New Genes Involved in the Synthesis of Diphthamide, a Modification of Eukaryotic Translation Elongation Factor 2 with Roles in Diphtheria Disease and Ovarian Cancer Formation

Diphthamide, the target of Diphtheria toxin, is a unique post-translational modification on His[subscript 699] (S. cerevisiae) of translation elongation factor 2 (eEF2) found in eukarya and archaea. It serves as the unique target for bacterial ADP-ribosylating toxins such as Diphtheria Toxin, Exotoxin A and Cholix toxin. So far six genes have been known to be involved in the complex three-step biosynthesis pathway: bona fide diphthamide genes DPH1-DPH5 and the recently identified YBR246w. While the latter was shown to be involved in the final step of the pathway, its exact role remains unclear. Dph1-Dph4 facilitate the initial step of the pathway and the methytransferase, Dph5, the second step. Surprisingly, after almost four decades of intensive research the enzyme catalyzing the final step, the conversion of the intermediate diphthine into the final product diphthamide, has remained elusive. We sought to exploit yeast genetic interaction and chemical genomic databases in order to identify novel diphthamide biosynthesis genes. A novel candidate gene YLR143w was identified and we here present genetic, phenotypic and biochemical analyses that clearly identify YLR143w as a novel diphthamide biosynthesis gene. Our observations implicate that YLR143w is the main catalytic enzyme necessary for the third step of the pathway, while YBR246w has a regulatory role involving Dph5-EF2 interaction. Furthermore, we demonstrate that Dph1 is likely the primary catalytic enzyme which generates the initial modification on the His[subscript 699] residue. In addition to the implications in bacterial pathogenesis, diphthamide and the biosynthesis genes DPH1, DPH3 and DPH4 are associated with cancer formation as well as defects in embryonic development and cell proliferation control. We here demonstrate that diphthamide deficient yeast cells display a significant increase in -1 frameshifting during translation and propose that this is the underlying cause of the phenotypes seen in mammalian organisms

Insights into Diphthamide, key Diphtheria Toxin effector

Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and interrelation among the Dph proteins, we found, by expression in E. coli and co-immune precipitation in yeast, that Dph1 and Dph2 interact and that they form a complex with Dph3. Protein-protein interaction mapping shows that Dph1-Dph3 complex formation can be dissected by progressive DPH1 gene truncations. This identifies N- and C-terminal domains on Dph1 that are crucial for diphthamide synthesis, DT action and cytotoxicity of sordarin, another microbial eEF2 inhibitor. Intriguingly, dph1 truncation mutants are sensitive to overexpression of DPH5, the gene necessary to synthesize diphthine from the first diphthamide pathway intermediate produced by Dph1-Dph3. This is in stark contrast to dph6 mutants, which also lack the ability to form diphthamide but are resistant to growth inhibition by excess Dph5 levels. As judged from site-specific mutagenesis, the amidation reaction itself relies on a conserved ATP binding domain in Dph6 that, when altered, blocks diphthamide formation and confers resistance to eEF2 inhibition by sordarin

The amidation step of Diphthamide biosynthesis in yeast requires <em>DPH6</em>, a gene identified through mining the <em>DPH1-DPH5</em> interaction network

Diphthamide is a highly modified histidine residue in eukaryal translation elongation factor 2 (eEF2) that is the target for irreversible ADP ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step-amidation of the intermediate diphthine to diphthamide-is ill-defined. Here we mine the genetic interaction landscapes of DPH1-DPH5 to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants maintain eEF2 forms that evade inhibition by DT and sordarin, a diphthamide-dependent antifungal. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Consistent with an expected requirement for ATP in diphthine amidation, Dph6 contains an essential adenine nucleotide hydrolase domain and binds to eEF2. Dph6 is therefore a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal -1 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation

The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network.

Diphthamide is a highly modified histidine residue in eukaryal translation elongation factor 2 (eEF2) that is the target for irreversible ADP ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step-amidation of the intermediate diphthine to diphthamide-is ill-defined. Here we mine the genetic interaction landscapes of DPH1-DPH5 to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants maintain eEF2 forms that evade inhibition by DT and sordarin, a diphthamide-dependent antifungal. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Consistent with an expected requirement for ATP in diphthine amidation, Dph6 contains an essential adenine nucleotide hydrolase domain and binds to eEF2. Dph6 is therefore a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal -1 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation

Both the Alpha_ANH_like_IV and YjgF-YER057c-UK114 domains in Dph6 are essential for its functionality.

<p>(A) Diagram showing the <i>DPH6</i> wild-type and mutant constructs tested in (B), indicating the Alpha_ANH_like_IV (ANH) and YjgF-YER057c-UK114 (UK114) domains and the position of point mutations, an in-frame deletion (- - - - -) and triple myc epitope tag (<i>myc</i>₃) as appropriate. (B) Ten-fold serial cell dilutions of a <i>dph6</i> deletion strain carrying the constructs shown in (A) or the corresponding empty vector (top panel, pSU6 [wt <i>DPH6</i>]; lower panel, pSU7 [wt <i>DPH6</i>]: <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003334#pgen.1003334.s012" target="_blank">Table S3</a>) were spotted onto SCD-Leu plates with or without 10 µg/ml sordarin and grown at 30°C for 3 days.</p

The biosynthetic pathway for modification of eEF2 by diphthamide.

<p>For roles played by the <i>bona fide</i> diphthamide genes <i>DPH1–DPH5</i> in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified <i>DPH</i> gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.</p

Model for the diphthamide pathway incorporating the proposed novel roles of Dph5, Dph6, and Dph7.

<p>(A) Diphthamide pathway showing interaction of Dph5 with unmodified eEF2 and the proposed role of Dph7 in displacement of Dph5 prior to diphthine amidation. (B) Elimination of the trimethylamino group in the absence of the proposed amidase Dph6 suggesting lability of diphthine in its absence.</p

<i>DPH6</i> and <i>DPH7</i> deletion strains copy traits typically related to the <i>bona fide</i> diphthamide mutants <i>dph1-dph5</i>.

<p>(A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its <i>dph1-dph7</i> gene deletion derivatives (upper panels) as well an MKK-derived <i>eft1 eft2</i> double deletion background maintaining plasmid p<i>EFT2</i> wild-type or H₆₉₉ substitution (H₆₉₉ N and H₆₉₉I) alleles of <i>EFT2</i> (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml⁻¹ sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from <i>dph1</i>, <i>dph5</i>, <i>dph6</i> and <i>dph7</i> mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast <i>dph</i> mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003334#s4" target="_blank">Materials and Methods</a>). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.</p

MS/MS spectra of diphthamide-, ACP-, and diphthine-modified EF2 peptide 686-VNILDVTLHADAIHR-700 from wild-type and mutant yeast strains.

<p>Spectra are shown for (A) diphthamide-modified peptide from the wild-type yeast strain; (B) ACP-modified peptide from the <i>dph5Δ</i> mutant; (C) diphthine-modified peptide in the <i>dph7Δ</i> strain; (D) diphthine-modified peptide in the <i>dph6Δ</i> strain; (E) diphthine-modified peptide in the <i>dph6Δ</i> strain with loss of the trimethylamino group before analysis in the mass spectrometer indicated by the parent ion m/z. In each case the parent ion m/z and charge state is indicated. In (A), (C) and (D), * indicates neutral loss of trimethylamino during MS/MS. The inset in (C) shows greater detail for the more crowded part of the MS/MS spectrum. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003334#pgen.1003334.s002" target="_blank">Figure S2A</a> indicates how the B and Y ions are derived from the peptide sequence.</p