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Futoshi Sekiguchi, Jafar Nasiri, Maryam Sedghi, Mansoor Salehi, Majid Hosseinzadeh, Nobuhiko Okamoto, Takeshi Mizuguchi, Mitsuko Nakashima, Satoko Miyatake, Atsushi Takata, Noriko Miyake, Naomichi Matsumoto
Biallelic mutations of the gene encoding diphthamide biosynthesis 1 (DPH1, NM_001383.3) cause developmental delay, dysmorphic features, sparse hair, and short stature (MIM *603527). Only two missense DPH1 mutations have been reported to date. Here, we describe a consanguineous family with two siblings both showing developmental delay, agenesis of the corpus callosum, dysmorphic facial features, sparse hair, brachycephaly, and short stature. By wholeexome sequencing, a homozygous frameshift mutation in DPH1 (c...
February 6, 2018: Journal of Human Genetics
Ziyan Wang, Baozhen Sun, Fei Zhu
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...
January 30, 2018: Fish & Shellfish Immunology
Junya Nakajima, Shingo Oana, Tomohiro Sakaguchi, Mitsuko Nakashima, Hironao Numabe, Hisashi Kawashima, Naomichi Matsumoto, Noriko Miyake
The diphthamide biosynthesis 1 (DPH1) gene encodes one of the essential components of the enzyme catalyzing the first step of diphthamide formation on eukaryotic elongation factor 2 (EEF2). Diphthamide is the posttranslationally modified histidine residue on EEF2 that promotes protein chain elongation in the ribosome. DPH1 defects result in a failure of protein synthesis involving EEF2, leading to growth defects, embryonic lethality, and cell death. In humans, DPH1 mutations cause developmental delay with a short stature, dysmorphic features, and sparse hair, and are inherited in an autosomal recessive manner (MIM#616901)...
January 23, 2018: Journal of Human Genetics
Minghui Liu, Kai Yin, Xu Guo, Huijin Feng, Min Yuan, Yanqing Liu, Jianguo Zhang, Baoliang Guo, Chen Wang, Guangxin Zhou, Zhen Zhou, Chen-Yu Zhang, Xi Chen
BACKGROUND/AIMS: This study focused on the oncogenic role of Diphthamide biosynthesis 1 (DPH1) in colorectal cancer (CRC) cells. METHODS: The expression of DPH1 was determined by quantitative RT-PCR analysis and western blotting in CRC tissues. The role of DPH1 in CRC cells was investigated via cell viability and invasion assays under the condition of DPH1 silencing or overexpression. Bioinformatics analysis and luciferase reporter analysis were used to identify the upstream microRNA which might regulate DPH1...
November 17, 2017: Cellular Physiology and Biochemistry
Tobias Killian, Steffen Dickopf, Alexander K Haas, Claudia Kirstenpfad, Klaus Mayer, Ulrich Brinkmann
We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation...
November 13, 2017: Scientific Reports
Hikari Hayashi, Riku Nagai, Taisho Abe, Miki Wada, Koichi Ito, Nono Takeuchi-Tomita
The stress-related protein Stm1 interacts with ribosomes, and is implicated in repressing translation. Stm1 was previously studied both in vivo and in vitro by cell-free translation systems using crude yeast lysates, but its precise functional mechanism remains obscure. Using an in vitro reconstituted translation system, we now show that Stm1 severely inhibits translation through its N-terminal region, aa 1 to 107, and this inhibition is antagonized by eEF3. We found that Stm1 stabilizes eEF2 on the 80S ribosome in the GTP-bound form, independently of eEF2's diphthamide modification, a conserved post-translational modification at the tip of domain IV...
October 23, 2017: Journal of Biochemistry
Desirée Villahermosa, Oliver Fleck
Efficient protein synthesis in eukaryotes requires diphthamide modification of translation elongation factor eEF2 and wobble uridine modifications of tRNAs. In higher eukaryotes, these processes are important for preventing neurological and developmental defects and cancer. In this study, we used Schizosaccharomyces pombe as a model to analyse mutants defective in eEF2 modification (dph1Δ), in tRNA modifications (elp3Δ), or both (dph3Δ) for sensitivity to cytotoxic agents and thermal stress. The dph3Δ and elp3Δ mutants were sensitive to a range of drugs and had growth defects at low temperature...
August 3, 2017: Scientific Reports
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...
December 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 26, 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...
May 9, 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
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