AcEFP is a genuine EF-1α. Still, it possesses many features of typical EF-1α reported so far, suggesting that AcEFP is involved in protein synthesis or its regulation. Genomic Southern analysis indicated that the sea urchin genome contains one or at most two copies of the AcEFP gene. A single transcript of 2.2 kb is expressed ubiquitously in adult tissues examined and, during embryogenesis, zygotically after the blastula stage. Whole mount in situ hybridization showed that the AcEFP gene is also widely expressed in embryos, with relatively high expression in the gut and oral ectoderm, both of which are proliferating tissues in embryos. The expression of AcEFP was not affected in embryos by agents that destabilize the extracellular matrix (ECM), suggesting that expression of AcEFP is relatively independent of the integrity of ECM in sea urchin embryos.
Eukaryotic protein synthesis is the highly conserved, complex mechanism of translating genetic information into proteins. Although this process is essential for cellular homeostasis, dysregulations are associated with cellular malfunctions and diseases including cancer and diabetes. In the challenging and ongoing search for adequate treatment possibilities, natural products represent excellent research tools and drug leads for new interactions with the translational machinery and for influencing mRNA translation. In this review, bacterial-, marine- and plant-derived natural compounds that interact with different steps of mRNA translation, comprising ribosomal assembly, translation initiation and elongation, are highlighted. Thereby, the exact binding and interacting partners are unveiled in order to accurately understand the mode of action of each natural product. The pharmacological relevance of these compounds is furthermore assessed by evaluating the observed biological activities in the light of translational inhibition and by enlightening potential obstacles and undesired side-effects, e.g. in clinical trials. As many of the natural products presented here possess the potential to serve as drug leads for synthetic derivatives, structural motifs, which are indispensable for both mode of action and biological activities, are discussed. Evaluating the natural products emphasizes the strong diversity of their points of attack. Especially the fact that selected binding partners can be set in direct relation to different diseases emphasizes the indispensability of natural products in the field of drug development. Discovery of new, unique and unusual interacting partners again renders them promising tools for future research in the field of eukaryotic mRNA translation.
Each cell type in an organism comes to acquire a unique pattern of gene expression through differentiation during development. It is obvious that this pattern of gene expression must be maintained for the normal functioning of cells and for the survival of the organism. Since aging is considered to be a result of failure of maintenance at all levels, attempts have been made to look for an age-related drifting-away of cells in terms of changes in the pattern of gene expression during aging. In most cases, mRNA levels of different genes have been estimated by RNA-DNA hybridization, using cDNA or genomic probes for specific genes. More recently, the availability of gene array technology has made it possible to compare the expression of thousands of genes. The results obtained show that during aging the expression of some genes increases, of some it decreases and of others it remains constant [1–7]. In all such studies on measuring the levels of mRNA in young and old cells and tissues, it is assumed that this estimate is a direct measure of gene activity. This is a simplistic notion, because it is well known that post-transcriptional changes, such as the processing, transport and turnover of RNA, change significantly the levels of mRNA.
Two cytosolic proteins of murine epidermis or porcine spleen with molecular masses of 37 kDa (p37) and 50 kDa (p50) are differentially phosphorylated in vitro by the purified protein kinase C (PKC) isoenzymes alpha, beta, gamma (cPKC) and PKC delta. p37, identified as annexin I, is preferentially phosphorylated by cPKC, whereas p50, identified as elongation factor eEF-1 alpha, is phosphorylated with much greater efficacy by PKC delta than by cPKC. Using the recombinant PKC isoenzymes alpha, beta, gamma, delta, epsilon, eta, and zeta, we could show that purified eEF-1 alpha is indeed a specific substrate of PKC delta. It is not significantly phosphorylated by PKC epsilon, -eta and -zeta and only slightly by PKC alpha, -beta, and -gamma. PKC delta phosphorylates eEF-1 alpha at Thr-431 (based on the murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a sequence corresponding to that of 422-443 from murine eEF-1 alpha and containing Thr-431 is an absolutely specific substrate for the delta-type of PKC. The single basic amino acid close to Thr-431 (Arg-429) is essential for recognition of the peptide as a substrate by PKC delta and for the selectivity of this recognition. Substitution of Arg-429 by alanine abolishes the ability of PKC delta to phosphorylate the peptide, and insertion of additional basic amino acids in the vicinity of Thr-431 causes a complete loss of selectivity.
Elongation factor 1 (EF-1) regulates the specific interaction of aminoacyl-tRNA with the ribosome during the elongation phase of protein biosynthesis. Although individual functions of its separate chains have been well defined, to date there is hardly information about the structure and function of the whole complex. We describe here the complete subunit structure of elongation factor 1, and discuss its change during development of Artemia. Elongation factor 1 consists of a pentameric complex, composed of four different subunits alpha, beta, gamma, and delta in a molar ratio of 2:1:1:1, Although one molecule of EF-1 alpha dissociates easily from the complex EF-1 alpha,beta gamma delta under the influence of aminoacyl-tRNA and GTP, the second molecule of EF-1 alpha was found to remain firmly attached. Thus, in eukaryotic protein synthesis, movement of transfer RNAs to the ribosome seems under the influence of two distinct molecules of EF-1 alpha, a result possibly related to the presumed consumption of two molecules of GTP by EF-Tu during the elongation step of prokaryotic protein synthesis.
Data about possible participation of the eukaryotic elongation factors in the organization and regulation of microtubular and microfilament networks of cellular cytoskeleton are reviewed. Potential role of the factors to coordinate protein syntesis and polymerization/depolymerization of cytoskeleton under different cellular conditions is suggested.
Senescence of cells in culture is characterized by a loss in proliferative potential and a wide variety of structural and metabolic changes (see Table 1, p.368). Not all of these changes need have causal implications in senescence, and the challenge is to sort out causal from correlative phenomena associated with aging. The latter may serve as useful biomarkers of aging but may not reveal much about the underlying causative mechanisms leading eventually to cell and organismic failure and death. The articles in this issue describe many age-related changes in DNA replication and gene expression, and we hope some of these will, in fact, lead to insights into the critical mechanisms of senescence.
Ageing is a highly complex process of genetic and epigenetic interactions at all levels of organization. There is no unified theory which could make universal statements about the principles, mechanisms and evolution of ageing. In spite of various claims made for the ‘anti-ageing’ products, none has been shown to affect the ageing process per se. Recent developments in the understanding of molecular mechanisms of repair, maintenance and fidelity of information transfer will open up new approaches for modulating the ageing process with the help of various tools of biotechnology already in use.
Eukaryotic translation elongation factor 1A (eEF1A) is one of the main components of the translation apparatus.The protein also fulfills a number of non-canonical functions not related directly to protein synthesis. In this work the homogenous elongation factor 1A was obtained from hen liver by several chromatographic procedures with different carriers. The purified protein was used to develop monoclonal antibodies. The anti-eEF1A antibodies obtained are species-unspecific, they recognize both eEF1A and eEF1A2 isoforms in ELJSA and immunoblotting. The antibodies have been successfully used for immunohistological studies on human tissues.
Elongation factor 1α (EF-1α) is an abundant cellular protein and its amino-acid sequence has been inferred from numerous organisms, including bacteria, archaebacteria, plants and animals. In large measure, it would appear that the overall structure has probably been maintained given the 33% identity and 56% similarity of Escherichia coli EF-Tu with human EF-1α. Chemical sequencing of EF-Tu and EF-1α has revealed that these proteins are post-translationally modified. In order to assess the possible function of these modifications, we have chemically sequenced the EF-1α from the lower eukaryote Saccharomyces cerevisiae (yeast). To our surprise, the methylation pattern of yeast EF-1α was quite different from either rabbit or brine shrimp EF-1α with only the trimethyllysine at position 79 conserved although the yeast protein is 81% identical to rabbit EF-1α. A dimethyllysine was observed at position 316 which corresponds to a trimethyllysine in brine shrimp and rabbit EF-1α. The other positions in yeast EF-1α which were methylated were unrelated to the other six possible positions for modification observed in brine shrimp or rabbit EF-1α. In addition, the unique glycerylphosphorylethanolamine observed in mammalian EF-1α and suspected in brine shrimp EF-1α was not found in yeast EF-1α.
ELONGATION factor-lalpha (EF-lalpha), an essential component of the eukaryotic translational apparatus, is a GTP-binding protein that catalyses the binding of aminoacyl-transfer RNAs to the ribosome1-3. Expression of the EF-lalpha gene decreases towards the end of the lifespans of mouse and human fibroblasts4,5, but forced expression of EF-lalpha prolongs the lifespan of Drosophila melanogaster6. Eukaryotic initiation factor-4E, another component of the translational machinery, is mitogenic or oncogenic when constitutively expressed in some mammalian cells7-9. Thus, components of the protein synthesis apparatus seem to be involved in the control of cell proliferation. Using expression cloning, we have isolated a complementary DNA clone from a BALB/c 3T3 mouse fibroblast variant, A31-I-13 (ref. 10), which specifies a factor determining the susceptibility of BALB/c 3T3 to chemically and physically induced transformation. Here we report that the factor is EF-lalpha and that its constitutive expression causes BALB/c 3T3 A31-I-1 (ref. 10), C3H10T1/2 (ref. 11) and Syrian hamster SHOK12 fibroblasts to become highly susceptible to transforma-tion induced by 3-methylcholanthrene and ultraviolet light. EF-lalpha messenger RNA is also constitutively expressed in a quiescent culture of the highly susceptible variant A31-1-13. We conclude that the removal of regulation of the expression of these com-ponents of the translational machinery may predispose cells to become more susceptible to malignant transformation.
Two cytosolic proteins of murine epidermis or porcine spleen with molecular masses of 37 kDa (p37) and 50 kDa (p50) are differentially
phosphorylated in vitro by the purified protein kinase C (PKC) isoenzymes α, β, (cPKC) and PKC. p37, identified as annexin I, is preferentially phosphorylated by cPKC, whereas p50, identified as elongation factor eEF-1α,
is phosphorylated with much greater efficacy by PKC than by cPKC. Using the recombinant PKC isoenzymes α, β, , , , , and , we could show that purified eEF-1α is indeed a specific substrate of PKC. It is not significantly phosphorylated by PKC, -, and - and only slightly by PKCα, -β, and -. PKC phosphorylates eEF-1α at Thr-431 (based on the murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a sequence
corresponding to that of 422-443 from murine eEF-1α and containing Thr-431 is an absolutely specific substrate for the -type of PKC. The single basic amino acid close to Thr-431 (Arg-429) is essential for recognition of the peptide as a substrate
by PKC and for the selectivity of this recognition. Substitution of Arg-429 by alanine abolishes the ability of PKC to phosphorylate the peptide, and insertion of additional basic amino acids in the vicinity of Thr-431 causes a complete
loss of selectivity.
Translation elongation factor 1A (eEF1A) is a factor critically involved in the process of protein synthesis. The activity of eEF1A has been shown by several studies to be regulated by post-translational modifications such as phosphorylation and dephosphorylation. However, until now less research has focused on other post-translational modifications of eEF1A, especially acetylation. In this report, we provide new evidence for the existence of eEF1A acetylation in PLC5 cells by immunoprecipitation and Western blotting. Using the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), we found that the deacetylation of eEF1A is mainly attributable to classes I and II HDAC rather than class III HDAC, and, furthermore, that the antitumour agent etoposide phosphate (VP 16) enhances the acetylation of eEF1A in a synergistic way with TSA. Our data suggest the possibility that the increased acetylation of eEF1A could be a new mechanism for the antitumour effect of etoposide.
A human pancreatic adenocarcinoma λgt11 expression library was differentially screened with mRNA derived from normal and cancerous pancreatic tissues. Five clones preferentially hybridized with pancreatic adenocarcinoma mRNA. cDNA inserts from 4 of these clones were amplified by PCR, labelled with 32P and used in Northern blot analysis against mRNA prepared from a variety of tumour and normal tissues. λGER-4 identified a pancreas-associated mRNA (> 10 kb) with no homology with known sequences at either the nucleic or amino-acid level. λGER-2 identified a 1.7-kb mRNA transcript that was over-expressed in mRNA prepared from pancreas, colon, breast, lung and gastric tumours relative to normal tissues. Sequence analysis and restriction-enzyme mapping showed that this clone was completely homologous with the active form of human elongation factor EF-1. This high level of EF-1-mRNA expression in tumour tissues lends support to the increasing evidence that EF-1 is an important regulator of the cell cycle.
The sections in this article are:
Transcription
RNA Synthesis
m RNA Levels
Transcriptional and Posttranscriptional Processing of m RNA
Factors That Regulate Transcription
Effect of Dietary Restriction on Transcription
Translation
Protein Synthesis
Effect of Dietary Restriction on Protein Synthesis
Fidelity of Protein Synthesis
Various Steps of Protein Synthesis
Protein Degradation
Degradation of Mixed Protein Populations
Degradation of Individual Proteins
Degradation of Abnormal Proteins
Effect of Dietary Restriction on Protein Degradation
Summary and Conclusions
Background:
Elongation factor-1 (EF-1) is a cellular protein that plays a role in protein synthesis by mediating the transfer of aminoacyl-tRNA to 80S ribosomes. It is comprised of four subunits: alpha, beta, gamma, and delta. EF-1gamma is a substrate for the maturation-promoting factor, which determines entry into the M-phase of the cell cycle in all eukaryotic cells. Previously, the authors showed that EF-1gamma RNA is overexpressed in a high proportion of colorectal carcinomas. At that time, there were no antibodies to EF-1gamma, so the EF-1gamma protein could not be examined. Because levels of RNA do not always parallel the levels of the protein it encodes, it was important to develop antibodies to EF-1gamma to examine its expression at the protein level in colorectal carcinoma.
Methods:
Twenty-nine patients undergoing surgical resection for colorectal adenocarcinoma were studied. A polyclonal antibody to EF- 1gamma in rabbit was prepared. Tumors and normal-appearing mucosa distant from the tumor (> or = 10 cm) were obtained from each patient. Cytosolic proteins were extracted from the tissues and examined by Western blot analysis with the EF-1gamma antibody. Colonic tumors also were studied by immunohistochemical analysis with another EF-1gamma polyclonal antibody.
Results:
Using Western blot analysis, the authors observed greater expression of EF-1gamma in the tumors than in the more distal normal-appearing mucosa. Overexpression was not observed in the patients with the two Dukes Stage A tumors, but was observed in four of ten patients with Dukes Stage B tumors, seven of eight patients with Dukes Stage C tumors, and six of nine patients with Dukes Stage D tumors. Overall, 17 of 29 patients (59%) were found to have overexpression of EF-1gamma. Using immunohistochemical analysis, EF-1gamma protein was shown to be located predominantly in tumor epithelium rather than the stroma or infiltrating mononuclear cells.
Conclusions:
Previous studies showed that EF-1gamma mRNA frequently is overexpressed in colorectal adenocarcinoma. This study showed that EF-1gamma also was overexpressed at the protein level in colorectal adenocarcinoma relative to more distal normal-appearing mucosa from the same patient. Immunohistochemical analysis demonstrated that this protein was expressed predominantly in the tumor epithelial cells and therefore was not derived from cells involved in the desmoplastic response.
This article describes some functional properties of eukaryotic EF-1A, a core component of the protein synthesis machinery, at the onset of cell transformation. EF-1A is shown to be upregulated in cell death moreover, it seems to be involved in the regulation of ubiquitin-mediated protein degradation. In addition, EF-1A undergoes several post-translational modifications such as phospho-rylation and methylation that generally influence its activity. Here, the behavior of EF-1A in a hepidermoid cancer cell line (H1355) is reported. In these cells, interferon α (IFNα) induces both apoptosis and a counteracting EGF-->Erk-dependent survival response. Treatment of H1355 cells with IFNα increased EF-1A protein expression by inhibiting its degradation via proteasome-dependent pathway. In particular, the increment of EF-1A was mainly due to the EF-1A2 isoform rather than the EF-1A1 one. If the expression level of EF-1A was decreased by RNA interference, an enhanced apoptosis induced by IFNα was instead observed. In addition, treatment of H1355 cells with IFNα induced also both serine and threonine C-Raf-mediated phosphorylation of EF-1A. The suppression of C-Raf activity with BAY 43-9006 C-Raf inhibitor completely antagonized the increase of EF-1A expression and enhanced apoptosis induced by IFNα. In conclusion, the interaction between EF-1A and C-Raf increases EF-1A stability and induces a survival activity. These results suggest that the extra-translational roles of EF-1A could offer tools for cancer intervention.
Several components of the eukaryotic protein synthesis apparatus have been associated with oncogenic transformation of cells. Altered expression of translation elongation factor 1α (EF-1α), a core component of protein synthesis and closely related sequences have been linked with transformed phenotypes by several independent studies, in diverse systems. A dominant acting oncogene, prostate tumor inducing gene-1 (PTI-1) has provided further evidence for this link. PTI-1 appears to be a hybrid molecule with components derived from both prokaryotic and eukaryotic origins. The predicted protein coding moiety represents an EF-1α molecule, truncated N-terminal to amino acid residue 68 and having six additional point mutations. This coding sequence is fused to a 5′ untranslated region (UTR) showing strongest homology to ribosomal RNA derived from Mycoplasma hyopneumoniae. Expression studies using the cloned cDNA in nude mouse tumor formation assays have confirmed the oncogenic nature of the molecule. A broad spectrum of tumor derived cell lines, from varied tissue sources and blood samples from patients having confirmed prostate carcinoma, all scored positive for expression of PTI-1, while corresponding normal tissues or blood samples were negative. Based on its near identity to EF-1α, it is proposed that PTI-1 represents a new class of oncogene whose transforming capacity probably arises through mechanisms including: (i) protein translational infidelity, resulting in the synthesis of mutant polypeptides due to loss of proofreading function during peptide chain elongation, (ii) by its association with and alteration of the cytoskeleton, (iii) by impinging on one particular or several different signal transduction pathways through its properties as a G-protein.
The eukaryotic translation elongation factor 1A (eEF1A), besides to its canonical role in protein synthesis, is also involved in several other cellular processes, depending on changes in cellular location, cell type, concentration of ligands, substrates or cofactors. Therefore eEF1A is a moonlighting protein that participates to a network of molecular interactions involving its structural domains. Since the identification of novel protein-protein interactions represents important tasks in post-genomic era, the interactome of eEF1A1 M-domain was investigated by using a proteomic approach. To this purpose, the eEF1A1 M-domain was fused with glutathione-S-transferase (GST) and Strep-tag (ST) at it's N- and C-terminal, respectively. The recombinant protein (GST-M-ST) was purified and incubated with a mouse embryo lysate by applying an affinity chromatography strategy. The interacting proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Besides the known partners, the pool of interacting proteins contained sorbin, a polypeptide of 153 amino acids present in SH3 domain-containing adaptor proteins, such as SORBS2. This interaction was also assessed by Western blot on immunoprecipitate from mouse embryo or H1355 cell lysates with anti-eEF1A or anti-SORBS2 antibodies and on eEF1A1-His pull-down from H1355 cell lysate with antibody anti-SORBS2. Furthermore, the interaction between eEF1A and SORBS2 was also confirmed by confocal microscopy and FRET analysis. Interestingly, a co-localization of SORBS2 and eEF1A was evidenced at level of plasma membrane, thus suggesting the involvement of eEF1A1 in novel key signal transduction complexes.
The immunosuppressant cyclosporin A (CsA) is reported to have a strong anti-ischemic effect. Although this neuroprotective effect is speculated to be related to the blockade of a mitochondrial permeability transition pore (mPTP), the underlying molecular mechanism remains to be elucidated. This study focused on the effect of CsA on transcriptional regulation in brain cells.
CsA and a control substance were injected into rat brains and purified extracted mRNA. Both mRNAs were compared using a cDNA subtraction technique.
Nine significantly up-regulated genes and seven significantly down-regulated genes were detected following CsA administration. All of the up-regulated genes are neurotrophic or reported to have roles in regeneration of brain tissue. Among the down-regulated genes, three are known to be detrimental to neuronal cells and are also reported to facilitate the pathology of Alzheimer's disease (AD) and four genes are related to oxidative metabolism.
Strong immunosuppression would present as a side-effect during CsA use as a neuroprotectant. The results of this study will help to discriminate between the CsA immunosuppressive effect and the neuroprotective effect at the molecular level and may lead to the development of new conceptual and pharmacological tools.
Aging is a complex process that has been shown to be linked to accumulation of DNA damage. Telomere shortening represents a cell-intrinsic mechanism leading to DNA damage accumulation and activation of DNA damage checkpoints in aging cells. Activation of DNA damage checkpoints in response to telomere dysfunction results in induction of cellular senescence-a permanent cell cycle arrest. Senescence represents a tumor suppressor mechanism protecting cells from evolution of genomic instability and transformation. As a drawback, telomere shortening may also limit tissue renewal and regenerative capacity of tissues in response to aging and chronic disease. In aged organs, telomere shortening may also increase the cancer risk by initiation of chromosomal instability, loss of proliferative competition of aging stem cells, and selection of aberrant growing clones. Consequently, aged individuals are more susceptible and vulnerable to various diseases and show an increased cancer risk. Recently, proteins were discovered, which are induced by telomere dysfunction and DNA damage. It was shown that these proteins represent new biomarkers of human aging and disease. Here, we review the scientific background and experimental data on these newly discovered biomarkers.
Despite the broad anti-tumour potential of the proteasome inhibitor bortezomib, partial information is available with regard to its effects on hepatocellular carcinoma (HCC) cells. Here we studied the effects of bortezomib on two human HCC cell lines displaying a different phenotype, hepatocyte-like for HepG2 and undifferentiated for JHH6. Bortezomib induced a dose- and time-dependent increase in cell toxicity and decrease of cell viability, with JHH6 being less sensitive than HepG2. Moreover, a differential influence on major cell cycle regulatory genes was responsible for the observed decrease of S and increase of G(2)-M phase cells. In HepG2, bortezomib induced a post-transcriptional increase of cyclin E1 together with a transcriptional-mediated decrease of the transcription factor E2F1. This in turn resulted in the reduction of the hyper-phosphorylated form of pRB and in the transcriptional down-regulation of the E2F1 targets cyclin D1, cyclin A2 and CdK2 but not cyclin E1. Up-regulation of LRH1, a liver specific cyclin E1 transcription factor, accounted for the unvaried cyclin E1 mRNA levels. Additionally, bortezomib induced both transcriptional and post-translational increase of p21(waf1/cip1) and p27(kip1). In JHH6, an overall more contained variation in cell cycle mediators was observed with the reduction of E2F1, cyclin A2, LRH1 and the increase of p21(waf1/cip1) being the most evident. In conclusion, the presented data show the mechanisms regulating cell proliferation inhibition by bortezomib in two different HCC cell lines. Despite a certain phenotype-dependent effect, the potent action exerted by bortezomib makes this drug attractive for future experimentation in animal models, possibly leading to novel treatments for HCC.
To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochondria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.
The human gene sequences encoding the translation-associated functions of alpha-subunit of elongation factor 1 (EF-1 alpha) and the ubiquitin carboxyl extension protein (HUBCEP80) have been isolated by differential cDNA screening, and found to have significantly higher levels of expression in fibroadenomas (benign) compared with carcinomas (malignant) of the breast. These data parallel our previous findings that the acidic ribosomal phosphoprotein P2 also has higher expression levels in the benign breast tumours (Sharp et al., 1990). In situ hybridisation has shown these genes to be expressed predominantly in the epithelium of breast tumours.
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Elongation factor-1 alpha (EF-1 alpha) is an ubiquitous protein that functions in peptide elongation during mRNA translation. We previously reported the isolation of a rat S1 protein that is antigenically related to statin, a nonproliferation-specific protein; this S1 gene shares a high degree of homology to EF-1 alpha. We constructed specific riboprobes to the two genes, based on the difference in the 3' noncoding regions of both S1 and EF-1 alpha mRNAs. Northern analysis and RNase protection assays have revealed that S1 mRNA is present only in brain, heart, and muscle, while EF-1 alpha mRNA has been detected in all tissues surveyed so far. The same tissue specificity has been observed in mouse, suggesting that S1 expression is conserved between these two mammalian species. S1 transcript was detected in late brain embryogenesis (day 20), but in lower amounts than in 3-month-old adult brain. We show that the relative levels of both S1 and EF-1 alpha transcripts and their respective tissue abundances remain unchanged during the aging process. The function of S1 is not yet known; but these results suggest that it may be involved in specific control mechanisms for protein synthesis in tissues where cells (i.e. neurons and myocytes) are permanently locked in a state of nonproliferation.
Posttranslational modifications of proteins are involved in determining their activities, stability, and specificity of interaction. More than 140 major and minor modifications of proteins have been reported. Of these, only a few have been studied in relation to the aging of cells, tissues, and organisms. These include phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and deamidation. Several of these modifications occur on proteins involved in crucial cellular processes, such as DNA synthesis, protein synthesis, protein degradation, signal transduction, cytoskeletal organization, and the components of extracellular matrix. Some of the modifications are the markers of abnormal and altered proteins for rapid degradation. Others make them less susceptible to degradation by normal proteolytic enzymes, and hence these accumulate during aging.
Phorbol esters induce drastic morphological alterations in cells of different origin by altering the conformation and the interrelationship of the elements of the cytoskeletal system. Treatment of early passage (young) and late passage (senescent) human fibroblasts MRC-5 with phorbol-12-myristate-13-acetate (PMA) results in the rearrangement of actin and tubulin filaments. PMA brings about the disorientation and diffusion of the heavily criss-crossed network of actin and microtubulin fibres characteristic of senescent cells suggesting thereby an increased sensitivity of senescent cells to phorbol esters. Since phorbol esters are known to be specific activators of protein kinase C (PKC), the PMA-induced modulation of the cytoskeleton patterns in ageing fibroblasts provides further support for the view that the effectiveness of the signalling mechanisms is retained during cellular ageing.
A human pancreatic adenocarcinoma lambda gt11 expression library was differentially screened with mRNA derived from normal and cancerous pancreatic tissues. Five clones preferentially hybridized with pancreatic adenocarcinoma mRNA. cDNA inserts from 4 of these clones were amplified by PCR, labelled with alpha 32P and used in Northern blot analysis against mRNA prepared from a variety of tumour and normal tissues. lambda GER-4 identified a pancreas-associated mRNA (greater than 10 kb) with no homology with known sequences at either the nucleic or amino-acid level. lambda GER-2 identified a 1.7-kb mRNA transcript that was over-expressed in mRNA prepared from pancreas, colon, breast, lung and gastric tumours relative to normal tissues. Sequence analysis and restriction-enzyme mapping showed that this clone was completely homologous with the active form of human elongation factor EF-1 alpha. This high level of EF-1 alpha-mRNA expression in tumour tissues lends support to the increasing evidence that EF-1 alpha is an important regulator of the cell cycle.
Polypeptide assembly rates during in vivo hepatic protein synthesis were studied as a function of age and restricted feeding in male rats. With ageing the time to assemble the average peptide in the liver of fully-fed rats significantly increased. In young rats maintained on a restricted feeding regime known to retard ageing, the time to assemble the average polypeptide was increased 2.5 times. With ageing the rate of peptide elongation increased so that at 2 years of age the underfed animals assembled peptides at a significantly faster rate than their age-matched controls. The rate of elongation of peptides during hepatic protein synthesis was shown to be directly dependent upon circulating T3 levels rather than the dietary status of the animal. On refeeding young diet restricted rats, polypeptide assembly kinetics did not immediately return to control values although the rate of protein synthesis was significantly increased. Total liver RNA content increased significantly in refed animals allowing for a greater rate of chain initiation to offset the slow rate of chain elongation. A period of 28 days of ad libitum feeding was required before assembly kinetics returned to control values and is probably indicative of a persistent impaired monodeiodination of T4 to T3.
Fixed mortality of normal somatic cells is a well-established fact though the mechanism underlying this universal phenomenon remains unknown. Use of immortal cells in conjunction with their normal mortal counterparts has delineated the dominant genetic nature of the senescent phenotype over immortalization. Although the involvement of proteins in determining the entry/exit/arrest of cells in the cell cycle is evident from the literature, none of them has been confirmed for its role in senescence-associated irreversible cell cycle exit/arrest. The identification of true mortality markers might be possible by selecting a system of natural and conditional aging achieved by the fusion of mortal and spontaneously immortalized cells of the same origin. We report here a few such protein markers which might serve as useful handles to tease out the molecular events determining mortality/immortality of cultured cells.
The slowing down of protein synthesis is a change widely observed during the aging of organisms. It has also been claimed that a decline in the rate of protein synthesis occurs during cellular aging. However, the evidence in favour of this view is not clear-cut, and reliable estimates of rates of protein synthesis during cellular aging have yet to be made. Studies on various components of the protein synthetic machinery during cellular aging have revealed a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors. Detailed studies on the structure and function of ribosomes, tRNA isoacceptor profiles, activities of aminoacyl-tRNA synthetases, levels and activities of initiation factors, rates of protein elongation, and the accuracy of protein synthesis will be needed before the molecular mechanisms of the regulation of protein synthesis during cellular aging can be understood.
The developmental regulation of the translational elongation factor EF-1 alpha has been analyzed in tobacco. A gene fusion was constructed consisting of the 5' and 3' regions of the tomato genomic clone LeEF-A from the EF-1 alpha gene family and the beta-glucuronidase coding region. Analysis of the transgenic plants containing this chimeric gene demonstrated that the tomato LeEF-A flanking sequences were sufficient to confer expression patterns similar to those of the endogenous tobacco EF-1 alpha gene. The patterns of beta-glucuronidase activity in this system indicated that during plant growth and development EF-1 alpha is regulated with increased expression corresponding to regions of high protein synthesis, including meristems, rapidly growing tissues, and developing gametophytes. In addition, EF-1 alpha expression responds rapidly to changes in growth patterns induced by hormone treatment. Our results are in agreement with studies in animals indicating that EF-1 alpha expression may be rate limiting for protein synthesis and demonstrate that the analysis of EF-1 alpha is of value for studying interrelationships between protein synthesis and developmental control.
Dietary calorie restriction of rats has been previously shown to increase protein synthetic rates in liver and kidney cells during ageing. Here we have compared the activity and amounts of active elongation factors EF-1 alpha and EF-2 in cell-free extracts prepared from livers isolated from male Fischer 344 rats of different ages. Although there is some age-related decline in the catalytic activity and amounts of active EF-1 alpha during ageing, no differences between freely-fed and calorie-restricted animals were observed. In the case of EF-2, the amounts of ADP-ribosylatable EF-2 neither declined during ageing nor differed between freely-fed and calorie-restricted animals. Thus differences in the protein synthetic rates in calorie-restricted and freely-fed rats are not reflected at the level of protein elongation factors, and may involve some other mechanisms of regulation.
The elongation step is involved in the regulation of protein synthesis during the cell cycle, environmental stress, ageing and transformation. Using a diphtheria toxin-mediated assay for measuring the levels of ADP-ribosylatable elongation factor EF-2, we have observed an irreversible decrease of up to 64% in the amount of ADP-ribosylatable EF-2 in normal diploid human fibroblasts MRC-5 undergoing ageing in vitro. However, a similar decrease in low serum-associated G0/G1-arrested cells is reversible both in MRC-5 cells and in their SV40-transformed counterparts. Reduced levels of ADP-ribosylatable EF-2 could account for the slowing-down of protein synthesis during cell cycle arrest and during cellular ageing in culture.
In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2. EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome. Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation. Yeast contains a third factor, EF-3, whose structure and function is not yet well understood.
We have undertaken an immunological and biochemical analysis of the most abundant soluble protein of previtellogenic Xenopus oocytes, 42S p48. We show that this protein shares immunological cross-reactivity with elongation factor 1 alpha (EF-1 alpha). Direct assays of both 42S fractions and purified 42S p48 show that this cross-reactivity is of functional significance since 42S p48, like EF-1 alpha, can transfer charged amino acids to ribosomes. We further demonstrate that 42S p48 is degraded soon after the onset of vitellogenesis, while the EF-1 alpha concentration remains essentially unchanged during this transition. These properties of 42S p48 are discussed with regard to its role in oogenesis.
A cDNA library was constructed from poly(A)+ RNA derived from late passage WI-38 cells and differentially screened with cDNA probes from early and late passage cells. From a number of clones which exhibited differences in intensity of hybridization to the early or late passage probes, one was chosen for further analysis because of its large increase in hybridization to the late passage probe. This clone accounted for approximately 1% of the recombinants in the library. The partial cDNA clone shows complete sequence homology to elongation factor I alpha (EF-I alpha). Northern analysis of poly(A)+ RNA from cells at various population doublings suggested that a 2.2-kb transcript, homologous to EF-I alpha accumulates as cells near the end of their replicative life span (phase III). When early and late passage cells were reexposed to serum after serum starvation, this transcript decreased in abundance. Additionally, a lower molecular weight transcript (1.6 kb) was detected 18 h following serum stimulation in early passage cells and 9 h after stimulation in late passage cells.
We have isolated a senescence-specific clone (pSEN) from a cDNA library constructed from late passage WI-38 human diploid fibroblast that accounts for approximately 1% of the recombinants. Nucleotide sequence analysis of the partial cDNA clone has led to the identification of pSEN as elongation factor I alpha. Northern analysis of poly(A)+ RNA from various intermediate population doubling levels shows that a 2.2 kb transcript hybridizes to pSEN but is expressed prior to PDL-40 at very low levels. This transcript begins to accumulate at PDL-40 and is induced approximately 50-fold just prior to senescence. Furthermore, this transcript was shown to be specific to Go of the cell cycle whereas a second, lower molecular weight transcript (1.6 kb) was observed during S phase (Giordano and Foster, unpublished data). The 2.2 kb transcript is also detected in neonatal foreskin cells but very little increase in abundance is observed between early and late passage cells. Sucrose gradient fractionation of RNA from late passage WI-38 cells suggests that the lower molecular weight transcript is associated with the polysome fraction while the 2.2 kb transcript sediments with the nonpolysomal fraction. Thus, the possibility exists that the 1.6 kb transcript is derived from the 2.2 kb transcript.
In Drosophila melanogaster, the decrease in protein synthesis that accompanies aging is preceded by a decrease in elongation factor EF-1 alpha protein and mRNA. Here we show that Drosophila transformed with a P-element vector containing an EF-1 alpha gene under control of hsp70 regulatory sequences have a longer life-span than control flies.
The time at which human diploid cell strains can be expected to cease dividing in vitro (Phase III) is not a function of the number of subcultivations but rather of the number of potential cell doublings. Each clonable cell within the population is endowed with the same doubling potential (50 ±10). Cells of the same strain, but with different “doubling potentials”, were mixed. Phase III in such mixed populations occurs at that time when the “youngest” cell component is expected to reach Phase III. The “older” component has no effect on the time at which Phase III is expected to take place in the “younger” component. An ancillary conclusion that Phase III cannot be explained by the presence of a latent virus, mycoplasma or media composition is confirmed. Human diploid cell strains derived from adult lung have a significantly lower doubling potential in vitro than do fetal strains. The Phase III phenomenon may be related to senescence in vivo. The cellular theory of aging must be related to normal cells in vitro and not to heteroploid cell lines. The former have a finite period of multiplication; the latter are indefinitely cultivable. In vivo experiments also indicate that transplanted normal tissue has a finite lifetime. Chromosome anomalies occurring in Phase III may be related to such anomalies occurring in the cells of older animals, including man. The survival curves obtained with human diploid cell strains are comparable to “multiple-hit” or “multiple-target” curves obtained with other biological systems where an initial threshold dose is required before an exponential form of the curve is established. Whatever cell component(s) may be involved in the finite lifetime of human diploid cell strains, the ultimate accumulation of nondividing cells could be the result of accumulated damage to a single cellular target or to inactivation of many targets.
Malignant transformation of cultured cells is often characterized by changes in growth properties as well as of cell morphology (1–3). These alterations reflect changes in gene expression that develop through a series of progressive events (4, 5; see also article by L.M. Franks in this volume). Working with the hypothesis that malignant transformation may be due to the abnormal expression of normal genes (6–10) we have carried out a detailed and systematic study of the polypeptides synthesized by normal and transformed cells under a variety of physiological conditions in an effort to reveal cellular proteins that are involved in regulating cell proliferation and that could be used as general markers for malignant transformation (6–11). In this article we will review our studies of normal and transformed human cultured cells. These studies have so far revealed 58 transformation sensitive polypeptides that are present both in normal and transformed cells and that are common to many cell types analyzed (9). Some of these polypeptides may correspond to “oncogenes” (12–14; see also other articles in this volume), and the elucidation of their function may lead a better understanding of the processes that control cell proliferation.
Polypeptide chain elongation is conveniently described in three separate steps. Much is now known about the functioning and structures of molecular components involved in each step but little is known about the kinetics of the process and its motive force. This is the second article in our three-part series on protein synthesis.
Two-dimensional gel electrophoretic (NEPHGE) analysis of proteins from mouse 3T3B and 3T3B/SV40 cells labelled with [methyl-3H]methionine in the presence of cycloheximide have revealed that the elongation factor 1α (EF-1α) in these cells is methylated and that the extent of methylation is higher in the SV40 transformed cell type. It is suggested that methylation may account for differences in growth properties for the different cell types.
The translational control of protein synthesis during early postnatal neural development and aging was examined in the mouse and the rat. The activity of brain elongation factor 1 (bEF-1) was found to decrease exponentially with age and to decline parallel to the age-dependent decrease in total protein synthesis in both rodents. This decrement in bEF-1 activity fell within the range of reported age-related decreases in protein synthesis in in vitro systems. The factor was present in multiple forms; the lighter species predominated in older animals, whereas the young light form apparently disappeared with increasing age, and was replaced by others arising from the heavy form. Elongation factor 1 derived from young brains functioned as a rate-limiting component in polypeptide synthesis in previously saturated adult systems. The data suggest that bEF-1 has an important modulatory effect on total brain protein synthesis.
1. The published results on protein synthesis during aging are contradictory. Possible sources of error and variability include: an insufficient number of different animal ages used; use of whole organs that are cytologically highly heterogeneous; different animal strains; neglecting to measure the specific activity of the precursor pool for protein synthesis; and inadequate methodology for measurement of in vivo rates of protein synthesis.
2. In general, protein synthesis rates in mammals have been reported to decline 4–70% with age. In insects and other organisms, greater losses (60–90%) have been observed.
3. Limited evidence indicates that in some systems a decline in the rate of protein synthesis may be due to alterations (as yet of unknown nature) in the initiation components of the protein synthetic apparatus. Futhermore, some studies suggest that in some organisms aging affects the expression of specific parts of the genome.
4. The significance of results on protein metabolism obtained from some studies with nematodes is at present unknown, owing to problems associated with age-synchronization methods. Also, the in vitro fibroblast system for the study of human cellular aging has not been met with universal acceptance; it is generally believed that this system has not yet been established as a valid analogy to mammalian aging in vivo.
5. Failure to detect defective enzymes in many old organisms indicates at least that not all proteins are altered during aging. The complete thermal stability of purified enzymes from old organisms suggests that the observed thermolability of the same enzymes in crude cell extracts is not an intrinsic property of those enzymes. Post-translational modifications (partial denaturation) may constitute the primary mechanism for the production of altered cell polypeptides during aging.
6. The available evidence does not support the concept of an age-dependent decline in translational accuracy. The future purification to absolute homogeneity of an altered enzyme and its ‘young’ (unaltered) counterpart, and their sequencing, should resolve the question of translational errors.
7. Some degree of age-related ribosome loss appears to occur in fixed postmitotic cells. In general, the published polyribosomal profiles may represent artefacts due to insufficiently suppressed ribonuclease activity during extraction.
8. The published studies on protein degradation during aging are also contradictory. Some investigators have neglected the possibility of reutilization of labelled amino acid. It is possible that some of the observed age-related alterations in protein degradation rates are due to altered endocrine status of the animals used, rather than to defects in the protein degradative pathways. The studies utilizing cell culture systems are also contradictory, probably due to different experimental designs.
9. Limited evidence suggests that protein degradation may slow down with age in mammals and nematodes. An inefficient protein degradation system in old organisms could provide an explanation for the accumulation of altered macromolecules in some organisms. Virtually nothing is known about regulatory mechanisms of protein degradation during senescence.
10. There is a need to examine which proteins are synthesized and degraded at selectively different rates as a function of age and what their physiological role is. This approach would be more informative than the study of total protein turnover with age.
11. The physiological significance, and the causes of the observed declines in protein synthesis and degradation rates during aging and senescence, remain to be established.
Previous studies have indicated that the high-molecular-weight form of elongation factor 1 (EF-1H) contained four subunits (α, β, γ, and δ). Using the conventional methods of gel-filtration and ion-exchange chromatography, various forms of elongation factor 1 (EF-1α, EF-βδ, EF-1βγδ) have been purified from rabbit reticulocyte lysate. The procedure described allows one to purify these factors from a single batch of lysate in sufficient amounts for physical and biochemical studies. EF-1α is a single polypeptide of Mr 52,000, and has an isoelectric point of 9.1. EF-1βδ and EF-1βγδ are composed of two and three nonidentical polypeptides, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both proteins can form stable aggregates in native conditions that can reach more than 2,000,000 Da. The isoelectric point for each polypeptide was determined; 5.8 for EF-1β, 5.5 for EF-1γ, and 4.8 for EF-1δ. The activity of both proteins was compared on a molecular basis by their ability to stimulate EF-1α in the poly(U)-directed synthesis of polyphenylalanine. On the basis of this assay EF-1βγδ is slightly more active than EF-1βδ. The similarity of the amino acid composition of EF-1γ and EF-1δ and the molar ratio of α:β:γ:δ in EF-1H of approximately 1:1:0.5:0.5 have led to the conclusion that EF-1δ is probably a breakdown product of EF-1γ, and that the native form of EF-1H probably contains only the α, β, and γ subunits.
A cell-free system devoid of polysomes, which translates natural mRNA, has been prepared from rat liver. It contains ribosomal subunits, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and protein factors necessary for translation. Protein synthesis required an energy-generating system, mRNA, and 3 mM Mg2+ concentration, and it was inhibited by 7-methylguanylic acid. The total extent and the rate of protein synthesis were approximately 30% greater when the translating system was prepared from livers of 3-month-old rats, as compared to 30-month-old rats. A ribosome-free fraction containing the protein factors required for translation was also prepared from 3-month-old and 30-month-old rat livers and brains, by extraction with 0.5 M KCl. The high-salt extracts were analyzed for elongation factors EF-1 and EF-2 in a poly(U) translating system. Although the activity of EF-2 was similar in preparations from young and old rats, the EF-1 activity in the 3-month-old rat livers and brains was 30 to 40% greater than in 30-month-old animals. The protein synthesizing activity of high salt-washed ribosomes stripped of endogenous peptidyl-tRNA and mRNA, from livers and brains of young and old animals, was the same.
The specific activities of elongation factor 1, RNA and DNA polymerase, as well as fructose-1,6-diphosphate aldolase were determined as a function of age in the nematode Turbatrix aceti. It was found that the specific activities of both DNA polymerase and aldolase declined constantly as the animal aged. In contrast, both elongation factor 1 and RNA polymerase showed sharp increases in their specific activities at 5 and 15 days, respectively, before ultimately declining. It was also shown that elongation factor 1, which exists mainly as a high molecular weight species in the young animal, undergoes a conversion to a lower molecular weight species as the organism ages. In addition there is a progressive accumulation of inactive or partially active elongation factor 1 molecules in older animals. The addition of α-tocopherol to the growth medium of these nematodes resulted in an increased life-span as well as alterations in the patterns of the enzyme activities.
Vero M3 cells, a line derived from the kidney of an African Green Monkey, display certain alterations in their protein synthetic apparatus as a function of time during a growth cycle. (Growth cycle here refers to exponential growth of unsynchronized cells in culture and their subsequent passage into the stationary phase.) The capacity of cytoplasmic extracts of these cells to promote endogeneous mRNA-mediated polypeptide synthesis or poly U-mediated polyphenylalanine synthesis declines from the second day after the initiation of the growth cycle. The ribosome sedimentation profile indicates that after the second day of growth a decrease also occurs in the total amount of ribosomes per cell, and that a shift occurs from predominantly polyribosome structures to predominantly subunits and monoribosomes structures. The activity of the translation factor, elongation factor 1, also progressively decreases after the second day of growth. Furthermore, when crude factor preparations from cells in the second day of growth (Exponential phase) and from cells in the fifth day of growth (Stationary phase) are compared for leucyl-tRNA synthetase and prolyl-tRNA synthetase activities, it is found that the extracts from fifth-day cells have significantly less activity. The activity of another enzyme, acid phosphatase, remains relatively unaffected as a function of time during the cell growth cycle. When HeLa S3 plating cells are grown under the same conditions, they do not display the same responses.
We have investigated the regulation of protein synthesis in animal cells by serum factors. Withdrawal of serum from the medium of actively dividing Vero cells resulted in an immediate decline in the rate of peptide chain elongation (Hassell and Engelhardt, 1973). Assay of elongation factor I (EFI) activity in the post-ribosomal supernatant as well as that associated with the ribosomes revealed that serum deprivation resulted also in reduction in the activity of this factor. The decline in the activity of EFI after serum deprivation occurred to the same extent and at the same time as the decline in the in vivo rate of protein synthesis and the in vitro peptide synthetic capacity of cell-free extracts. A temporal correlation therefore exists among the in vivo rate of protein synthesis, the peptide synthetic activity of cell-free extracts, and the activity of EFI. The activity of peptidyl transferase was not altered by serum deprivation. The loss of extract peptide synthetic activity resulting from serum deprivation was reversible since serum addition to previously serum-starved cultures resulted in full restoration of activity for polyphenylalanine (polyPhe) synthesis within 3 h. Moreover, RNA synthesis was not required for this turn-on of polyPhe synthesis. Vased on these data we conclude that a translational control mechanism is operative in Vero cells deprived of serum.
Age-related changes of polypeptide chain elongation factor 2 (EF-2) in mouse and rat livers were investigated. The percentage of heat-labile components in EF-2 was 5 to 10% in young animals and 15 to 45% in old ones. These results and other findings (Takahashi et al., in press) support the idea that decrease in translational activity in old animals is due, in part, to alterations of protein components of the translational system.
The age-related reduction in cell-free protein synthesis in the free-living nematode Turbatrix aceti is due to a defect in the ribosomes. Addition of young ribosomal wash or use of young medium does not improve the activity of old, run-off ribosomes in the presence of phenylalanine and poly(U). It appears that some of the old ribosomes are incapable of binding the EF-1-GTP-aminoacyl-tRNA complex. These ineffective ribosomes are present in the 80 S (monosomal) fraction. Old ribosomes obtained from polysomes appear to bind normally.
An antibody (anti-p21ser) was raised against a ras p21-related synthetic peptide and was able to recognize specifically the substitution of serine for glycine at amino acid 12 of p21. This substitution causes oncogenic activation of p21. Anti-p21ser was found to immunoprecipitate v-Ki-ras p21 and to strongly inhibit its ability to autophosphorylate and to bind GTP in an immunoabsorption assay. Furthermore, binding of the antibody to p21 was specifically inhibited by GTP or GDP, suggesting that amino acids around position 12 are part of the GTP/GDP binding site. These results, taken together with the observation that the microinjection of anti-p21ser into cells transformed by v-Ki-ras p21 causes a transient reversion of the cells to a normal phenotype [Feramisco, J. R., Clark, R., Wong, G., Arnheim, N., Milley, R. & McCormick, F. (1985) Nature (London) 314, 639-642], support the idea that interaction of p21 with guanine nucleotides is crucial to the transforming function of this protein.
The protein synthesis elongation factor EF-1 alpha of Mucor racemosus hyphae contained eight or nine methylated amino acids
per molecule, whereas the factor from sporangiospores was nonmethylated. During the course of spore germination, the specific
activity of the factor in crude extracts increased sixfold. This increase in activity was accompanied by a constant level
of EF-1 alpha-specific mRNA and a constant level of EF-1 alpha protein. Methylation of the protein, however, accelerated during
the germination process, in parallel with the increase in specific activity of the factor. We propose that the activity of
EF-1 alpha is regulated during germination through methylation of the protein and does not involve transcriptional regulation.
Embryos of the brine shrimp, Artemia salina, were used in a study of polypeptide chain initiation in an in vitro system from a eukaryote. A protein, isolated from the high-speed supernatant, has been highly purified and shown to have properties that suggest it is the eukaryotic equivalent of the Escherichia coli initiation factor F(2): It promotes the AUG-dependent binding of fMet-tRNA (E. coli) to the Artemia 40S ribosomal subunit, but not to either the 60S or 80S species; the bound fMet-tRNA is placed in a site on the smaller subunit from which it reacts with puromycin upon addition of the 60S subunit; and the activity is sensitive to aurintricarboxylic acid and edeine, specific inhibitors of initiation. The factor, a basic protein of molecular weight about 100,000, is inactivated by N-ethylmaleimide, an SH-binding reagent, and is clearly distinct from the Artemia elongation factors, T(1) and T(2). In addition, the factor stimulates the poly(U)-dependent binding of Phe-tRNA (E. coli) to the Artemia 40S ribosomal subunit. This reaction, though similar to the fMet-tRNA-binding reaction, differs in that the bound Phe-tRNA is largely resistant to release by puromycin.
The molecular basis was sought for the previously observed drop in the synthesis of elongation factor one (EF-1) and subsequent decline in overall protein synthesis in aging Drosophila melanogaster. It has been found that translatable poly(A+)RNA for EF-1 disappears at about the same time that EF-1 synthesis decreases. This disappearance is specific for EF-1, since overall poly(A+)RNA levels and their translation to cellular proteins remain constant over the life-span of the organism. The disappearance of translatable RNA is not the result of a specific loss of the polyadenylate segment of poly(A+)RNA, since poly(A-)RNA exhibits a similar specific loss of translation ability for EF-1.
Two forms of EF-1 are present in the high-speed supernatant fraction from wheat embryo homogenate. In embryos from dry seeds EF-1H is 55% of the total amount of EF-1, while after 40 h of germination this form completely disappears. When germination and protein synthesis are accelerated by means of 6-benzyladenine, the rate of conversion of EF-1H is increased. On the other hand, the block of germination and of the evolution of protein synthesis by abscisic acid, block this conversion; the block of water uptake, that stops germination and causes a decrease in protein synthesis, reverses the conversion of EF-1H to EF-1L, increasing EF-1H from 15% to 40% of the total.
Protein synthetic activity was determined in postmitochondrial preparations from heart, brain, kidney, liver and skeletal muscle of 5-26-month-old female C57B1/6J mice. An age-dependent decrease in the rate of protein synthesis was exhibited by all preparations except heart muscle. A 65% decrease in translational rate was found in liver, with the greatest decrease appearing after 21 months. Translation in the brain preparation declined little during the first 20 months, but dropped 33% between 20 and 26 months. The kidney preparation decreased 30% during the first 16 months and 70% by the end of 26 months of age. Skeletal muscle showed an overall decrease of 85% in translation rate. In contrast, heart muscle decreased no more than 10% over the life-span of the mice. From these results, it appears that aging has a differential effect on protein synthesis in different kinds of cells.
During the two decades since Orgel (1963) first pointed out the risk of instability in the cellular translation process, this concept has generated a great deal of interest. Models of the dynamics of error propagation have a useful role to play in elucidating the general stability characteristics of the translation process, and they may also aid in the design and interpretation of critical experiments. Present models are of limited realism, however, and future models will be needed which can take account of error feedback at different levels of information transfer and which represent more flexibly the effect of protein synthetic errors on enzyme activity and specificity. A recent, interesting application of error feedback theory was made by Hasegawa et al. (1984) who argued that the more rapid evolution of tRNAs and ribosomal proteins in animal mitochondria than in their cytoplasmic counterparts may have arisen because animal mitochondria import information-handling enzymes from the cytoplasm and are, therefore, less at risk of error catastrophe. This places the mitochondrial translation process under less strict evolutionary constraint.
The decrease in the rate of protein synthesis in aging adult Drosophila melanogaster was found previously to be due, to a great extent, to a drop in the rate of peptide chain elongation, and principally to lowered activity of elongation factor one (EF-1). This decrease does not appear to be caused by appearance of an inhibitor of peptide chain elongation. Instead, the synthesis of EF-1 declines markedly early in adult life. This decrease is followed by lowered activity of EF-1 and by a drop in the synthesis of most of the cellular proteins.
The effects of age on the initiation and elongation stages of protein synthesis were measured in cell-free preparations from brain, liver, kidney and skeletal muscle of young (3-5 months) and senescent (23-27 months) female C57B1/6J mice. The ability to form initiation complexes from isolated 40 S and 60 S ribosomal subunits decreased only slightly with age. In contrast, the rate of peptide chain elongation decreased by 67% in brain preparations, 80% in liver, 81% in kidney and 85% in skeletal muscle of the senescent mice when compared with the young mice.
Protein synthesis in cell-free systems of rat liver and kidney decreases markedly with age. Examination of activity changes of the different steps revealed for both types of organs that reduced binding of aminoacyl-tRNA to ribosomes and reduced peptidyl transfer might be of major importance for the decrease in overall protein synthesis whereas ageing has only little effect on translocation as well as on initiation and termination.
Cultured normal human and animal cells are predestined to undergo irreversible functional decrements that mimic age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 +/- 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occurs in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.
A large, transient reduction in the population size of human fibroblasts in early passages significantly increases the variability of the life-spans of cultures in comparison to control cultures, as predicted by the commitment theory of cellular aging. The theory also predicts that a constant population of noncycling cells will appear in the later part of the culture life-span. This was confirmed by labeling the cells in culture with tritiated thymidine.
Chinese hamster ovary cells growing exponentially in serum-containing media cease to grow at relatively high cell density due to the depletion of a component or components provided in the serum. When exponentially or rapidly growing cells are incubated with radioactive amino acid, incorporation into proteins is markedly greater than in incubations containing stationary or resting phase cells. Amino acid incorporation in postmitochondrial extracts, in incubations designed to measure translation of endogenous mRNAs, is also significantly greater with preparations obtained from exponential phase cells than from stationary phase cells. This observation suggests that the level of mRNA or the ability to translate mRNA differs in stationary as compared to exponential phase cells. When extracts are depleted of endogenous mRNA and analyzed for their ability to translate exogenous natural (globin) mRNA, the activity of exponential cell extracts is much greater than that of stationary cell preparations. These results indicate that the translational system differs in stationary phase cells as compared to exponential phase cells. The formation of a complex containing 40S ribosomal subunits and [35S]methionine in the absence of mRNA and the transfer of radioactive methionine to the 80S initiation complex in the presence of mRNA and cycloheximide are quantitatively similar in extracts from exponential phase and stationary phase cells. These findings suggest that transition from the expo-nential to the stationary phase does not significantly affect reactions involved in chain initiation such as the formation of ternary complex containing eIF-2, GTP, and Met-tRNAf, the association of the ternary complex with 40S subunits, the binding of mRNA, and the joining of 60S subunits. When mRNA-depleted extracts are incubated with poly(uridylic acid), the synthesis of poly(phenylalanine) in exponential cell preparations is considerably greater than in stationary cells. Since the poly(U)-dependent synthesis of poly(phenylalanine) requires only components for polypeptide chain elongation, these results suggest that the activity of a component or components required for chain elongation, such as elongation factors or ribosomes, is altered. Analysis of individual elongation factors reveals that whereas the activity of EF-2 is similar in both preparations, the activity of EF-1 in stationary phase cells is markedly lower than in exponentially growing cells. Differences in activity between the two cell phases were not detected in analyses of chain termination and release. Thus, passage from the exponential to the stationary phase in Chinese hamster ovary cells is accompanied by a decrease in the amount of polysome-associated translatable mRNA and in the activity of EF-1; all of the other components involved in initiation, elongation, and termination of protein synthesis appear to be unaffected.
- Huschtscha