Comparative Genomic Analysis of Genes Encoding Translation Elongation Factor 1Bα in Human and Mouse Shows EEF1B1 to Be a Recent Retrotransposition Event

We have characterized genomic loci encoding translation elongation factor 1Bα (eEF1Bα) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca²⁺/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.     

Cloning and nucleotide sequence of the gene for an archaebacterial protein synthesis elongation factor Tu

Summary A restriction fragment enrichment procedure was devised for the identification and cloning of the gene for protein synthesis elongation factor Tu (EF-Tu) from Methanococcus vannielii, employing hybridisation with an internal tufB gene probe from Escherichia coli. Methanococcus contains a single tuf gene on its chromosome; it is expressed in E. coli and it codes for a polypeptide of 46.5 kDa. The overall architecture of the protein bears a striking resemblance to that of eukaryotic elongation factor 1 (EF-1). The close similarity to EF-1 is supported by the sequence homology values which are in the range of 34% to 35% with eubacterial, plastid and mitochondrial EF-Tu sequences and as high as 52% to 54% with those from eukaryotic EF-1.     

Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.     

Deer mice: "The Drosophila of North American mammalogy"

Summary: Oocyte‐somatic cell communication is necessary for normal ovarian function. However, the identities of the majority of oocyte‐secreted proteins remain unknown. A novel cDNA encoding mouse oo cyte‐s ecreted p rotein 1 (OOSP1) was identified using a modified subtractive hybridization screen. The Oosp1 cDNA encodes a 202‐amino acid protein that contains a 21‐amino acid signal peptide sequence, 5 putative N‐linked glycosylation consensus sequences, and 6 cysteines that are predicted to form 3 disulfide bonds. OOSP1 shares amino acid identity with placental‐specific protein 1 (PLAC1), a secreted protein expressed in the placenta and the ectoplacental cone. The Oosp1 mRNA is approximately 1.0 kb and is present at high levels in the oocytes of adult ovaries and at lower levels in the spleen. The mouse Oosp1 gene is 5 exons, spans greater than 16.4 kb, and localizes to chromosome 19 at a position that shares synteny with human chromosome 11q12–11q13. The identification of OOSP1 as a new oocyte‐secreted protein permits future in vitro and in vivo functional analyses to define its role in ovarian folliculogenesis. genesis 31:105–110, 2001. © 2001 Wiley‐Liss, Inc.     

Comparative genomic analysis of genes encoding translation elongation factor 1B(alpha) in human and mouse shows EEF1B1 to be a recent retrotransposition event

We have characterized genomic loci encoding translation elongation factor 1B(alpha) (eEF1B(alpha)) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

Unbiased functional proteomics strategy for protein kinase inhibitor validation and identification of bona fide protein kinase substrates: application to identification of EEF1D as a substrate for CK2

Protein kinases have emerged as attractive targets for treatment of several diseases prompting large-scale phosphoproteomics studies to elucidate their cellular actions and the design of novel inhibitory compounds. Current limitations include extensive reliance on consensus predictions to derive kinase-substrate relationships from phosphoproteomics data and incomplete experimental validation of inhibitors. To overcome these limitations in the case of protein kinase CK2, we employed functional proteomics and chemical genetics to enable identification of physiological CK2 substrates and validation of CK2 inhibitors including TBB and derivatives. By 2D electrophoresis and mass spectrometry, we identified the translational elongation factor EEF1D as a protein exhibiting CK2 inhibitor-dependent decreases in phosphorylation in (32)P-labeled HeLa cells. Direct phosphorylation of EEF1D by CK2 was shown by performing CK2 assays with EEF1D -FLAG from HeLa cells. Dramatic increases in EEF1D phosphorylation following λ-phosphatase treatment and phospho- EEF1D antibody recognizing EEF1D pS162 indicated phosphorylation at the CK2 site in cells. Furthermore, phosphorylation of EEF1D in the presence of TBB or TBBz is restored using CK2 inhibitor-resistant mutants. Collectively, our results demonstrate that EEF1D is a bona fide physiological CK2 substrate for CK2 phosphorylation. Furthermore, this validation strategy could be adaptable to other protein kinases and readily combined with other phosphoproteomic methods.     

Chromosome instability region analysis and identification of the driver genes of the epithelial ovarian cancer cell lines A2780 and SKOV3

Epithelial ovarian cancer (EOC) is one of the most prevalent gynaecological cancers worldwide. The molecular mechanisms of serous ovarian cancer (SOC) remain unclear and not well understood. SOC cases are primarily diagnosed at the late stage, resulting in a poor prognosis. Advances in molecular biology techniques allow us to obtain a better understanding of precise molecular mechanisms and to identify the chromosome instability region and key driver genes in the carcinogenesis and progression of SOC. Whole-exome sequencing was performed on the normal ovarian cell line IOSE80 and the EOC cell lines SKOV3 and A2780. The single-nucleotide variation burden, distribution, frequency and signature followed the known ovarian mutation profiles, without chromosomal bias. Recurrently mutated ovarian cancer driver genes, including LRP1B, KMT2A, ARID1A, KMT2C and ATRX were also found in two cell lines. The genome distribution of copy number alterations was found by copy number variation (CNV) analysis, including amplification of 17q12 and 4p16.1 and deletion of 10q23.33. The CNVs of MED1, GRB7 and MIEN1 located at 17q12 were found to be correlated with the overall survival of SOC patients (MED1: p = 0.028, GRB7: p = 0.0048, MIEN1: p = 0.0051), and the expression of the three driver genes in the ovarian cell line IOSE80 and EOC cell lines SKOV3 and A2780 was confirmed by western blot and cell immunohistochemistry.     

Loss of heterozygosity atBRCA1/2 loci in hereditary and sporadic ovarian cancers

Loss of heterozygosity atBRCA1/2 loci in breast and ovarian tumors is a suggested risk factor for germlineBRCA1/2 mutation status. We evaluated the presence of losses of selected microsatellite markers localized on chromosomes 17 and 13q in hereditary and sporadic ovarian tumors. 151 consecutive primary ovarian tumors (including 21 withBRCA1/2 mutations and 130 without the mutations) were screened for loss of heterozygosity at loci on chromosomes 17 and 13q. Losses of heterozygosity of at least one microsatellite marker localized on chromosomes 17 and 13q were revealed in 123 (81.5%) and 104 (68.9%) tumors, respectively. Losses of all informative markers on chromosomes 17 and 13 occurred in 30 (19.9%) and 31 (20.5%) tumors, respectively. There was no difference in the frequency of losses atBRCA1 intragenic markers (D17S855 and D17S1323) between BRCA1-positive and BRCA1-negative patients. The frequency of losses on chromosome 17 was higher in high-grade than in low-grade carcinomas. Loss of heterozygosity on chromosomes 17 and 13q is a frequent phenomenon in both hereditary and sporadic ovarian cancers. The frequency of losses atBRCA1 intragenic markers in the ovarian tumor tissue is not strongly related to the presence ofBRCA1 germline mutations.     

The expression levels of the translational factors eEF1A 1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade

Despite the involvement of the elongation factors eEF1A (eEF1A1 and eEF1A2) in the development of different cancers no information is available on their possible contribution to the biology of hepatocellular carcinoma (HCC). We investigated the expression of both forms of eEF1A in HepG2 and JHH6 cell lines considered to be a good in vitro model of HCC at different stage of differentiation. Our data indicate that the mRNA amount of eEF1A1 is increased in both cell lines as compared to normal liver tissue, but eEF1A2 mRNA level is markedly increased only in JHH6. Moreover, the less differentiated cell line JHH6 displays higher EEF1A1 and EEF1A2 mRNAs levels and an higher nuclear-enriched/cytoplasm ratio of EEF1A protein compared to the better differentiated HepG2 cell line. Over-expression depends only partially on gene amplification. The more abundant mRNA levels and the higher nuclear-enriched/cytoplasm ratio of eEF1A in JHH6 neither correlate with apoptosis resistance nor with proliferation rate in sub-confluent cells. However, in confluent cells, a clear tendency to maintain JHH6 into the cell cycle was observed. In conclusion, we document the increased mRNA levels of EEF1A genes in HCC cell lines compared to normal liver. Additionally, we show the increased nuclear-enriched/cytoplasmic protein ratio of eEF1A and the marked raise of the expression of both eEF1A forms in JHH6 compared to HepG2, suggesting the possibility that eEF1A forms might become a relevant markers related to HCC tumor phenotype.     

Characterization of a novel annexin gene from cotton (Gossypium hirsutum cv CRI 35) and antioxidative role of its recombinant protein

Plant annexins represent a multigene family involved in cellular elongation and development. A cDNA encoding a novel annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library and designated GhAnx1. This gene encodes a 316 amino acid protein with a theoretical molecular mass of 36.06 kDa and a theoretical pI of 6.19. At the amino acid level, it shares high sequence similarity and has evolutionary relationships with annexins from higher plants. The purified recombinant protein expressed in Escherichia coli was used to investigate its physicochemical properties. Circular dichroism spectrum analyses showed a positive peak rising to the maximum at 196 nm and a broad negative band rounding 215 nm, suggesting that the GhAnx1 protein was prominently α‐helical. The fluorescence measurements indicated that it could bind to Ca²⁺ in vitro. These results demonstrated that GhAnx1 was a typical annexin protein in cotton. A bioassay experiment was conducted to analyze its potential function and showed that E. coli cells expressing GhAnx1 were protected from tert‐butyl hydroperoxide (tBH) stress, suggesting that it had a potential antioxidative role. Northern blot analyses revealed that GhAnx1 was highly expressed in fibers, especially during the elongation stage, suggesting that it might be important for fiber elongation.     

Role of Cks1 amplification and overexpression in breast cancer

Gain of chromosome 1q is a common event in many kinds of carcinomas. The Cks1 gene, located at 1q21, is required for p27 ubiquitination by the SCF^skp2 ubiquitinating machinery. In the present study, we found that Cks1 gene amplification was highly correlated with protein overexpression. Statistical analysis showed that amplification and overexpression of Cks1 were strongly associated with lymph node metastasis and poor prognosis. At the molecular level, knockdown of Cks1 expression by RNA interference inhibited the growth of MDA-MB-231 cells, damaged cell migration and invasion ability. Knockdown of Cks1 expression promoted apoptosis of breast cancer cells and a wobble mutant of Cks1 that was resistant to Cks1 siRNA can rescue this effect. Overexpression of Cks1 inhibited the apoptosis of breast cancer cells through the MEK-Erk pathway. These data suggest that Cks1 is an oncogene in the 1q21 amplicon and plays an important role for breast cancer development.     

Structural analysis of the elongation factor G protein from the low-temperature-adapted bacterium Arthrobacter globiformis SI55

The first structural analysis of elongation factor G (EF-G) from a cold-adapted bacterium is presented. EF-G is an essential protein involved in the elongation process during protein synthesis and is therefore thought to play a crucial role in the low-temperature adaptation of cold-adapted microorganisms. To define its importance, the EF-G gene (fus) from the psychrotolerant bacterium Arthrobacter globiformis SI55 was cloned and sequenced. The deduced primary structure of the elongation factor is composed of 700 amino acids with a predicted molecular mass of 77.4 kDa. A three-dimensional model of the protein was constructed based on the known crystal structures of structurally homologous proteins. Structural features that might potentially be important for activity and flexibility at low temperature were deduced by comparisons with models of the EF-G proteins from the closely related mesophiles Micrococcus luteus and Mycobacterium tuberculosis. These features include a loss in the number of salt bridges in intradomain and interdomain positions, increased solvent interactions mediated by greater charge and polarity on domain surfaces, loop insertions, loss of proline residues in loop structures, and an increase of hydrophobicity in core regions. Specific changes have also been identified in the catalytic domain (G domain) and sites of potential ribosome interaction, which may directly affect guanosine triphosphate (GTP) hydrolysis and elongation rates at low temperature. Received: September 20, 1999 / Accepted: December 2, 1999     

MARCH5 RNA promotes autophagy,migration, and invasion of ovarian cancer cells

MARCH5 is a crucial regulator of mitochondrial fission. However, the expression and function of MARCH5 in ovarian cancer have not been determined. This study investigated the expression and function of MARCH5 in ovarian cancer with respect to its potential role in the tumorigenesis of the disease as well as its usefulness as an early diagnostic marker. We found that the expression of MARCH5 was substantially upregulated in ovarian cancer tissue in comparison with the normal control. Silencing MARCH5 in SKOV3 cells decreased TGFB1-induced cell macroautophagy/autophagy, migration, and invasion in vitro and in vivo, whereas the ectopic expression of MARCH5 in A2780 cells had the opposite effect. Mechanistic investigations revealed that MARCH5 RNA may function as a competing endogenous RNA (ceRNA) to regulate the expression of SMAD2 and ATG5 by competing for MIR30A. Knocking down SMAD2 or ATG5 can block the effect of MARCH5 in A2780 cells. Also, silencing the expression of MARCH5 in SKOV3 cells can inhibit the TGFB1-SMAD2/3 pathway. In contrast, the ectopic expression of MARCH5 in A2780 cells can activate the TGFB1-SMAD2/3 pathway. In turn, the TGFB1-SMAD2/3 pathway can regulate MARCH5 and ATG5 through MIR30A. Overall, the results of this study identified MARCH5 as a candidate oncogene in ovarian cancer and a potential target for ovarian cancer therapy.     

Changes in protein phosphorylation in wild-type and nickel-resistant cells and their involvement in morphological elongation

Summary Treatment of wild-type Balb/c-3T3 cells with NiCl₂ orN ⁶,2-O-dibutyl-adenosine 3,5-monophosphate (Bt₂-CAMP) resulted in a high degree and frequency of cellular elongation. Nickel-resistant Balb/c-3T3 cells (B200) treated with Bt₂-CAMP elongated at the same exposure concentration as wild-type cells. In contrast, treatment of the nickel-resistant cells with both non-cytotoxic and cytotoxic doses of NiCl₂ failed to induce elongation. Nickel-resistant cells had two-thirds of the total protein-phosphorylation activity of wild-type cells. Both cAMP and NiCl₂ enhanced phosphorylation of specific proteins in intact wild-type cells as detected by³²P autoradiography of these proteins separated on two-dimensional gels. A nickel-dependent phosphorylation of specific proteins is seen following NiCl₂ treatment of wild-type cells but was not observed in B200 cells. In contrast, the pattern of Bt₂-cAMP-stimulated protein phosphorylation was quite similar in both wild-type and nickel-resistant cells. Although it is unclear at present how nickel ions affect the cellular protein-phosphorylation system, these results suggested that targets controlling cellular elongation are sensitive to nickel, are altered in nickel-resistant cells and appear to involve protein phosphorylation. Further characterization of these targets may help in understanding the mechanisms of nickel carcinogenesis.     

EEF2 analysis challenges the monophyly of Archaeplastida and Chromalveolata

Background

Classification of eukaryotes provides a fundamental phylogenetic framework for ecological, medical, and industrial research. In recent years eukaryotes have been classified into six major supergroups: Amoebozoa, Archaeplastida, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. According to this supergroup classification, Archaeplastida and Chromalveolata each arose from a single plastid-generating endosymbiotic event involving a cyanobacterium (Archaeplastida) or red alga (Chromalveolata). Although the plastids within members of the Archaeplastida and Chromalveolata share some features, no nucleocytoplasmic synapomorphies supporting these supergroups are currently known.

Methodology/Principal Findings

This study was designed to test the validity of the Archaeplastida and Chromalveolata through the analysis of nucleus-encoded eukaryotic translation elongation factor 2 (EEF2) and cytosolic heat-shock protein of 70 kDa (HSP70) sequences generated from the glaucophyte Cyanophora paradoxa, the cryptophytes Goniomonas truncata and Guillardia theta, the katablepharid Leucocryptos marina, the rhizarian Thaumatomonas sp. and the green alga Mesostigma viride. The HSP70 phylogeny was largely unresolved except for certain well-established groups. In contrast, EEF2 phylogeny recovered many well-established eukaryotic groups and, most interestingly, revealed a well-supported clade composed of cryptophytes, katablepharids, haptophytes, rhodophytes, and Viridiplantae (green algae and land plants). This clade is further supported by the presence of a two amino acid signature within EEF2, which appears to have arisen from amino acid replacement before the common origin of these eukaryotic groups.

Conclusions/Significance

Our EEF2 analysis strongly refutes the monophyly of the Archaeplastida and the Chromalveolata, adding to a growing body of evidence that limits the utility of these supergroups. In view of EEF2 phylogeny and other morphological evidence, we discuss the possibility of an alternative eukaryotic supergroup.