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

Mateyak, Maria K.; Kinzy, Terri Goss

doi:10.1074/jbc.m113.488783

Cited by 27 publications

(24 citation statements)

References 52 publications

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“…ADPribosylation of eEF2 in such conditions was confirmed by native PAGE (Fig. S4A,B) (20). An ability of eEF2-ADPR to bind POST complexes was shown by western blotting of the POST complexes purified in sucrose density gradient (Fig.…”

Section: Adp-ribosylation Of Eef2 Prevents Reverse Translocationmentioning

confidence: 89%

“…The residue is modified into diphthamide in archaea and eukaryotes by a set of conserved enzymes (17). Various bacterial toxins, such as diphtheria toxin (18), exotoxin A (17), or cholera toxin (19) perform an ADP-ribosylation of this residue, which leads to translational inhibition (20) and cell death (18). For eEF2, it was shown that the ADP-ribosylation affected the translocation step but did not influence eEF2 binding to the ribosome (21).…”

Section: Introductionmentioning

confidence: 99%

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Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Susorov

Zakharov

Shuvalova

et al. 2018

Journal of Biological Chemistry

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During protein synthesis, a ribosome moves along the mRNA template and, using aminoacyl-tRNAs, decodes the template nucleotide triplets to assemble a protein amino acid sequence. This movement is accompanied by shifting of mRNA-tRNA complexes within the ribosome in a process called translocation. In living cells, this process proceeds in a unidirectional manner, bringing the ribosome to the 3′-end of mRNA, and is catalyzed by the GTPase translation elongation factor 2 (EF-G in prokaryotes, eEF2 in eukaryotes). Interestingly, the possibility of spontaneous backward translocation has been shown in vitro for bacterial ribosomes, suggesting a potential reversibility of this reaction. However, this possibility has not yet been tested in eukaryotic cells. Here, using a reconstituted mammalian translation system, we show that eukaryotic elongation factor eEF2 catalyzes ribosomal reverse translocation at one mRNA triplet. We found that this process requires a cognate tRNA in the ribosomal E-site and cannot occur spontaneously without eEF2. The efficiency of this reaction depended on the concentrations of eEF2 and cognate tRNAs and increased in the presence of nonhydrolyzable GTP analogues. Of note, ADP-ribosylation of eEF2 domain IV blocked reverse translocation, suggesting a crucial role of interactions of this domain with the ribosome for the catalysis of the reaction. In summary, our findings indicate that eEF2 is able to induce ribosomal translocation in forward and backward directions highlighting the universal mechanism of tRNA-mRNA movements within the ribosome.

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Section: Adp-ribosylation Of Eef2 Prevents Reverse Translocationmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Susorov

Zakharov

Shuvalova

et al. 2018

Journal of Biological Chemistry

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“…Domain IV can be enzymatically modified by ADP ribosyltransferases in a reaction that uses NAD + (nicotinamide adenine dinucleotide) as the ADP ribosyl donor, thereby inhibiting EF2 function and ultimately, blocking translation and protein biosynthesis (Honjo et al ., ; Mateyak and Kinzy, ). Strikingly, for ADP ribosylation to occur, EF2 must carry diphthamide (Van Ness et al ., 1980a,b), a post‐translationally modified histidine residue that is conserved between archaea and eukaryotes (Fig.…”

Section: Ef2 Function During Mrna Translation Elongationmentioning

confidence: 99%

The diphthamide modification pathway from Saccharomyces cerevisiae – revisited

Schaffrath

Abdel‐Fattah

Klassen

et al. 2014

Molecular Microbiology

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SummaryDiphthamide is a conserved modification in archaeal and eukaryal translation elongation factor 2 (EF2). Its name refers to the target function for diphtheria toxin, the disease-causing agent that, through ADP ribosylation of diphthamide, causes irreversible inactivation of EF2 and cell death. Although this clearly emphasizes a pathobiological role for diphthamide, its physiological function is unclear, and precisely why cells need EF2 to contain diphthamide is hardly understood. Nonetheless, the conservation of diphthamide biosynthesis together with syndromes (i.e. ribosomal frame-shifting, embryonic lethality, neurodegeneration and cancer) typical of mutant cells that cannot make it strongly suggests that diphthamidemodified EF2 occupies an important and translationrelated role in cell proliferation and development. Whether this is structural and/or regulatory remains to be seen. However, recent progress in dissecting the diphthamide gene network (DPH1-DPH7) from the budding yeast Saccharomyces cerevisiae has significantly advanced our understanding of the mechanisms required to initiate and complete diphthamide synthesis on EF2. Here, we review recent developments in the field that not only have provided novel, previously overlooked and unexpected insights into the pathway and the biochemical players required for diphthamide synthesis but also are likely to foster innovative studies into the potential regulation of diphthamide, and importantly, its ill-defined biological role.

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“…These bacterial proteins enter cells and catalyze ADP ribosylation of diphthamide using nictotinamide adenine dinucleotide (NAD) as substrate (20,21). This inactivates eEF2, arrests protein synthesis, and kills (14).…”

Section: Significancementioning

confidence: 99%

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

Proc. Natl. Acad. Sci. U.S.A.

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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

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

Cited by 27 publications

References 52 publications

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

The diphthamide modification pathway from Saccharomyces cerevisiae – revisited

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

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