Diphthamide promotes TOR signaling by increasing the translation of proteins in the TORC1 pathway.

Diphthamide, a modification found only on translation elongation factor 2 (EF2), was proposed to suppress -1 frameshifting in translation. Although diphthamide is conserved among all eukaryotes, exactly what proteins are affected by diphthamide deletion is not clear in cells. Through genome-wide profiling for a potential -1 frameshifting site, we identified that the target of rapamycin complex 1 (TORC1)/mammalian TORC1 (mTORC1) signaling pathway is affected by deletion of diphthamide. Diphthamide deficiency in yeast suppresses the translation of TORC1-activating proteins Vam6 and Rtc1. Interestingly, TORC1 signaling also promotes diphthamide biosynthesis, suggesting that diphthamide forms a positive feedback loop to promote translation under nutrient-rich conditions. Our results provide an explanation for why diphthamide is evolutionarily conserved and why diphthamide deletion can cause severe developmental defects.

Protein kinase A controls yeast growth in visible light.

BACKGROUND: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400-700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. RESULTS: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. CONCLUSION: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination.

Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1-4 (DPH1-DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for DPH1, DPH2, or DPH3, which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that Drosophila nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.

Interplay between reversible phosphorylation and irreversible ADP-ribosylation of eukaryotic translation elongation factor 2.

The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating toxins. We analyzed the interplay between phosphorylation and diphthamide-dependent ADP-ribosylation. Phosphorylation does not require diphthamide, eEF2 without it still becomes phosphorylated. ADP-ribosylation not only modifies the H715 diphthamide but also inhibits phosphorylation of S595 located in proximity to H715, and stimulates phosphorylation of T56. S595 can be phosphorylated by CDK2 and CDK1 which affects EEF2K-mediated T56-phosphorylation. Thus, ADP-ribosylation and S595-phosphorylation by kinases occur within the same vicinity and both trigger T56-phosphorylation. Diphthamide is surface-accessible permitting access to ADP-ribosylating enzymes, the adjacent S595 side chain extends into the interior. This orientation is incompatible with phosphorylation, neither allowing kinase access nor phosphate attachment. S595 phosphorylation must therefore be accompanied by structural alterations affecting the interface to ADP-ribosylating toxins. In agreement with that, replacement of S595 with Ala, Glu or Asp prevents ADP-ribosylation. Phosphorylation (starvation) as well as ADP-ribosylation (toxins) inhibit protein synthesis, both affect the S595/H715 region of eEF2, both trigger T57-phosphorylation eliciting similar transcriptional responses. Phosphorylation is short lived while ADP-ribosylation is stable. Thus, phosphorylation of the S595/H715 'modifier region' triggers transient interruption of translation while ADP-ribosylation arrests irreversibly.

The asymmetric function of Dph1-Dph2 heterodimer in diphthamide biosynthesis.

Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation elongation factor 2 (EF2). In the first step of diphthamide biosynthesis, a [4Fe-4S] cluster-containing radical SAM enzyme, Dph1-Dph2 heterodimer in eukaryotes or Dph2 homodimer in archaea, cleaves S-adenosylmethionine and transfers the 3-amino-3-carboxypropyl group to EF2. It was demonstrated previously that for the archaeal Dph2 homodimer, only one [4Fe-4S] cluster is necessary for the in vitro activity. Here, we demonstrate that for the eukaryotic Dph1-Dph2 heterodimer, the [4Fe-4S] cluster-binding cysteine residues in each subunit are required for diphthamide biosynthesis to occur in vivo. Furthermore, our in vitro reconstitution experiments with Dph1-Dph2 mutants suggested that the Dph1 cluster serves a catalytic role, while the Dph2 cluster facilitates the reduction of the Dph1 cluster by the physiological reducing system Dph3/Cbr1/NADH. Our results reveal the asymmetric functional roles of the Dph1-Dph2 heterodimer and may help to understand how the Fe-S clusters in radical SAM enzymes are reduced in biology.

Molecular characterization of diphthamide biosynthesis protein 7 in Marsupenaeus japonicus and its role in white spot syndrome virus infection.

Diphthamide biosynthesis protein 7 (Dph7) is a vital protein for diphthamide biosynthesis in archaea and eukaryotes. The 1143 bp cDNA sequence of Dph7 was cloned from the gills of Marsupenaeus japonicus using RT-PCR and RACE. Data showed that Dph7 was highly expressed in the gills and digestive gland of M. japonicus. Furthermore, the expression of dph7 was induced by infection with white spot syndrome virus (WSSV). When Dph7 was knocked down, immune genes such as toll, prophenoloxidase (proPO), p53, tumor necrosis factor-α (TNF-α) and signal transducer and activator of transcription (STAT) were significantly down-regulated (P < 0.01) in hemocytes. First, we demonstrated that Dph7 is very important in the progression of WSSV infection and that the time of death for WSSV-infected shrimp was significantly advanced following RNAi targeting of Dph7. We also investigated the effect of Dph7 on apoptosis rate in M. japonicas and found that Dph7-dsRNA treatment caused lower levels of apoptosis in hemocytes, both in the disease-free group and the WSSV group. Knock-down of Dph7 affected the activity of both phenoloxidase (PO) and superoxide dismutase (SOD), and total hemocyte count (THC) after infection with WSSV, indicating that Dph7 plays a regulatory role in the immunological reaction of shrimp in response to WSSV infection. Thus, we conclude that Dph7 may promote the anti-WSSV immune response of shrimp by regulating apoptosis, SOD and PO activity, and can influence the progression of WSSV infection.

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.

Diphthamide - a conserved modification of eEF2 with clinical relevance.

Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria. Diphthamide synthesis enzymes (DPH1 and DPH3) are associated with cancer, and DPH gene mutations can cause diphthamide deficiency syndrome (DDS). Finally, new analyses provide evidence that diphthamide may restrict propagation of viruses including SARS-CoV-2 and HIV-1, and that DPH enzymes are targeted by viruses for degradation to overcome this restriction. This review describes how diphthamide is synthesized and functions in translation, and covers its clinical relevance in human development, cancer, and infectious diseases.

Integrative analysis of the role of the DPH gene family in hepatocellular carcinoma and expression validation.

Background: The diphthamide (DPH) gene family is a group of genes that encode a set of enzymes that specifically modify eukaryotic elongation factor 2 (eEF2). Although previous studies have shown a link between the DPH genes (DPHs) and carcinogenesis, it is still unknown how the DPHs affect hepatocellular carcinoma (HCC). This study aimed to describe the expression, clinical significance, and potential mechanisms of DPHs in HCC. Methods: Real-time quantitative polymerase chain reaction (RT-qPCR), Genotype-Tissue Expression (GTEx), and The Cancer Genome Atlas (TCGA) databases were utilized to research the expression of DPHs in HCC. The relationship between the expression of DPHs and the clinicopathological characteristics of HCC patients was investigated using TCGA data, and their diagnostic value was evaluated using receiver operating characteristic (ROC) curves and their prognostic value was analyzed using Kaplan-Meier curves and univariate and multivariate Cox regression analyses. Potential reasons for the upregulation of DPH2 and DPH3 (DPH2,3) expression in HCC were analyzed using multiple databases. Additionally, this study also explored the potential biological functions of DPH2,3 in HCC via gene sets enrichment analysis (GSEA). Correlation analysis of DPH2,3 expression with immune-related genes and immune checkpoints was performed using Spearman's correlation analysis, and single-sample GSEA was used to assess the distribution of tumor-infiltrating immune cell types. Results: DPH1,7 expression was downregulated in tumor tissues while DPH2,3,5,6 expression was upregulated and showed a similar expression pattern in HCC. The results of the ROC analysis suggested that DPHs had valuable diagnostic properties in HCC. Kaplan-Meier analysis demonstrated that DPH2,3,7 had prognostic predictive value in HCC. Furthermore, univariate and multivariate Cox regression suggested that DPH2,3 was an independent predictive factor for HCC. GSEA analysis revealed that DPH2,3 might be tightly associated with Pathways in cancer, cell cycles, Fc gamma R mediated phagocytosis, etc. Additionally, DPH2,3 expression and numerous immune-related genes showed a positive connection, including chemokines receptor genes, immunosuppressive genes, chemokines genes, human leukocyte antigen (HLA) genes, and immunostimulatory genes. Further analysis of the association between 24 immune infiltrating cells and DPH2,3 revealed the greatest negative correlation between natural killer (NK) cells and Th17 cells, but the greatest positive correlation with Th2 cells. Conclusions: DPHs significantly influence the development and progression of HCC. DPH2,3 has significant diagnostic and prognostic potential and may be a promising target for immunotherapy.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

DPH1 and DPH2 variants that confer susceptibility to diphthamide deficiency syndrome in human cells and yeast models.

The autosomal-recessive diphthamide deficiency syndrome presents as intellectual disability with developmental abnormalities, seizures, craniofacial and additional morphological phenotypes. It is caused by reduced activity of proteins that synthesize diphthamide on human translation elongation factor 2. Diphthamide synthesis requires seven proteins (DPH1-DPH7), with clinical deficiency described for DPH1, DPH2 and DPH5. A limited set of variant alleles from syndromic patients has been functionally analyzed, but databases (gnomAD) list additional so far uncharacterized variants in human DPH1 and DPH2. Because DPH enzymes are conserved among eukaryotes, their functionality can be assessed in yeast and mammalian cells. Our experimental assessment of known and uncharacterized DPH1 and DPH2 missense alleles showed that six variants are tolerated despite inter-species conservation. Ten additional human DPH1 (G113R, A114T, H132P, H132R, S136R, C137F, L138P, Y152C, S221P, H240R) and two DPH2 (H105P, C341Y) variants showed reduced functionality and hence are deficiency-susceptibility alleles. Some variants locate close to the active enzyme center and may affect catalysis, while others may impact on enzyme activation. In sum, our study has identified functionally compromised alleles of DPH1 and DPH2 genes that likely cause diphthamide deficiency syndrome.

Yeast gene KTI13 (alias DPH8) operates in the initiation step of diphthamide synthesis on elongation factor 2.

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four biosynthetic steps encoded by the DPH1-DPH7 network and an ill-defined KTI13 function. On further examining the latter gene in yeast, we found that kti13Δ null-mutants maintain unmodified EF2 able to escape ADP-ribosylation by DT and to survive EF2 inhibition by sordarin, a diphthamide-dependent antifungal. Consistently, mass spectrometry shows kti13Δ cells are blocked in proper formation of amino-carboxyl-propyl-EF2, the first diphthamide pathway intermediate. Thus, apart from their common function in tRNA modification, both Kti11/Dph3 and Kti13 share roles in the initiation step of EF2 modification. We suggest an alias KTI13/DPH8 nomenclature indicating dual-functionality analogous to KTI11/DPH3.

DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1•Dph2 for Diphthamide Synthesis on EF2.

In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1•Dph2 from Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for the loss of diphthamide reveal that the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1•Dph2 from classical radical SAM enzymes, which generate canonical 5'-deoxyadenosyl (dAdo) radicals.

CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication.

During translation of the genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus in the COVID-19 pandemic, host ribosomes undergo programmed ribosomal frameshifting (PRF) at a conserved structural element. Although PRF is essential for coronavirus replication, host factors that regulate this process have not yet been identified. Here we perform genome-wide CRISPR-Cas9 knockout screens to identify regulators of SARS-CoV-2 PRF. These screens reveal that loss of ribosome recycling factors markedly decreases frameshifting efficiency and impairs SARS-CoV-2 viral replication. Mutational studies support a model wherein efficient removal of ribosomal subunits at the ORF1a stop codon is required for frameshifting of trailing ribosomes. This dependency upon ribosome recycling is not observed with other non-pathogenic human betacoronaviruses and is likely due to the unique position of the ORF1a stop codon in the SARS clade of coronaviruses. These findings therefore uncover host factors that support efficient SARS-CoV-2 translation and replication.

Targeting CD123 in AML.

An ideal cell surface target for therapy in leukemia would be: tumor-specific (not expressed on normal cells) or at least enriched on tumor cells, necessary for tumor but not for normal cell survival, internalized efficiently (if the surface-targeted agent is conjugated to chemotherapy or a toxin molecule), and recycled rapidly to the cell surface. While a single target that meets all of these criteria has not yet been discovered in AML, CD123 has emerged as an attractive candidate.1 The first-in-class CD123-targeting agent, tagraxofusp-erzs (SL-401) was approved in 2018 for patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN)2 and is currently in trials for several other hematologic malignancies, including AML. Several other CD123-targeted drugs are in development.

Methods for Studying the Radical SAM Enzymes in Diphthamide Biosynthesis.

Diphthamide is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. Biosynthesis of diphthamide was proposed to involve four steps. The first step is a CC bond forming reaction catalyzed by unique radical S-adenosylmethionine (SAM) enzymes. Classical radical SAM enzymes use SAM and [4Fe-4S] clusters to generate a 5'-deoxyadenynal radical and catalyze numerous reactions. Radical SAM enzymes in diphthamide biosynthesis cleave a different CS bond in SAM to generate a 3-amino-3-carboxypropyl radical and modify a histidine residue of substrate protein EF2. Here, we describe our investigations on these unique radical SAM enzymes, including the preparation, characterization, and activity assays we have developed.

Structural Insights into the Role of Diphthamide on Elongation Factor 2 in mRNA Reading-Frame Maintenance.

One of the most critical steps of protein biosynthesis is the coupled movement of mRNA, which encodes genetic information, with tRNAs on the ribosome. In eukaryotes, this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-electron microscopy structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights into the role of diphthamide in the mechanism of translation fidelity in eukaryotes.

Roles of Elongator Dependent tRNA Modification Pathways in Neurodegeneration and Cancer.

Transfer RNA (tRNA) is subject to a multitude of posttranscriptional modifications which can profoundly impact its functionality as the essential adaptor molecule in messenger RNA (mRNA) translation. Therefore, dynamic regulation of tRNA modification in response to environmental changes can tune the efficiency of gene expression in concert with the emerging epitranscriptomic mRNA regulators. Several of the tRNA modifications are required to prevent human diseases and are particularly important for proper development and generation of neurons. In addition to the positive role of different tRNA modifications in prevention of neurodegeneration, certain cancer types upregulate tRNA modification genes to sustain cancer cell gene expression and metastasis. Multiple associations of defects in genes encoding subunits of the tRNA modifier complex Elongator with human disease highlight the importance of proper anticodon wobble uridine modifications (xm5U34) for health. Elongator functionality requires communication with accessory proteins and dynamic phosphorylation, providing regulatory control of its function. Here, we summarized recent insights into molecular functions of the complex and the role of Elongator dependent tRNA modification in human disease.

Nutritional Control of Stem Cell Division through S-Adenosylmethionine in Drosophila Intestine.

The intestine has direct contact with nutritional information. The mechanisms by which particular dietary molecules affect intestinal homeostasis are not fully understood. In this study, we identified S-adenosylmethionine (SAM), a universal methyl donor synthesized from dietary methionine, as a critical molecule that regulates stem cell division in Drosophila midgut. Depletion of either dietary methionine or SAM synthesis reduces division rate of intestinal stem cells. Genetic screening for putative SAM-dependent methyltransferases has identified protein synthesis as a regulator of the stem cells, partially through a unique diphthamide modification on eukaryotic elongation factor 2. In contrast, SAM in nutrient-absorptive enterocytes controls the interleukin-6-like protein Unpaired 3, which is required for rapid division of the stem cells after refeeding. Our study sheds light upon a link between diet and intestinal homeostasis and highlights the key metabolite SAM as a mediator of cell-type-specific starvation response.

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.