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Desirée Villahermosa, Karen Knapp, Oliver Fleck
Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate...
May 29, 2017: Current Genetics
Min Dong, Masaki Horitani, Boris Dzikovski, Jack H Freed, Steven E Ealick, Brian M Hoffman, Hening Lin
S-Adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe-4S] cluster to cleave homolytically the C5',adenosine-S bond of SAM to generate a 5'-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the Cγ,Met-S bond of methionine to generate a 3-amino-3-carboxylpropyl radical. We here show radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM...
April 13, 2017: Journal of the American Chemical Society
Klaus Mayer, Anna Schröder, Jerome Schnitger, Sebastian Stahl, Ulrich Brinkmann
The diphthamide on eukaryotic translation elongation factor 2 (eEF2) is the target of ADPribosylating toxins and -derivatives that serve as payloads in targeted tumor therapy. Diphthamide is generated by seven DPH proteins; cells deficient in these (DPHko) lack diphthamide and are toxin-resistant. We have established assays to address the functionality of DPH1 (OVCA1) and DPH5 variants listed in dbSNP and cosmic databases: plasmids encoding wildtype and mutant DPHs were transfected into DPHko cells. Supplementation of DPH1 and DPH5 restores diphthamide synthesis and toxin sensitivity in DPH1ko and DPH5ko cells, respectively...
February 24, 2017: Toxins
Zhewang Lin, Min Dong, Yugang Zhang, Eunyoung Alisa Lee, Hening Lin
Diphthamide and the tRNA wobble uridine modifications both require diphthamide biosynthesis 3 (Dph3) protein as an electron donor for the iron-sulfur clusters in their biosynthetic enzymes. Here, using a proteomic approach, we identified Saccharomyces cerevisiae cytochrome b5 reductase (Cbr1) as a NADH-dependent reductase for Dph3. The NADH- and Cbr1-dependent reduction of Dph3 may provide a regulatory linkage between cellular metabolic state and protein translation.
December 2016: Nature Chemical Biology
Jean-Marc Billod, Patricia Saenz-Mendez, Anders Blomberg, Leif A Eriksson
The eukaryotic translation Elongation Factor 2 (eEF2) is an essential enzyme in protein synthesis. eEF2 contains a unique modification of a histidine (His699 in yeast; HIS) into diphthamide (DTA), obtained via 3-amino-3-carboxypropyl (ACP) and diphthine (DTI) intermediates in the biosynthetic pathway. This essential and unique modification is also vulnerable, in that it can be efficiently targeted by NAD(+)-dependent ADP-ribosylase toxins, such as diphtheria toxin (DT). However, none of the intermediates in the biosynthesis path is equally vulnerable against the toxins...
September 26, 2016: Journal of Chemical Information and Modeling
Min Dong, Masaki Horitani, Boris Dzikovski, Maria-Eirini Pandelia, Carsten Krebs, Jack H Freed, Brian M Hoffman, Hening Lin
Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster...
August 10, 2016: Journal of the American Chemical Society
Priyanka D Abeyrathne, Cha San Koh, Timothy Grant, Nikolaus Grigorieff, Andrei A Korostelev
Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel...
2016: ELife
Jason Murray, Christos G Savva, Byung-Sik Shin, Thomas E Dever, V Ramakrishnan, Israel S Fernández
Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2)...
May 9, 2016: ELife
Gabriele Picco, Consalvo Petti, Livio Trusolino, Andrea Bertotti, Enzo Medico
We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo...
2015: Scientific Reports
Evgeniya Denisova, Barbara Heidenreich, Eduardo Nagore, P Sivaramakrishna Rachakonda, Ismail Hosen, Ivana Akrap, Víctor Traves, Zaida García-Casado, José Antonio López-Guerrero, Celia Requena, Onofre Sanmartin, Carlos Serra-Guillén, Beatriz Llombart, Carlos Guillén, Jose Ferrando, Enrique Gimeno, Alfred Nordheim, Kari Hemminki, Rajiv Kumar
Recent reports suggested frequent occurrence of cancer associated somatic mutations within regulatory elements of the genome. Based on initial exome sequencing of 21 melanomas, we report frequent somatic mutations in skin cancers in a bidirectional promoter of diphthamide biosynthesis 3 (DPH3) and oxidoreductase NAD-binding domain containing 1 (OXNAD1) genes. The UV-signature mutations occurred at sites adjacent and within a binding motif for E-twenty six/ternary complex factors (Ets/TCF), at -8 and -9 bp from DPH3 transcription start site...
November 3, 2015: Oncotarget
Sebastian Stahl, Ana Rita da Silva Mateus Seidl, Axel Ducret, Sabine Kux van Geijtenbeek, Sven Michel, Tomas Racek, Fabian Birzele, Alexander K Haas, Ruediger Rueger, Michael Gerg, Gerhard Niederfellner, Ira Pastan, Ulrich Brinkmann
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...
August 25, 2015: Proceedings of the National Academy of Sciences of the United States of America
Miguel R Lugo, A Rod Merrill
The cholix toxin gene (chxA) was first identified in V. cholerae strains in 2007, and the protein was identified by bioinformatics analysis in 2008. It was identified as the third member of the diphtheria toxin group of mono-ADP-ribosyltransferase toxins along with P. aeruginosa exotoxin A and C. diphtheriae diphtheria toxin. Our group determined the structure of the full-length, three-domain cholix toxin at 2.1 Å and its C-terminal catalytic domain (cholixc) at 1.25 Å resolution. We showed that cholix toxin is specific for elongation factor 2 (diphthamide residue), similar to exotoxin A and diphtheria toxin...
August 2015: Toxins
Matteo Pasetto, Antonella Antignani, Pinar Ormanoglu, Eugen Buehler, Rajarshi Guha, Ira Pastan, Scott E Martin, David J FitzGerald
Immunotoxins (antibody-toxin fusion proteins) target surface antigens on cancer cells and kill these cells via toxin-mediated inhibition of protein synthesis. To identify genes controlling this process, an RNAi whole-genome screen (∼ 22,000 genes at three siRNAs per gene) was conducted via monitoring the cytotoxicity of the mesothelin-directed immunotoxin SS1P. SS1P, a Pseudomonas exotoxin-based immunotoxin, was chosen because it is now in clinical trials and has produced objective tumor regressions in patients...
March 10, 2015: Proceedings of the National Academy of Sciences of the United States of America
Olga Kolaj-Robin, Alastair G McEwen, Jean Cavarelli, Bertrand Séraphin
UNLABELLED: Modification of wobble uridines of many eukaryotic tRNAs requires the Elongator complex, a highly conserved six-subunit eukaryotic protein assembly, as well as the Killer toxin-insensitive (Kti) proteins 11-14. Kti11 was additionally shown to be implicated in the biosynthesis of diphthamide, a post-translationally modified histidine of translation elongation factor 2. Recent data indicate that iron-bearing Kti11 functions as an electron donor to the [4Fe-4S] cluster of radical S-Adenosylmethionine enzymes, triggering the subsequent radical reaction...
March 2015: FEBS Journal
Sebastian Glatt, Rene Zabel, Ivana Vonkova, Amit Kumar, Daili J Netz, Antonio J Pierik, Vladimir Rybin, Roland Lill, Anne-Claude Gavin, Jochen Balbach, Karin D Breunig, Christoph W Müller
The small, highly conserved Kti11 alias Dph3 protein encoded by the Kluyveromyces lactis killer toxin insensitive gene KTI11/DPH3 is involved in the diphthamide modification of eukaryotic elongation factor 2 and, together with Kti13, in Elongator-dependent tRNA wobble base modifications, thereby affecting the speed and accuracy of protein biosynthesis through two distinct mechanisms. We have solved the crystal structures of Saccharomyces cerevisiae Kti13 and the Kti11/Kti13 heterodimer at 2.4 and 2.9 Å resolution, respectively, and validated interacting residues through mutational analysis in vitro and in vivo...
January 6, 2015: Structure
Vicky M-H Sung, Chia-Lun Tsai
BACKGROUND: Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase. RESULTS: Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket...
2014: BMC Biochemistry
Raffael Schaffrath, Wael Abdel-Fattah, Roland Klassen, Michael J R Stark
Diphthamide 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...
December 2014: Molecular Microbiology
Yunki Y Yau, Rupert W L Leong, Sean Shin, Sonia Bustamante, Russell Pickford, Leila Hejazi, Beth Campbell, Valerie C Wasinger
INTRODUCTION: Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) characterized by variable phenotypes. Metabolites are signatures of biochemical activity that can reveal unknown pathogenic pathways. We employed untargeted mass spectrometry (MS) based metabolomics to identify novel inflammatory mechanisms in IBD and a targeted assay to quantify metabolites of the auto-immunomodulating kynurenine pathway (KP) in IBDs and health. MATERIALS AND METHODS: Metabolome analysis of CD, UC, and control plasmas was performed on a Liquid Chromatography (LC)-MS/MS system...
September 2014: Discovery Medicine
Zhewang Lin, Xiaoyang Su, Wei Chen, Bo Ci, Sheng Zhang, Hening Lin
Present on archaeal and eukaryotic translation elongation factor 2, diphthamide represents one of the most intriguing post-translational modifications on proteins. The biosynthesis of diphthamide was proposed to occur in three steps requiring seven proteins, Dph1-7, in eukaryotes. The functional assignments of Dph1-5 in the first and second step have been well established. Recent studies suggest that Dph6 (yeast YLR143W or human ATPBD4) and Dph7 (yeast YBR246W or human WDR85) are involved in the last amidation step, with Dph6 being the actual diphthamide synthetase catalyzing the ATP-dependent amidation reaction...
April 30, 2014: Journal of the American Chemical Society
Min Dong, Xiaoyang Su, Boris Dzikovski, Emily E Dando, Xuling Zhu, Jintang Du, Jack H Freed, Hening Lin
Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from S-adenosyl-l-methionine (SAM) to the histidine residue of EF2, forming a C-C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1-Dph4. However, the exact molecular functions for the four proteins are unknown...
February 5, 2014: Journal of the American Chemical Society
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