The diphthamide modification on elongation factor-2 renders mammalian cells resistant to ricin

Diphthamide is a post-translational derivative of histidine in protein synthesis elongation factor-2 (eEF-2) that is present in all eukaryotes with no known normal physiological role. Five proteins Dph1–Dph5 are required for the biosynthesis of diphthamide. Chinese hamster ovary (CHO) cells mutated in the biosynthetic genes lack diphthamide and are resistant to bacterial toxins such as diphtheria toxin. We found that diphthamide-deficient cultured cells were threefold more sensitive than their parental cells towards ricin, a r ibosome- i nactivating p rotein (RIP). RIPs bind to ribosomes at the same site as eEF-2 and cleave the large ribosomal RNA, inhibiting translation and causing cell death. We hypothesized that one role of diphthamide may be to protect ribosomes, and therefore all eukaryotic life forms, from RIPs, which are widely distributed in nature. A protective role of diphthamide against ricin was further demonstrated by complementation where dph mutant CHO cells transfected with the corresponding DPH gene acquired increased resistance to ricin in comparison with the control-transfected cells, and resembled the parental CHO cells in their response to the toxin. These data show that the presence of diphthamide in eEF-2 provides protection against ricin and suggest the hypothesis that diphthamide may have evolved to provide protection against RIPs.     

Elongation factor 2 diphthamide is critical for translation of two IRES-dependent protein targets,XIAP and FGF2, under oxidative stress conditions

Elongation factor-2 (eEF2) catalyzes the movement of the ribosome along the mRNA. A single histidine residue in eEF2 (H715) is modified to form diphthamide. A role for eEF2 in the cellular stress response is highlighted by the fact that eEF2 is sensitive to oxidative stress and that it must be active to drive the synthesis of proteins that help cells to mitigate the adverse effects of oxidative stress. Many of these proteins are encoded by mRNAs containing a sequence called an “internal ribosomal entry site” (IRES). Under high oxidative stress conditions diphthamide-deficient cells were significantly more sensitive to cell death. These results suggest that diphthamide may play a role in protection against the degradation of eEF2. This protection is especially important in those situations in which eEF2 is necessary for the reprogramming of translation from global to IRES synthesis. Indeed, we found that the expression of X-linked inhibitor of apoptosis (XIAP) and fibroblast growth factor 2 (FGF2), two proteins synthesized from mRNAs with IRESs that promote cell survival, is deregulated in diphthamide-deficient cells. Our findings therefore suggest that eEF2 diphthamide controls the selective translation of IRES-dependent protein targets XIAP and FGF2, critical for cell survival under conditions of oxidative stress.     

OVCA1: tumor suppressor gene

OVCA1, also known as DPH2L1, is a tumor suppressor gene associated with ovarian carcinoma and other tumors. Ovca1 homozygous mutant mice die at birth with developmental delay and cell-autonomous proliferation defects. Ovca1 heterozygous mutant mice are tumor-prone but rarely develop ovarian tumors. OVCA1 appears to be the homolog of yeast DPH2, which participates in the first biosynthetic step of diphthamide, by modification of histidine on translation elongation factor 2 (EF-2). Yeast dph2 mutants are resistant to diphtheria toxin, which catalyses ADP ribosylation of EF-2 at diphthamide. Thus, there appears to be growing evidence implicating alterations in protein translation with tumorigenesis.     

Carbonyl reduction by YmfI in Bacillus subtilis prevents accumulation of an inhibitory EF‐P modification state

Translation elongation factor P (EF‐P) in Bacillus subtilis is required for a form of surface migration called swarming motility. Furthermore, B. subtilis EF‐P is post‐translationally modified with a 5‐aminopentanol group but the pathway necessary for the synthesis and ligation of the modification is unknown. Here we determine that the protein YmfI catalyzes the reduction of EF‐P‐5 aminopentanone to EF‐P‐5 aminopentanol. In the absence of YmfI, accumulation of 5‐aminopentanonated EF‐P is inhibitory to swarming motility. Suppressor mutations that enhanced swarming in the absence of YmfI were found at two positions on EF‐P, including one that changed the conserved modification site (Lys 32) and abolished post‐translational modification. Thus, while modification of EF‐P is thought to be essential for EF‐P activity, here we show that in some cases it can be dispensable. YmfI is the first protein identified in the pathway leading to EF‐P modification in B. subtilis, and B. subtilis encodes the first EF‐P ortholog that retains function in the absence of modification.     

The involvement of Ca2+ and integrins in directional responses of zebrafish keratocytes to electric fields

Many cells respond directionally to small DC electrical fields (EFs) by an unknown mechanism, but changes in intracellular Ca²⁺ are widely assumed to be involved. We have used zebrafish (Danio rerio) keratocytes in an effort to understand the nature of the EF‐cell interaction. We find that the adult zebrafish integument drives substantial currents outward through wounds produced by scale removal, establishing that keratocytes near the wound will experience endogenous EFs. Isolated keratocytes in culture turn toward the cathode in fields as small as 7 mV mm^?1, and the response is independent of cell size. Epidermal sheets are similarly sensitive. The frequency of intracellular Ca²⁺ spikes and basal Ca²⁺ levels were increased by EFs, but the spikes were not a necessary aspect of migration or EF response. Two‐photon imaging failed to detect a pattern of gradients of Ca²⁺ across the lamellipodia during normal or EF‐induced turning but did detect a sharp, stable Ca²⁺ gradient at the junction of the lamellipodium and the cell body. We conclude that gradients of Ca²⁺ within the lamellipodium are not required for the EF response. Immunostaining revealed an anode to cathode gradient of integrin β1 during EF‐induced turning, and interference with integrin function attenuated the EF response. Neither electrophoretic redistribution of membrane proteins nor asymmetric perturbations of the membrane potential appear to be involved in the EF response, and we propose a new model in which hydrodynamic forces generated by electro‐osmotic water flow mediate EF‐cell interactions via effects on focal adhesions. J. Cell. Physiol. 219: 162–172, 2009. © 2008 Wiley‐Liss, Inc.     

Dph3, a small protein required for diphthamide biosynthesis, is essential in mouse development

The translation elongation factor 2 in eukaryotes (eEF-2) contains a unique posttranslationally modified histidine residue, termed diphthamide, which serves as the only target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Diphthamide biosynthesis is carried out by five highly conserved proteins, Dph1 to Dph5, and an as-yet-unidentified amidating enzyme. The evolutionary conservation of the complex diphthamide biosynthesis pathway throughout eukaryotes implies a key role for diphthamide in normal cellular physiology. Of the proteins required for diphthamide synthesis, Dph3 is the smallest, containing only 82 residues. In addition to having a role in diphthamide biosynthesis, Dph3 is also involved in modulating the functions of the Elongator complex in yeast. To explore the physiological roles of Dph3 and to begin to investigate the function of diphthamide, we generated dph3 knockout mice and showed that dph3+/- mice are phenotypically normal, whereas dph3-/- mice, which lack the diphthamide modification on eEF-2, are embryonic lethal. Loss of both dph3 alleles causes a general delay in embryonic development accompanied by lack of allantois fusion to the chorion and increased degeneration and necrosis in neural tubes and is not compatible with life beyond embryonic day 11.5. The dph3-/- placentas also developed abnormally, showing a thinner labyrinth lacking embryonic erythrocytes and blood vessels. These results attest to the physiological importance of Dph3 in development. The biological roles of Dph3 are also discussed.     

Stall no more at polyproline stretches with the translation elongation factors EF‐P and IF‐5A

Synthesis of polyproline proteins leads to translation arrest. To overcome this ribosome stalling effect, bacteria depend on a specialized translation elongation factor P (EF‐P), being orthologous and functionally identical to eukaryotic/archaeal elongation factor e/aIF‐5A (recently renamed ‘EF5’). EF‐P binds to the stalled ribosome between the peptidyl‐tRNA binding and tRNA‐exiting sites, and stimulates peptidyl‐transferase activity, thus allowing translation to resume. In their active form, both EF‐P and e/aIF‐5A are post‐translationally modified at a positively charged residue, which protrudes toward the peptidyl‐transferase center when bound to the ribosome. While archaeal and eukaryotic IF‐5A strictly depend on (deoxy‐) hypusination (hypusinylation) of a conserved lysine, bacteria have evolved diverse analogous modification strategies to activate EF‐P. In Escherichia coli and Salmonella enterica a lysine is extended by β‐lysinylation and subsequently hydroxylated, whereas in Pseudomonas aeruginosa and Shewanella oneidensis an arginine in the equivalent position is rhamnosylated. Inactivation of EF‐P, or the corresponding modification systems, reduces not only bacterial fitness, but also impairs virulence. Here, we review the function of EF‐P and IF‐5A and their unusual posttranslational protein modifications.     

A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi

Translation elongation factor G (EF‐G) in bacteria plays two distinct roles in different phases of the translation system. EF‐G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post‐termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF‐G are distributed over two different EF‐G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF‐G is also found in bacteria. Two EF‐G paralogues are found in the spirochaete Borrelia burgdorferi, EF‐G1 and EF‐G2. We demonstrate that EF‐G1 is a translocase, while EF‐G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF‐G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF‐3) is present in the reaction. These results indicate that two B. burgdorferi EF‐G paralogues are close relatives to mitochondrial EF‐G paralogues rather than the conventional bacterial EF‐G, in both their phylogenetic and biochemical features.     

Modulation of diphthamide synthesis by 5′-deoxy-5′-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of an aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5′-deoxy-5′-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Suppressive role of regucalcin in liver cell proliferation: involvement in carcinogenesis

Regucalcin (RGN/SMP30) was discovered in 1978 and is a unique calcium‐binding protein contains no EF‐hand motif calcium‐binding domain. Its name, regucalcin, was proposed as it suppresses activation of enzymes related to calcium signalling. The regucalcin gene (rgn) is localized on the X chromosome. Regucalcin plays its role of suppressor protein in intracellular signalling pathways, including of protein kinases and protein phosphatase activities, protein synthesis, and DNA and RNA synthesis in liver cells. Overexpression of endogenous regucalcin has a suppressive effect on cell proliferation in modelled rat hepatoma H4‐II‐E cells, which are induced by various signalling stimulations in vitro. This suppressive effect is independent of apoptosis. Endogenous regucalcin plays a suppressive role on overproduction of proliferating cells in regenerating rat liver in vivo. Regucalcin mRNA expression is uniquely down‐regulated in development of carcinogenesis in liver of rats in vivo. Regucalcin mRNA and protein expressions are also depressed in human hepatoma HepG2 cells, MCF‐7 breast cancer cells, and prostate cancer LNCaP cells. Depression of regucalcin expression may be associated with activity progression of carcinogens. Regucalcin may be a key molecule suppressor protein in cell proliferation and carcinogenesis.     

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.     

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP^R) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP^R-eEF2 to examine the effects of DT in vivo. ADP^R of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP^R-eEF2.     

Nucleolar retention of a translational C/EBPα isoform stimulates rDNA transcription and cell size

The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N‐terminal sequences. Here, we describe the function of an N‐terminally extended protein isoform of CCAAT enhancer‐binding protein α (C/EBPα) that is translated from an alternative non‐AUG initiation codon. We show that a basic amino‐acid motif within its N‐terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended‐C/EBPα occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I‐specific factors u pstream‐b inding f actor 1 (UBF‐1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL‐60 cells, endogenous expression of extended‐C/EBPα is lost concomitantly with nucleolar C/EBPα immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended‐C/EBPα induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPα adding to its role as key regulator of cell growth and proliferation.     

Translation elongation factor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin resistance

Eukaryotic elongation factor 2 (eEF2) mediates translocation in protein synthesis. The molecular mimicry model proposes that the tip of domain IV mimics the anticodon loop of tRNA. His-699 in this region is post-translationally modified to diphthamide, the target for Corynebacterium diphtheriae and Pseudomonas aeruginosa toxins. ADP-ribosylation by these toxins inhibits eEF2 function causing cell death. Mutagenesis of the tip of domain IV was used to assess both functions. A H694A mutant strain was non-functional, whereas D696A, I698A, and H699N strains conferred conditional growth defects, sensitivity to translation inhibitors, and decreased total translation in vivo. These mutant strains and those lacking diphthamide modification enzymes showed increased -1 frameshifting. The effects are not due to reduced protein levels, ribosome binding, or GTP hydrolysis. Functional eEF2 forms substituted in domain IV confer dominant diphtheria toxin resistance, which correlates with an in vivo effect on translation-linked phenotypes. These results provide a new mechanism in which the translational machinery maintains the accurate production of proteins, establishes a role for the diphthamide modification, and provides evidence of the ability to suppress the lethal effect of a toxin targeted to eEF2.     

Regulation of adipose‐tissue‐derived stromal cell orientation and motility in 2D‐ and 3D‐cultures by direct‐current electrical field

Cell alignment and motility play a critical role in a variety of cell behaviors, including cytoskeleton reorganization, membrane‐protein relocation, nuclear gene expression, and extracellular matrix remodeling. Direct current electric field (EF) in vitro can direct many types of cells to align vertically to EF vector. In this work, we investigated the effects of EF stimulation on rat adipose‐tissue‐derived stromal cells (ADSCs) in 2D‐culture on plastic culture dishes and in 3D‐culture on various scaffold materials, including collagen hydrogels, chitosan hydrogels and poly(L‐lactic acid)/gelatin electrospinning fibers. Rat ADSCs were exposed to various physiological‐strength EFs in a homemade EF‐bioreactor. Changes of morphology and movements of cells affected by applied EFs were evaluated by time‐lapse microphotography, and cell survival rates and intracellular calcium oscillations were also detected. Results showed that EF facilitated ADSC morphological changes, under 6 V/cm EF strength, and that ADSCs in 2D‐culture aligned vertically to EF vector and kept a good cell survival rate. In 3D‐culture, cell galvanotaxis responses were subject to the synergistic effect of applied EF and scaffold materials. Fast cell movement and intracellular calcium activities were observed in the cells of 3D‐culture. We believe our research will provide some experimental references for the future study in cell galvanotaxis behaviors.     

The role of COBRA‐LIKE 2 function,as part of the complex network of interacting pathways regulating Arabidopsis seed mucilage polysaccharide matrix organization

The production of hydrophilic mucilage along the course of seed coat epidermal cell differentiation is a common adaptation in angiosperms. Previous studies have identified COBRA‐LIKE 2 (COBL2), a member of the COBRA‐LIKE gene family, as a novel component required for crystalline cellulose deposition in seed coat epidermal cells. In recent years, Arabidopsis seed coat epidermal cells (SCEs), also called mucilage secretory cells, have emerged as a powerful model system for the study of plant cell wall components biosynthesis, secretion, assembly and de muro modification. Despite accumulating data, the molecular mechanism of COBL function remains largely unknown. In the current research, we utilized genetic interactions to study the role of COBL2 as part of the protein network required for seed mucilage production. Using correlative phenotyping of structural and biochemical characteristics, unique features of the cobl2 extruded mucilage are revealed, including: ‘unraveled’ ray morphology, loss of primary cell wall ‘pyramidal’ organization, reduced Ruthenium red staining intensity of the adherent mucilage layer, and increased levels of the monosaccharides arabinose and galactose. Examination of the cobl2cesa5 double mutant provides insight into the interface between COBL function and cellulose deposition. Additionally, genetic interactions between cobl2 and fei1fei2 as well as between each of these mutants to mucilage‐modified 2 (mum2) suggest that COBL2 functions independently of the FEI‐SOS pathway. Altogether, the presented data place COBL2 within the complex protein network required for cell wall deposition in the context of seed mucilage and introduce new methodology expending the seed mucilage phenotyping toolbox.