Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Many genes are of unknown functions in any sequenced genome. A combination of chemical and genetic perturbations has been used to investigate gene functions. Here we present a case that such "chemogenomics" information can be effectively used to identify missing genes in a defined biological pathway. In particular, we identified the previously unknown enzyme diphthamide synthetase for the last step of diphthamide biosynthesis. We found that yeast protein YLR143W is the diphthamide synthetase catalyzing the last amidation step using ammonium and ATP. Diphthamide synthetase is evolutionarily conserved in eukaryotes. The previously uncharacterized human gene ATPBD4 is the ortholog of yeast YLR143W and fully rescues the deletion of YLR143W in yeast.

Section: Discussionmentioning

confidence: 59%

Chemogenomic approach identified yeast YLR143W as diphthamide synthetase

Su¹,

Lin²,

Chen

et al. 2012

“…Because the diphthamide on eEF2 is highly conserved in all eukaryotes as well as in archea (41), it is surprising that lack of diphthamide synthesis has little overall impact on cell growth. Animals with homozygous DPHko, however, do not survive beyond embryonic stages (13,(16)(17)(18). This finding suggests that the diphthamide may be necessary for development.…”

Section: Discussionmentioning

confidence: 95%

“…Many reports related to diphthamide synthesis of mammalian cells describe "partial knockouts" and "partial phenotypes" (i.e., reduced levels but not complete loss of diphthamide modification or toxin sensitivities) (7)(8)(9) So far, the function of diphthamide on eEF2 also remained rather elusive. Reports indicate that it contributes to translation fidelity (10)(11)(12)(13). On the other hand, DPH genes or eEF2 can be mutated to prevent diphthamide attachment, yet cells carrying such mutations are viable (5,11,14,15).…”

mentioning

confidence: 99%

“…On the other hand, DPH genes or eEF2 can be mutated to prevent diphthamide attachment, yet cells carrying such mutations are viable (5,11,14,15). Animals with heterozygous DPH knockouts (DPHko) can be generated, but homozygous DPH1ko, DPH3ko, and DPH4ko are embryonic lethal (13,(16)(17)(18). Because these studies are based on inactivation of individual genes, it is difficult to discriminate between phenotypes caused by gene loss and phenotypes as a consequence of loss of diphthamide.…”

mentioning

confidence: 99%

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Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

Stahl¹,

Seidl²,

Ducret

et al. 2015

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP-(diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.ADP-ribosylation of eEF2 | Pseudomonas exotoxin | diphtheria toxin | translation | DPH gene knockout

“…S6). FP59 is a fusion protein of LF amino acids 1-254 and the catalytic domain of Pseudomonas aeruginosa exotoxin A that kills all cells by ADP ribosylation of eEF2 after delivery to cytosol by PA (35,36). To examine the cytotoxic effects of the toxin on tumor endothelial cells, the cells were treated with PA-L1/LF for 48 h and 72 h, respectively, followed by annexin V plus propidium iodide (PI) staining to identify apoptotic cells by flow cytometry.…”

Section: Engineered Anthrax Lethal Toxins Inhibit Proliferation Of Tumormentioning

confidence: 99%

Solid tumor therapy by selectively targeting stromal endothelial cells

Liu

et al. 2016

Self Cite

Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors.anthrax toxin | tumor targeting | angiogenesis | CMG2 | TEM8 R ecognition that aberrant activation of the RAS and PI3K pathways is often the mechanism of human tumorigenesis has inspired development of many small molecule inhibitors of these pathways and has led to improved treatments in certain cancers (1). However, these therapies are effective only in patients having defects in the specific targets of these drugs, and the therapies are rarely curative due to the development of resistance through acquisition of additional oncogenic mutations (2). Therefore, strategies are needed that are effective against a broad spectrum of cancers and that act through features that are not subject to development of resistance. This unmet need has fostered continued interest in strategies that target host-derived tumor vasculature.Anthrax toxin, a major virulence factor of Bacillus anthracis, consists of three individually nontoxic proteins: the cellular binding component, protective antigen (PA), and two enzymatic moieties, lethal factor (LF) and edema factor (EF) (3). PA binds to two host cell-surface integrin-like proteins: tumor endothelium marker-8 (TEM8) [also termed anthrax toxin receptor 1 (ANTXR1)] and capillary morphogenesis protein-2 (CMG2 or ANTXR2) (4, 5). Receptor-bound PA is processed by the ubiquitous cell-associated furin protease to a 63-kDa fragment (PA63), which then forms LFand EF-binding competent PA oligomers. Three or four molecules of LF or EF bind to the PA oligomers, and the complexes are then endocytosed (6-8). The acidic pH within the endosomes causes the PA oligomer to form a pore in the endosomal membrane, allowing translocation of LF or EF into the cytosol of cells to exert their cytotoxic actions (9). Thus, LF plus PA forms lethal toxin and EF plus PA forms edema toxin, with both toxins playing essenti...