1,159 results on '"Peptide Elongation Factor Tu"'
Search Results
2. Nuclear encoded elongation factor EF-Tu is required for chloroplast development in rice grown under low-temperature conditions
- Author
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Liang Cai, Zongkai Liu, Long Cai, Xiaofeng Yan, Yuan Hu, Benyuan Hao, Zhuang Xu, Yunlu Tian, Xi Liu, Linglong Liu, Ling Jiang, Shirong Zhou, and Jianmin Wan
- Subjects
Chloroplasts ,Temperature ,Genetics ,Oryza ,Peptide Elongation Factor Tu ,Peptide Elongation Factors ,Molecular Biology - Published
- 2022
3. Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors
- Author
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Jing Wang, Yuka Yashiro, Yuriko Sakaguchi, Tsutomu Suzuki, and Kozo Tomita
- Subjects
RNA, Transfer ,Enterohemorrhagic Escherichia coli ,Escherichia coli Proteins ,Genetics ,Membrane Proteins ,Guanosine Triphosphate ,Peptide Elongation Factor Tu ,RNA, Transfer, Amino Acyl ,Growth Inhibitors - Abstract
Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA–CT) into neighboring bacteria. The CdiA–CT of enterohemorrhagic Escherichia coli EC869 (CdiA–CTEC869) cleaves the 3′-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA–CTEC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA–CTEC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA–CTEC869 to the same extent in the presence of Tu/Ts, and the CdiA–CTEC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA–CTEC869 interacts with domain II of Tu, thereby preventing the 3′-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA–CTEC869:Tu structure suggests that the 3′-portion of tRNA relocates into the CdiA–CTEC869 active site, located on the opposite side to the CdiA–CTEC869 :Tu interface, for tRNA cleavage. Thus, CdiA–CTEC869 is recruited to Tu:GTP:Ts, and CdiA–CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA–CTEC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA–CTEC869.
- Published
- 2022
4. A soybean EF-Tu family protein GmEF8, an interactor of GmCBL1, enhances drought and heat tolerance in transgenic Arabidopsis and soybean
- Author
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Hui-Yuan, Zhang, Ze-Hao, Hou, Yan, Zhang, Zhi-Yong, Li, Jun, Chen, Yong-Bin, Zhou, Ming, Chen, Jin-Dong, Fu, You-Zhi, Ma, Hui, Zhang, and Zhao-Shi, Xu
- Subjects
Thermotolerance ,Arabidopsis ,General Medicine ,Peptide Elongation Factor Tu ,Plants, Genetically Modified ,Biochemistry ,Droughts ,Gene Expression Regulation, Plant ,Stress, Physiological ,Structural Biology ,Soybean Proteins ,Soybeans ,Molecular Biology ,Plant Proteins - Abstract
A soybean elongation factor Tu family (EF-Tu) protein, GmEF8, was determined to interact with GmCBL1, and GmEF8 expression was found to be induced by various abiotic stresses such as drought and heat. An ortholog of GmEF8 was identified in Arabidopsis, a T-DNA knockout line for which exhibited hypersensitivity to drought and heat stresses. Complementation with GmEF8 rescued the sensitivity of the Arabidopsis mutant to drought and heat stresses, and GmEF8 overexpression conferred drought and heat tolerance to transgenic Arabidopsis plants. In soybean, plants with GmEF8-overexpressing hairy roots (OE-GmEF8) exhibited enhanced drought and heat tolerance and had higher proline levels compared to plants with RNAi GmEF8-knockdown hairy roots (MR-GmEF8) and control hairy roots (EV). A number of drought-responsive genes, such as GmRD22 and GmP5CS, were induced in the OE-GmEF8 line compared to MR-GmEF8 and EV under normal growth conditions. These results suggest that GmEF8 has a positive role in regulating drought and heat stresses in Arabidopsis and soybean. This study reveals a potential role of the soybean GmEF8 gene in response to abiotic stresses, providing a foundation for further investigation into the complexities of stress signal transduction pathways.
- Published
- 2022
5. FUNDC1 protects against doxorubicin-induced cardiomyocyte PANoptosis through stabilizing mtDNA via interaction with TUFM
- Author
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Yaguang Bi, Haixia Xu, Xiang Wang, Hong Zhu, Junbo Ge, Jun Ren, and Yingmei Zhang
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Cancer Research ,Immunology ,Membrane Proteins ,Apoptosis ,Cell Biology ,Peptide Elongation Factor Tu ,DNA, Mitochondrial ,Cardiotoxicity ,Mitochondria ,Mitochondrial Proteins ,Mice ,Cellular and Molecular Neuroscience ,Doxorubicin ,Animals ,Myocytes, Cardiac - Abstract
Doxorubicin (DOX) is an effective anthracycline chemotherapeutic anticancer drug with its life-threatening cardiotoxicity severely limiting its clinical application. Mitochondrial damage-induced cardiomyocyte death is considered an essential cue for DOX cardiotoxicity. FUN14 domain containing 1 (FUNDC1) is a mitochondrial membrane protein participating in the regulation of mitochondrial integrity in multiple diseases although its role in DOX cardiomyopathy remains elusive. Here, we examined whether PANoptosis, a novel type of programmed cell death closely associated with mitochondrial damage, was involved in DOX-induced heart injury, and FUNDC1-mediated regulation of cardiomyocyte PANoptosis, if any. FUNDC1 was downregulated in heart tissues in patients with dilated cardiomyopathy (DCM) and DOX-challenged mice. FUNDC1 deficiency aggravated DOX-induced cardiac dysfunction, mitochondrial injury, and cardiomyocyte PANoptosis. Further examination revealed that FUNDC1 countered cytoplasmic release of mitochondrial DNA (mtDNA) and activation of PANoptosome through interaction with mitochondrial Tu translation elongation factor (TUFM), a key factor in the translational expression and repair of mitochondrial DNA, via its 96–133 amino acid domain. TUFM intervention reversed FUNDC1-elicited protection against DOX-induced mtDNA cytosolic release and cardiomyocyte PANoptosis. Our findings shed light toward a beneficial role of FUNDC1 in DOX cardiotoxicity and cardiomyocyte PANoptosis, thus offering therapeutic promises in DOX-induced cardiotoxicity.
- Published
- 2022
6. A natural small molecule induces MAPT clearance via mTOR-independent autophagy
- Author
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Hui Yun Hwang, Dasol Kim, and Ho Jeong Kwon
- Subjects
0301 basic medicine ,Tau protein ,Biophysics ,tau Proteins ,Peptide Elongation Factor Tu ,Biochemistry ,Mitochondrial Proteins ,03 medical and health sciences ,0302 clinical medicine ,Autophagy ,medicine ,Humans ,Kaempferols ,Cytotoxicity ,Molecular Biology ,PI3K/AKT/mTOR pathway ,biology ,Chemistry ,TOR Serine-Threonine Kinases ,Cell Biology ,medicine.disease ,Small molecule ,Cell biology ,HEK293 Cells ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein ,TFEB ,Tauopathy ,Flux (metabolism) - Abstract
Autophagy, the process of lysosomal degradation of biological materials within cells, is often halted abnormally in proteopathies, such as tauopathy and amyloidopathy. Thus, autophagy regulators that rescue dysregulated autophagy have great potential to treat proteopathies. We previously reported that the natural small molecule kaempferide (Kaem) induces autophagy without perturbing mTOR signaling. Here, we report that Kaem promotes lysosomal degradation of microtubule-associated protein tau (MAPT) in inducible MAPT cells. Kaem enhanced autophagy flux by mitigating microtubule-associated protein 1 light chain 3 (LC3) accumulation when MAPT expression was induced. Kaem also promoted activation of transcription factor EB (TFEB) without inhibiting mTOR and without mTOR inhibition-mediated cytotoxicity. In addition, Kaem-induced MAPT degradation was abolished in the absence of mitochondrial elongation factor Tu (TUFM), which was previously shown to be a direct binding partner of Kaem. Collectively, these results demonstrate that Kaem could be a potential therapeutic for tauopathy and reveal that TUFM can be a drug target for autophagy-driven disorders.
- Published
- 2021
7. Structural insights of the elongation factor EF-Tu complexes in protein translation of Mycobacterium tuberculosis
- Author
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Bowen Zhan, Yanqing Gao, Wenqing Gao, Ye Li, Zhengyang Li, Qi Qi, Xin Lan, Hongbo Shen, Jianhua Gan, Guoping Zhao, and Jixi Li
- Subjects
X-Ray Diffraction ,Protein Biosynthesis ,Scattering, Small Angle ,BCG Vaccine ,Escherichia coli ,Medicine (miscellaneous) ,Mycobacterium tuberculosis ,Peptide Elongation Factor Tu ,Peptide Elongation Factors ,General Agricultural and Biological Sciences ,Guanosine Diphosphate ,General Biochemistry, Genetics and Molecular Biology - Abstract
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second-deadliest infectious disease worldwide. Emerging evidence shows that the elongation factor EF-Tu could be an excellent target for treating Mtb infection. Here, we report the crystal structures of Mtb EF-Tu•EF-Ts and EF-Tu•GDP complexes, showing the molecular basis of EF-Tu’s representative recycling and inactive forms in protein translation. Mtb EF-Tu binds with EF-Ts at a 1:1 ratio in solution and crystal packing. Mutation and SAXS analysis show that EF-Ts residues Arg13, Asn82, and His149 are indispensable for the EF-Tu/EF-Ts complex formation. The GDP binding pocket of EF-Tu dramatically changes conformations upon binding with EF-Ts, sharing a similar GDP-exchange mechanism in E. coli and T. ther. Also, the FDA-approved drug Osimertinib inhibits the growth of M. smegmatis, H37Ra, and M. bovis BCG strains by directly binding with EF-Tu. Thus, our work reveals the structural basis of Mtb EF-Tu in polypeptide synthesis and may provide a promising candidate for TB treatment.
- Published
- 2022
8. Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation
- Author
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Yoshihiko Iwane, Hiroyuki Kimura, Hiroaki Suga, and Takayuki Katoh
- Subjects
AcademicSubjects/SCI00010 ,Stereochemistry ,Oligonucleotides ,NAR Breakthrough Article ,Peptide ,Peptide Elongation Factor Tu ,RNA, Transfer, Amino Acyl ,Biology ,010402 general chemistry ,Methylation ,01 natural sciences ,Ribosome ,03 medical and health sciences ,Escherichia coli ,Genetics ,Amino Acids ,Thermus ,Base Pairing ,Peptide sequence ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,Base Sequence ,Translation (biology) ,0104 chemical sciences ,Amino acid ,Elongation factor ,Kinetics ,chemistry ,Protein Biosynthesis ,Transfer RNA ,Nucleic Acid Conformation ,Thermodynamics ,Peptidomimetics ,Genetic Engineering ,EF-Tu ,Protein Binding - Abstract
In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (MeAAs), is less efficient, especially when MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of MeAAs, achieving the incorporation of nine distinct MeAAs into both linear and thioether-macrocyclic peptide scaffolds.
- Published
- 2021
9. ProteoVision: web server for advanced visualization of ribosomal proteins
- Author
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Maria Ahmad, Vasanta L Chivukula, Caeden D. Meade, Aparna Maddala, Holly M McCann, Aakash Sharma, Anton S. Petrov, Burak Gulen, Claudia Alvarez-Carreño, Chad R. Bernier, Loren Dean Williams, and Petar I. Penev
- Subjects
Models, Molecular ,Ribosomal Proteins ,Internet ,Web server ,Multiple sequence alignment ,Information retrieval ,AcademicSubjects/SCI00010 ,Protein Conformation ,Protein domain ,Sequence alignment ,Peptide Elongation Factor Tu ,Biology ,computer.software_genre ,Visualization ,Acetolactate Synthase ,Upload ,Protein structure ,Bacterial Proteins ,Scripting language ,Web Server Issue ,Genetics ,Sequence Alignment ,computer ,Software - Abstract
ProteoVision is a web server designed to explore protein structure and evolution through simultaneous visualization of multiple sequence alignments, topology diagrams and 3D structures. Starting with a multiple sequence alignment, ProteoVision computes conservation scores and a variety of physicochemical properties and simultaneously maps and visualizes alignments and other data on multiple levels of representation. The web server calculates and displays frequencies of amino acids. ProteoVision is optimized for ribosomal proteins but is applicable to analysis of any protein. ProteoVision handles internally generated and user uploaded alignments and connects them with a selected structure, found in the PDB or uploaded by the user. It can generate de novo topology diagrams from three-dimensional structures. All displayed data is interactive and can be saved in various formats as publication quality images or external datasets or PyMol Scripts. ProteoVision enables detailed study of protein fragments defined by Evolutionary Classification of protein Domains (ECOD) classification. ProteoVision is available at http://proteovision.chemistry.gatech.edu/., Graphical Abstract Graphical AbstractProteoVision is a webserver designed to visualize phylogenetic, structural, and physicochemical properties of proteins by integration of four mutually synchronized applets that visually represent map data for each amino-acid simultaneously on levels of a (i) multiple sequence alignment; (ii) secondary structure; (iii) 3D structure and (iv) frequency box plot graph.
- Published
- 2021
10. Transfer RNA Synthesis-Coupled Translation and DNA Replication in a Reconstituted Transcription/Translation System
- Author
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Ryota Miyachi, Yoshihiro Shimizu, and Norikazu Ichihashi
- Subjects
DNA Replication ,RNA, Transfer ,Protein Biosynthesis ,Biomedical Engineering ,Escherichia coli ,General Medicine ,Peptide Elongation Factor Tu ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Ribosomes - Abstract
Transfer RNAs (tRNAs) are key molecules involved in translation. In vitro synthesis of tRNAs and their coupled translation are important challenges in the construction of a self-regenerative molecular system. Here, we first purified EF-Tu and ribosome components in a reconstituted translation system of
- Published
- 2022
11. Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus
- Author
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Delphine Patin, Mélanie Etheve-Quelquejeu, Camélia Kitoun, Michel Arthur, Matthieu Fonvielle, Laura Iannazzo, Lauriane Rietmeyer, Chloé Le Fournis, Dominique Mengin-Lecreulx, Nicolas Fix-Boulier, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Gestionnaire, Hal Sorbonne Université, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), École Pratique des Hautes Études (EPHE), and ANR-17-CE07-0041,SyntRNA,Chemo-biologie pour la characterization structurale et fonctionnelle de complexes ARN-protéines(2017)
- Subjects
Models, Molecular ,Staphylococcus aureus ,Lineage (genetic) ,GTP' ,AcademicSubjects/SCI00010 ,[SDV]Life Sciences [q-bio] ,Peptidoglycan ,Peptide Elongation Factor Tu ,Biology ,medicine.disease_cause ,Binding, Competitive ,environment and public health ,Ribosome ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Chemical Biology and Nucleic Acid Chemistry ,Cell Wall ,Genetics ,medicine ,030304 developmental biology ,0303 health sciences ,Base Sequence ,030302 biochemistry & molecular biology ,RNA, Transfer, Gly ,[SDV] Life Sciences [q-bio] ,RNA, Bacterial ,Biochemistry ,chemistry ,Transfer RNA ,Glycine ,Nucleic Acid Conformation ,Guanosine Triphosphate ,Protein Binding - Abstract
The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNAGly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNAGly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these ‘non-proteogenic’ tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of ‘proteogenic’ aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNAGly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.
- Published
- 2020
12. Molecular evolutionary and 3D protein structural analyses of Lactobacillus fermentum elongation factor Tu, a novel brain health promoting factor
- Author
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Yen-Wenn Liu, Wai Yee Low, Min-Tze Liong, Ying-Chieh Tsai, Jia Sin Ong, and Sy Bing Choi
- Subjects
0106 biological sciences ,Genetics ,Limosilactobacillus fermentum ,0303 health sciences ,Protein Conformation ,Lactobacillus fermentum ,Pseudogene ,Circular bacterial chromosome ,Brain ,Proteins ,Chromosome ,Peptide Elongation Factor Tu ,Biology ,biology.organism_classification ,01 natural sciences ,Genome ,Evolution, Molecular ,03 medical and health sciences ,Humans ,Binding site ,Gene ,EF-Tu ,030304 developmental biology ,010606 plant biology & botany - Abstract
The role of microbiota in gut-brain communication has led to the development of probiotics promoting brain health. Here we report a genomic study of a Lactobacillus fermentum PS150 and its patented bioactive protein, elongation factor Tu (EF-Tu), which is associated with cognitive improvement in rats. The L. fermentum PS150 circular chromosome is 2,238,401 bp and it consists of 2281 genes. Chromosome comparisons with other L. fermentum strains highlighted a cluster of glycosyltransferases as potential candidate probiotic factors besides EF-Tu. Molecular evolutionary analyses on EF-Tu genes (tuf) in 235 bacteria species revealed one to three copies of the gene per genome. Seven tuf pseudogenes were found and three species only possessed pseudogenes, which is an unprecedented finding. Protein variability analysis of EF-Tu showed five highly variable residues (40 K, 41G, 42 L, 44 K, and 46E) on the protein surface, which warrant further investigation regarding their potential roles as binding sites.
- Published
- 2020
13. Extraintestinal pathogenic
- Author
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Yu, Sun, Xuhang, Wang, Jin, Li, Feng, Xue, Fang, Tang, and Jianjun, Dai
- Subjects
Extraintestinal Pathogenic Escherichia coli ,Escherichia coli Proteins ,Iron ,Escherichia coli ,Transferrin ,Humans ,Peptide Elongation Factor Tu ,Escherichia coli Infections - Abstract
Extraintestinal pathogenic
- Published
- 2022
14. In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity
- Author
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Alexander Wagner, Laura I. Weise, and Hannes Mutschler
- Subjects
viruses ,Q beta Replicase ,RNA ,Medicine (miscellaneous) ,DNA-Directed RNA Polymerases ,Peptide Elongation Factor Tu ,General Agricultural and Biological Sciences ,General Biochemistry, Genetics and Molecular Biology ,Levivirus - Abstract
Host proteins are identified which are required to reconstitute a functional RNA phage MS2 replication machinery, providing a promising platform for cell-free gene expression. The RNA phage MS2 is one of the most important model organisms in molecular biology and virology. Despite its comprehensive characterisation, the composition of the RNA replication machinery remained obscure. Here, we characterised host proteins required to reconstitute the functional replicase in vitro. By combining a purified replicase sub-complex with elements of an in vitro translation system, we confirmed that the three host factors, EF-Ts, EF-Tu, and ribosomal protein S1, are part of the active replicase holocomplex. Furthermore, we found that the translation initiation factors IF1 and IF3 modulate replicase activity. While IF3 directly competes with the replicase for template binding, IF1 appears to act as an RNA chaperone that facilitates polymerase readthrough. Finally, we demonstrate in vitro formation of RNAs containing minimal motifs required for amplification. Our work sheds light on the MS2 replication machinery and provides a new promising platform for cell-free evolution.
- Published
- 2022
15. Whole exome sequencing revealed a heterozygous elongation factor Tu GTP-binding domain containing 2 ( EFTUD2 ) mutation in a couple experiencing recurrent pregnancy loss
- Author
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Mei, Yang, Huaqin, Sun, Yanyan, Liu, and Ting, Hu
- Subjects
Abortion, Habitual ,Pregnancy ,Mutation ,Exome Sequencing ,Humans ,Female ,Guanosine Triphosphate ,Peptide Elongation Factor Tu ,Peptide Elongation Factors ,Ribonucleoprotein, U5 Small Nuclear - Published
- 2022
16. Probing the Molecular Basis of Cofactor Affinity and Conformational Dynamics of
- Author
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Navneet, Kumar and Prabha, Garg
- Subjects
Binding Sites ,Escherichia coli ,Guanosine Triphosphate ,Mycobacterium tuberculosis ,Molecular Dynamics Simulation ,Peptide Elongation Factor Tu ,Peptide Elongation Factors ,Guanosine Diphosphate - Abstract
The emergence of multidrug-resistant and extensively drug-resistant tuberculosis strains is the reason that the infectious tuberculosis pathogen is still the most common cause of death. The quest for new antitubercular drugs that can fit into multidrug regimens, function swiftly, and overcome the ever-increasing prevalence of drug resistance continues. The crucial role of
- Published
- 2022
17. Phosphorylation of mammalian mitochondrial EF-Tu by Fyn and c-Src kinases
- Author
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Emine C, Koc, Caroline A, Hunter, and Hasan, Koc
- Subjects
CSK Tyrosine-Protein Kinase ,Mitochondrial Proteins ,Mammals ,Mice ,src-Family Kinases ,Animals ,Cell Biology ,Peptide Elongation Factor Tu ,Phosphorylation ,Proto-Oncogene Proteins c-fyn ,Oxidative Phosphorylation - Abstract
Src Family Kinases (SFKs) are tyrosine kinases known to regulate glucose and fatty acid metabolism as well as oxidative phosphorylation (OXPHOS) in mammalian mitochondria. We and others discovered the association of the SFK kinases Fyn and c-Src with mitochondrial translation components. This translational system is responsible for the synthesis of 13 mitochondrial (mt)-encoded subunits of the OXPHOS complexes and is, thus, essential for energy generation. Mitochondrial ribosomal proteins and various translation elongation factors including Tu (EF-Tu
- Published
- 2023
18. Chiral checkpoints during protein biosynthesis
- Author
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Rajan Sankaranarayanan, Shobha P Kruparani, and Santosh Kumar Kuncha
- Subjects
0301 basic medicine ,Peptide Elongation Factor Tu ,Biochemistry ,Ribosome ,Amino Acyl-tRNA Synthetases ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Cell Wall ,Protein biosynthesis ,Amino Acids ,Molecular Biology ,chemistry.chemical_classification ,Bacteria ,030102 biochemistry & molecular biology ,Chemistry ,Aminoacyl tRNA synthetase ,JBC Reviews ,Stereoisomerism ,Translation (biology) ,Cell Biology ,Genetic code ,Amino acid ,030104 developmental biology ,Protein Biosynthesis ,Protein folding ,Homochirality ,Ribosomes - Abstract
Protein chains contain only l-amino acids, with the exception of the achiral glycine, making the chains homochiral. This homochirality is a prerequisite for proper protein folding and, hence, normal cellular function. The importance of d-amino acids as a component of the bacterial cell wall and their roles in neurotransmission in higher eukaryotes are well-established. However, the wider presence and the corresponding physiological roles of these specific amino acid stereoisomers have been appreciated only recently. Therefore, it is expected that enantiomeric fidelity has to be a key component of all of the steps in translation. Cells employ various molecular mechanisms for keeping d-amino acids away from the synthesis of nascent polypeptide chains. The major factors involved in this exclusion are aminoacyl-tRNA synthetases (aaRSs), elongation factor thermo-unstable (EF-Tu), the ribosome, and d-aminoacyl-tRNA deacylase (DTD). aaRS, EF-Tu, and the ribosome act as "chiral checkpoints" by preferentially binding to l-amino acids or l-aminoacyl-tRNAs, thereby excluding d-amino acids. Interestingly, DTD, which is conserved across all life forms, performs "chiral proofreading," as it removes d-amino acids erroneously added to tRNA. Here, we comprehensively review d-amino acids with respect to their occurrence and physiological roles, implications for chiral checkpoints required for translation fidelity, and potential use in synthetic biology.
- Published
- 2019
19. The Pregnancy Zone Protein (PZP) is significantly downregulated in the placenta of preeclampsia and HELLP syndrome patients
- Author
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Löb, Sanja, Vattai, Aurelia, Kuhn, Christina, Mittelberger, Johanna, Herbert, Saskia-Laureen, Wöckel, Achim, Schmoeckel, Elisa, Mahner, Sven, and Jeschke, Udo
- Subjects
HELLP Syndrome ,Fetal Growth Retardation ,Placenta ,Immunology ,Carcinoma ,Obstetrics and Gynecology ,Peptide Elongation Factor Tu ,Pregnancy Proteins ,Peptide Elongation Factors ,Reproductive Medicine ,Pre-Eclampsia ,Pregnancy ,Immunology and Allergy ,Humans ,Female ,Guanosine Triphosphate ,Ribonucleoprotein, U5 Small Nuclear - Abstract
Preeclampsia is characterized by maternal hypertension and multi-organ injury. Elongation factor Tu GTP binding domain containing 2 (EFTUD 2) and the Pregnancy Zone Protein (PZP) seem to be important immunomodulatory factors in early gestation. Little is known about the role of EFTUD2 and PZP in disorders of late pregnancy like preeclampsia, HELLP syndrome and intrauterine growth restriction (IUGR). PZP, EFTUD2 and hCG expression was investigated by immunohistochemistry in the placenta of healthy pregnancies (n = 13), preeclampsia (n = 11), HELLP syndrome (n = 12) and IUGR (n = 8). Correlation analysis of protein expression was performed via Spearman correlation coefficient. The characterization of EFTUD2 and PZP expressing cells was evaluated by double-immunofluorescence. After cultivation of the chorion carcinoma cell line BeWo with hCG the expression of PZP and EFTUD2 was investigated by immunocytochemistry. PZP expression was significantly downregulated in the syncytiotrophoblast (ST) and extravillous trophoblast (EVT) of preeclampsia (ST: p 0.001, EVT:p = 0.019) and HELLP syndrome (ST: p = 0.004, EVT: p = 0.035). The expression of EFTUD2 was significantly lower in preeclampsia (ST: p = 0.003, EVT: p 0.001), HELLP syndrome (ST: p = 0.021, EVT: = 0.001, EVT: p = 0.001). EVTs were identified as EFTUD2 and PZP expressing cells by double-immunofluorescence. Stimulation of BeWo chorion carcinoma cells with hCG 1000 IU/mL for 48 h resulted in a significant upregulation of PZP expression (p = 0.027). Our results indicate that PZP and EFTUD2 might be involved in the development of placental dysfunction in preeclampsia and HELLP syndrome.
- Published
- 2021
20. Association of Elongation Factor Tu GTP-binding Domain-containing 2 Gene
- Author
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Anran, Tian, Yuwen, Li, Haozhi, Fan, Pingping, Hu, Ruirui, Xu, Hui, Yuan, Jinyuan, Cai, Wen, Zhang, Ming, Yue, Jun, Li, Chen, Dong, and Chuanlong, Zhu
- Subjects
Male ,Hepatitis B virus ,Genotype ,Peptide Elongation Factor Tu ,Hepatitis B ,Peptide Elongation Factors ,Polymorphism, Single Nucleotide ,Hepatitis B, Chronic ,Gene Frequency ,Case-Control Studies ,Humans ,Female ,Genetic Predisposition to Disease ,Guanosine Triphosphate ,Alleles ,Ribonucleoprotein, U5 Small Nuclear - Abstract
The elongation factor Tu GTP-binding domain-containing 2 gene (In total, 448 control subjects and 379 patients with chronic HBV infection from Zhangjiagang First People's Hospital (Jiangsu, China) were enrolled. Sequenom iPLEX assay was used to detect genotypes of four SNPs (rs1071682, rs2277617, rs2289674, and rs3809756). Dual-luciferase reporter vectors with wild-type A and mutant-type C alleles ofOnly rs3809756 was significantly associated with HBV infection susceptibility (
- Published
- 2021
21. Differential Contribution of Protein Factors and 70S Ribosome to Elongation
- Author
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Stanislav V Kirillov, Andrey L. Konevega, Daria S. Vinogradova, Elena Maksimova, Pavel Kasatsky, and Alena Paleskava
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QH301-705.5 ,Peptide Chain Elongation, Translational ,translation ,Peptide Elongation Factor Tu ,medicine.disease_cause ,Ribosome ,Catalysis ,Article ,antibiotics ,Inorganic Chemistry ,chemistry.chemical_compound ,Bacterial Proteins ,RNA, Transfer ,medicine ,Escherichia coli ,Physical and Theoretical Chemistry ,Biology (General) ,Molecular Biology ,QD1-999 ,Spectroscopy ,rapid kinetics ,biology ,Chemistry ,Thermus thermophilus ,Organic Chemistry ,Translation (biology) ,General Medicine ,biology.organism_classification ,Peptide Elongation Factor G ,Computer Science Applications ,elongation factor ,Elongation factor ,RNA, Bacterial ,Viomycin ,Biophysics ,heterologous system ,70S ribosome ,Elongation ,Hygromycin B ,Ribosomes ,medicine.drug - Abstract
The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.
- Published
- 2021
22. Elongation Factor Tu’s Nucleotide Binding Is Governed by a Thermodynamic Landscape Unique among Bacterial Translation Factors
- Author
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Katherine E. Gzyl, Dylan Girodat, Evan Mercier, and Hans-Joachim Wieden
- Subjects
Molecular switch ,chemistry.chemical_classification ,Binding Sites ,Guanosine ,Chemistry ,General Chemistry ,GTPase ,Molecular Dynamics Simulation ,Peptide Elongation Factor Tu ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,GTP Phosphohydrolases ,0104 chemical sciences ,Biological pathway ,Colloid and Surface Chemistry ,Prokaryotic translation ,Escherichia coli ,Biophysics ,Thermodynamics ,Nucleotide ,EF-Tu - Abstract
Molecular switches such as GTPases are powerful devices turning "on" or "off" biomolecular processes at the core of critical biological pathways. To develop molecular switches de novo, an intimate understanding of how they function is required. Here we investigate the thermodynamic parameters that define the nucleotide-dependent switch mechanism of elongation factor (EF) Tu as a prototypical molecular switch. EF-Tu alternates between GTP- and GDP-bound conformations during its functional cycle, representing the "on" and "off" states, respectively. We report for the first time that the activation barriers for nucleotide association are the same for both nucleotides, suggesting a guanosine nucleoside or ribose-first mechanism for nucleotide association. Additionally, molecular dynamics (MD) simulations indicate that enthalpic stabilization of GDP binding compared to GTP binding originates in the backbone hydrogen bonding network of EF-Tu. In contrast, binding of GTP to EF-Tu is entropically driven by the liberation of bound water during the GDP- to GTP-bound transition. GDP binding to the apo conformation of EF-Tu is both enthalpically and entropically favored, a feature unique among translational GTPases. This indicates that the apo conformation does not resemble the GDP-bound state. Finally, we show that antibiotics and single amino acid substitutions can be used to target specific structural elements in EF-Tu to redesign the thermodynamic landscape. These findings demonstrate how, through evolution, EF-Tu has fine-tuned the structural and dynamic features that define nucleotide binding, providing insight into how altering these properties could be exploited for protein engineering.
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- 2019
23. The Laminin Interactome: A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization
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Yu-Ching Su, Kristian Riesbeck, Emma Mattsson, Farshid Jalalvand, Birendra Singh, and Timothy F. Murphy
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0301 basic medicine ,Gene isoform ,Haemophilus Infections ,Lipoproteins ,Protein subunit ,030106 microbiology ,Plasma protein binding ,Peptide Elongation Factor Tu ,medicine.disease_cause ,Interactome ,Bacterial Adhesion ,Basement Membrane ,Haemophilus influenzae ,Major Articles and Brief Reports ,03 medical and health sciences ,Bacterial Proteins ,Laminin ,otorhinolaryngologic diseases ,medicine ,Humans ,Immunology and Allergy ,Adhesins, Bacterial ,Laminin binding ,Haemophilus Vaccines ,Binding Sites ,L-Lactate Dehydrogenase ,biology ,Heparin ,Chemistry ,Epithelial Cells ,Immunoglobulin D ,Molecular biology ,Bacterial adhesin ,030104 developmental biology ,Infectious Diseases ,A549 Cells ,biology.protein ,Carrier Proteins ,Bacterial Outer Membrane Proteins ,Protein Binding - Abstract
Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilus influenzae (NTHi) colonization in the airway mucosa. In this study, we identified elongation factor thermo-unstable (EF-Tu), l-lactate dehydrogenase (LDH), protein D (PD), and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (protein 4 [P4], protein E [PE], protein F [PF], and Haemophilus adhesion and penetration protein [Hap]), EF-Tu, LDH, PD, and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. More importantly, the NTHi laminin interactome consisting of the well-studied and novel Lbps recognized laminin LG domains from the subunit α chains of laminin-111 and -332, the latter isoform of which is the main laminin in the airway BM. The NTHi interactome mainly targeted multiple heparin-binding domains of laminin. In conclusion, the NTHi interactome exhibited a high plasticity of interactions with different laminin isoforms via multiple heparin-binding sites.
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- 2019
24. A novel TUFM homozygous variant in a child with mitochondrial cardiomyopathy expands the phenotype of combined oxidative phosphorylation deficiency 4
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Alina Kurolap, Hagit Baris Feldman, John D. Overton, Ann Saada, Adi Mory, Tova Hershkovitz, Tamar Paperna, Sarah Wolf, Alan R. Shuldiner, Claudia Gonzaga-Jauregui, and Hanna Mandel
- Subjects
Male ,0301 basic medicine ,Mitochondrial DNA ,Mitochondrial Diseases ,Mitochondrial disease ,Disease ,Oxidative phosphorylation ,Peptide Elongation Factor Tu ,030105 genetics & heredity ,Bioinformatics ,DNA, Mitochondrial ,Oxidative Phosphorylation ,Mitochondrial Proteins ,Consanguinity ,03 medical and health sciences ,Exome Sequencing ,Genetics ,medicine ,Humans ,Missense mutation ,Amino Acid Sequence ,Genetics (clinical) ,business.industry ,Homozygote ,Infant ,Dilated cardiomyopathy ,medicine.disease ,Phenotype ,Mitochondria ,030104 developmental biology ,Lactic acidosis ,Mutation ,Acidosis, Lactic ,Female ,Cardiomyopathies ,business ,Metabolism, Inborn Errors - Abstract
Translation of mitochondrial-specific DNA is required for proper mitochondrial function and energy production. For this purpose, an elaborate network of dedicated molecular machinery including initiation, elongation and termination factors exists. We describe a patient with an unusual phenotype and a novel homozygous missense variant in TUFM (c.344A>C; p.His115Pro), encoding mtDNA translation elongating factor Tu (EFTu). To date, only four patients have been reported with bi-allelic mutations in TUFM, leading to combined oxidative phosphorylation deficiency 4 (COXPD4) characterized by severe early-onset lactic acidosis and progressive fatal infantile encephalopathy. The patient presented here expands the phenotypic features of TUFM-related disease, exhibiting lactic acidosis and dilated cardiomyopathy without progressive encephalopathy. This warrants the inclusion of TUFM in differential diagnosis of metabolic cardiomyopathy. Cases that further refine genotype-phenotype associations and characterize the molecular basis of mitochondrial disorders allow clinicians to predict disease prognosis, greatly impacting patient care, as well as provide families with reproductive planning options.
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- 2019
25. Broad range of missense error frequencies in cellular proteins
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Henning Urlaub, Marina V. Rodnina, Christof Lenz, Raffaella Garofalo, Ingo Wohlgemuth, and Michael Pearson
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Mutation, Missense ,Gene Expression ,Computational biology ,Genome Integrity, Repair and Replication ,Peptide Elongation Factor Tu ,Biology ,medicine.disease_cause ,03 medical and health sciences ,0302 clinical medicine ,Gene expression ,Escherichia coli ,Genetics ,medicine ,Homeostasis ,Missense mutation ,Amino Acids ,Cellular proteins ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Mutation ,Strain (chemistry) ,Escherichia coli Proteins ,Proteogenomics ,cellular proteins ,Broad range ,Amino acid ,Targeted mass spectrometry ,chemistry ,030217 neurology & neurosurgery - Abstract
Assessment of the fidelity of gene expression is crucial to understand cell homeostasis. Here we present a highly sensitive method for the systematic Quantification of Rare Amino acid Substitutions (QRAS) using absolute quantification by targeted mass spectrometry after chromatographic enrichment of peptides with missense amino acid substitutions. By analyzing incorporation of near- and non-cognate amino acids in a model protein EF-Tu, we show that most of missense errors are too rare to detect by conventional methods, such as DDA, and are estimated to be between
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- 2019
26. Ubiquitin specific peptidase 5 regulates colorectal cancer cell growth by stabilizing Tu translation elongation factor
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Qun Wang, Aiwen Huang, Xiaodan Hou, Yili Yang, Ximao Cui, Kunkun Han, Long Cui, and Xin Xu
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Male ,0301 basic medicine ,Immunoblotting ,Mice, Nude ,Medicine (miscellaneous) ,Peptide Elongation Factor Tu ,medicine.disease_cause ,Deubiquitinating enzyme ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Ubiquitin specific peptidase 5 ,Cell Line, Tumor ,medicine ,Animals ,Humans ,RNA, Small Interfering ,Pharmacology, Toxicology and Pharmaceutics (miscellaneous) ,Transcription factor ,Gene knockdown ,EBF1 ,biology ,Cell growth ,Lentivirus ,TUFM ,Middle Aged ,HCT116 Cells ,Colorectal cancer ,Deubiquitinase ,Cell Transformation, Neoplastic ,030104 developmental biology ,chemistry ,Cell culture ,USP5 ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,Female ,Ubiquitin-Specific Proteases ,Growth inhibition ,Colorectal Neoplasms ,Carcinogenesis ,HT29 Cells ,Research Paper - Abstract
Ubiquitin specific peptidase 5 (USP5) is a ubiquitous expressed deubiquitinating enzyme (DUB). It has been shown involved in DNA repair, apoptosis, inflammation, and tumor cell growth. However, the function and molecular mechanism of USP5 in colorectal cancer (CRC) are still unclear. In the present study, we asked how it affected the growth of colorectal cancer cells. Methods: A shRNA-based high-content screening was performed to identify DUBs affecting the growth of CRC cells. CCK-8 assay and xenografts were used to assess CRC cell growth, survival and tumorigenesis. RT-qPCR, immunoblotting and immunohistochemistry were carried out to quantitate USP5 expression in CRC tissues and cell lines. Immunoprecipitation and mass spectrometry analysis were performed to identify USP5-interacting proteins. Cycloheximide chase was performed to assess Tu translation elongation factor (TUFM) stability. Dual luciferase reporter assay was utilized for USP5 promoter analysis. Results: We found that USP5 was highly expressed in a group of primary CRC tissues, and the increased USP5 was correlated with clinical stages and shorter overall survival. While USP5 knockdown effectively inhibited CRC cell growth, overexpressed USP5 promoted the growth of CRC cells and made them more resistant to doxorubicin (DOX). TUFM was discovered as a substrate of USP5. USP5 deubiquitinated TUFM and increased its level in CRC cells. Enforced expression of TUFM was able to alleviate the growth inhibition induced by USP5 knockdown. Further analyses showed that EBF transcription factor 1 (EBF1) was a major regulator for USP5 transcription, and DOX inhibited EBF1-USP5-TUFM axis in CRC cells. Conclusions: USP5 was required for CRC cells and promoted their growth and resistance to chemotherapeutics. TUFM was a USP5 deubiquitinating substrate that mediated the cellular effects of USP5. The transcription of USP5 was regulated by EBF1. Thus, targeting EBF1-USP5-TUFM axis is a potential novel strategy for CRC treatment.
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- 2019
27. Kinetic Analysis Suggests Evolution of Ribosome Specificity in Modern Elongation Factor-Tus from 'Generalist' Ancestors
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Arindam, De Tarafder, Narayan Prasad, Parajuli, Soneya, Majumdar, Betül, Kaçar, and Suparna, Sanyal
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molecular evolution ,Thermus thermophilus ,AcademicSubjects/SCI01130 ,Peptide Elongation Factor Tu ,AcademicSubjects/SCI01180 ,Substrate Specificity ,Evolution, Molecular ,Kinetics ,Bacterial Proteins ,Protein Biosynthesis ,fast kinetics, specificity ,Escherichia coli ,ancestral sequence reconstruction ,generalist ,Ribosomes ,Discoveries ,translation machinery ,EF-Tu - Abstract
It has been hypothesized that early enzymes are more promiscuous than their extant orthologs. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing presteady state fast kinetics using quench flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3- to 3.3-Gy-old ancestral EF-Tus toward two unrelated bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. Although the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. In addition, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remains virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, “generalist” ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.
- Published
- 2021
28. TUFM is involved in Alzheimer’s disease‐like pathologies that are associated with ROS
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Li Song, Bi-Rou Zhong, Guojun Chen, Xiao-Juan Deng, Gui-Feng Zhou, Li-Tian Hu, Yuanlin Ma, and Qi-Xin Wen
- Subjects
0301 basic medicine ,Mitochondrial translation ,Mice, Transgenic ,Peptide Elongation Factor Tu ,Cycloheximide ,Biochemistry ,Amyloid beta-Protein Precursor ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Alzheimer Disease ,mental disorders ,Presenilin-1 ,Genetics ,Amyloid precursor protein ,Animals ,Humans ,Gene silencing ,Phosphorylation ,Molecular Biology ,Gene knockdown ,Messenger RNA ,biology ,Chemistry ,HEK 293 cells ,Translation (biology) ,Mitochondria ,Cell biology ,Disease Models, Animal ,030104 developmental biology ,biology.protein ,Amyloid Precursor Protein Secretases ,Reactive Oxygen Species ,030217 neurology & neurosurgery ,Biotechnology - Abstract
Mitochondrial Tu translation elongation factor (TUFM or EF-Tu) is part of the mitochondrial translation machinery. It is reported that TUFM expression is reduced in the brain of Alzheimer's disease (AD), suggesting that TUFM might play a role in the pathophysiology. In this study, we found that TUFM protein level was decreased in the hippocampus and cortex especially in the aged APP/PS1 mice, an animal model of AD. In HEK cells that stably express full-length human amyloid-β precursor protein (HEK-APP), TUFM knockdown or overexpression increased or reduced the protein levels of β-amyloid protein (Aβ) and β-amyloid converting enzyme 1 (BACE1), respectively. TUFM-mediated reduction of BACE1 was attenuated by translation inhibitor cycloheximide (CHX) or α-[2-[4-(3,4-Dichlorophenyl)-2-thiazolyl]hydrazinylidene]-2-nitro-benzenepropanoic acid (4EGI1), and in cells overexpressing BACE1 constructs deleting the 5' untranslated region (5'UTR). TUFM silencing increased the half-life of BACE1 mRNA, suggesting that RNA stability was affected by TUFM. In support, transcription inhibitor Actinomycin D (ActD) and silencing of nuclear factor κB (NFκB) failed to abolish TUFM-mediated regulation of BACE1 protein and mRNA. We further found that the mitochondria-targeted antioxidant TEMPO diminished the effects of TUFM on BACE1, suggesting that reactive oxygen species (ROS) played an important role. Indeed, cellular ROS levels were affected by TUFM knockdown or overexpression, and TUFM-mediated regulation of apoptosis and Tau phosphorylation at selective sites was attenuated by TEMPO. Collectively, TUFM protein levels were decreased in APP/PS1 mice. TUFM is involved in AD pathology by regulating BACE1 translation, apoptosis, and Tau phosphorylation, in which ROS plays an important role.
- Published
- 2021
29. Black carp TUFM collaborates with NLRX1 to inhibit MAVS-mediated antiviral signaling pathway
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Jun Zou, Zhaoyuan Chen, Jiayi Huang, Hui Wu, Hao Feng, and Yingyi Cao
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0301 basic medicine ,Carps ,Immunology ,Biology ,Peptide Elongation Factor Tu ,Cell Line ,Small hairpin RNA ,Mitochondrial Proteins ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,Interferon ,Rhabdoviridae Infections ,medicine ,Animals ,Humans ,RNA, Small Interfering ,NLRX1 ,Promoter Regions, Genetic ,Adaptor Proteins, Signal Transducing ,Reporter gene ,Gene knockdown ,Innate immune system ,Immunity, Innate ,Cell biology ,030104 developmental biology ,HEK293 Cells ,Interferon Type I ,RNA Interference ,Signal transduction ,Rhabdoviridae ,030215 immunology ,Developmental Biology ,medicine.drug ,Signal Transduction - Abstract
TUFM is a mitochondrial protein and serves as a regulator of antiviral signaling; nevertheless, the character of TUFM in teleosts remains unidentified. In this study, TUFM homologue of black carp (Mylopharyngodon piceus) has been characterized and its role in innate immunity has been explored. Black carp TUFM (bcTUFM) comprises 447 amino acids and shows the high similarity to human TUFM. bcTUFM was about 50 kDa in the Western blot assay and was determined as a cytosolic protein by immunofluorescent microscopy. Knockdown of bcTUFM by shRNA enhanced the antiviral ability of the host cells. The induction fold of interferon promoter transcription in the cells co-expressing bcTUFM and bcMAVS was much lower than that of the cells expressing bcMAVS alone. Our previous study has identified that bcNLRX1 interacted with bcMAVS and functioned as an inhibitor of bcMAVS. The interaction between bcTUFM and bcNLRX1, but not bcTUFM and bcMAVS, was detected through co-immunoprecipitation. The subsequent reporter assay and plaque assay demonstrated that the inhibition of bcMAVS-mediated interferon production and antiviral activity by bcNLRX1 was enhanced by co-expressed bcTUFM. Thus, our data suggests that bcTUFM cooperates with bcNLRX1 to inhibit bcMAVS-mediated antiviral signaling during host antiviral innate immune response against SVCV.
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- 2021
30. Structural basis for late maturation steps of the human mitoribosomal large subunit
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Genís Valentín Gesé, B. Martin Hallberg, Miriam Cipullo, Anas Khawaja, and Joanna Rorbach
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Models, Molecular ,0301 basic medicine ,RNA Folding ,Peptidyl transferase ,Science ,Protein subunit ,Ribosomal proteins ,General Physics and Astronomy ,RRNA methylation ,Peptide Elongation Factor Tu ,Mitochondrion ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Mitochondrial Ribosomes ,03 medical and health sciences ,0302 clinical medicine ,RNA, Ribosomal, 16S ,Electron microscopy ,Mitochondrial ribosome ,Humans ,Monomeric GTP-Binding Proteins ,Multidisciplinary ,biology ,Chemistry ,Cryoelectron Microscopy ,Methyltransferases ,General Chemistry ,Ribosome ,Cell biology ,Elongation factor ,030104 developmental biology ,Multiprotein Complexes ,Peptidyl Transferases ,Transfer RNA ,biology.protein ,RNA ,Translational elongation ,Ribosome Subunits, Large ,030217 neurology & neurosurgery ,Biogenesis ,Protein Binding ,Transcription Factors - Abstract
Mitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation., Mitochondrial ribosomes (mitoribosomes) are characterized by a distinct architecture and thus biogenesis pathway. Here, cryo-EM structures of mitoribosome large subunit assembly intermediates elucidate final steps of 16 S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, as well as functions of several mitoribosome assembly factors.
- Published
- 2021
31. A Set of Active Promoters with Different Activity Profiles for Superexpressing
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Anna O, Shemyakina, Elena G, Grechishnikova, Andrey D, Novikov, Andrey F, Asachenko, Tatyana I, Kalinina, Konstantin V, Lavrov, and Alexander S, Yanenko
- Subjects
Corynebacterium glutamicum ,Superoxide Dismutase ,Rhodococcus ,Gene Expression Regulation, Bacterial ,Peptide Elongation Factor Tu ,Promoter Regions, Genetic ,Isocitrate Lyase ,Hydro-Lyases ,Culture Media - Published
- 2021
32. Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction
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Ho Jeong Kwon, Jong Shin Yoo, Dasol Kim, Jin Young Kim, Eun Sun Ji, and Hui Yun Hwang
- Subjects
0301 basic medicine ,QH301-705.5 ,Drug Evaluation, Preclinical ,Regulator ,Medicine (miscellaneous) ,Cellular homeostasis ,Context (language use) ,Peptide Elongation Factor Tu ,Mechanism of action ,Article ,General Biochemistry, Genetics and Molecular Biology ,Autophagy-Related Protein 5 ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,0302 clinical medicine ,3T3-L1 Cells ,Lipid droplet ,Adipocytes ,Autophagy ,Animals ,Humans ,Kaempferols ,Biology (General) ,PI3K/AKT/mTOR pathway ,Metabolic Syndrome ,chemistry.chemical_classification ,Reactive oxygen species ,Molecular medicine ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,Chemistry ,Lipid Metabolism ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,030220 oncology & carcinogenesis ,TFEB ,General Agricultural and Biological Sciences ,Autophagy-Related Protein 12 ,HeLa Cells - Abstract
Disorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem’s biological activity, the target protein was identified via combined drug affinity responsive target stability and LC–MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload., Kim, Hwang et al. use in vitro and in vivo models of autophagy disorder/metabolic dysfunction to show that in this context, the natural compound kaempferide is an autophagy enhancer and reveal that one of the underlying mechanisms governing this is mediated by the mitochondrial elongation factor TUFM. This insight may have therapeutic value in the treatment of metabolic disorders.
- Published
- 2021
33. Activation loop phosphorylaton of a non-RD receptor kinase initiates plant innate immune signaling
- Author
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Yasuhiro Kadota, Paul Derbyshire, Vardis Ntoukakis, Daniel Couto, Kyle W. Bender, Lena Stransfeld, Maria Font Farre, Alberto P. Macho, Jan Sklenar, Cyril Zipfel, Annalise Petriello, Benjamin Schwessinger, Marta Bjornson, Frank L.H. Menke, Alexandra M. E. Jones, Thomas A. DeFalco, University of Zurich, and Zipfel, Cyril
- Subjects
Receptor complex ,Arabidopsis ,Gene Expression ,Peptide Elongation Factor Tu ,Protein Serine-Threonine Kinases ,580 Plants (Botany) ,Ligands ,Biochemistry ,Protein Domains ,10126 Department of Plant and Microbial Biology ,receptor kinase ,Plant Immunity ,10211 Zurich-Basel Plant Science Center ,Receptor ,Protein kinase A ,SB ,1000 Multidisciplinary ,Innate immune system ,Multidisciplinary ,Kinase ,Chemistry ,phosphorylation ,Arabidopsis Proteins ,Cell Membrane ,fungi ,QK ,Biological Sciences ,Ligand (biochemistry) ,Plants, Genetically Modified ,QP ,Immunity, Innate ,Cell biology ,Cytoplasm ,Receptors, Pattern Recognition ,Phosphorylation ,bacteria ,signaling ,Protein Kinases ,Protein Binding ,Signal Transduction - Abstract
Significance Protein kinases lacking Arg in the catalytic loop HxD motif (i.e., non-RD kinases) are associated with innate immune signaling across kingdoms. Phosphorylation activates plant immune receptor kinases (RKs), but the mechanistic details of activation are limited. Using the non-RD immune RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we investigated the role of the receptor cytoplasmic domain in immune signaling and found that the catalytic activity of EFR is dispensable for antibacterial immunity. Nevertheless, ligand-induced EFR-mediated signaling is initiated by activation loop phosphorylation, but not via the catalytic activity of the receptor protein kinase domain. We propose that leucine-rich repeat-receptor kinase complexes containing a non-RD kinase are activated through phosphorylation-dependent conformational changes of the receptor cytoplasmic domain., Receptor kinases (RKs) are fundamental for extracellular sensing and regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) are primarily peptide receptors that regulate responses to myriad internal and external stimuli. Phosphorylation of LRR-RK cytoplasmic domains is among the earliest responses following ligand perception, and reciprocal transphosphorylation between a receptor and its coreceptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in antibacterial immunity. These results reveal a noncatalytic role for EFR in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RKs with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor, which could initiate signaling either allosterically or through driving the dissociation of negative regulators of the complex.
- Published
- 2021
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34. Coding triplets in the tRNA acceptor-TΨC arm and their role in present and past tRNA recognition
- Author
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Itay Fayerverker, Ilana Agmon, and Tal Mor
- Subjects
Biophysics ,Aminoacylation ,Biology ,Peptide Elongation Factor Tu ,Biochemistry ,Amino Acyl-tRNA Synthetases ,Evolution, Molecular ,03 medical and health sciences ,RNA, Transfer ,Structural Biology ,Genetics ,Escherichia coli ,Nucleotide ,Amino Acids ,Codon ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Translation system ,030302 biochemistry & molecular biology ,Translation (biology) ,Cell Biology ,Genetic code ,Amino acid ,Elongation factor ,chemistry ,Genetic Code ,Transfer RNA ,Nucleic Acid Conformation - Abstract
The mechanism and evolution of the recognition scheme between key components of the translation system, that is, tRNAs, synthetases, and elongation factors, are fundamental issues in understanding the translation of genetic information into proteins. Statistical analysis of bacterial tRNA sequences reveals that for six amino acids, a string of 10 nucleotides preceding the tRNA 3' end carries cognate coding triplets to nearly full extent. The triplets conserved in positions 63-67 are implicated in the recognition by the elongation factor EF-Tu, and those conserved in positions 68-72, in the identification of cognate tRNAs, and their derived minihelices by class IIa synthetases. These coding triplets are suggested to have primordial origin, being engaged in aminoacylation of prebiotic tRNAs and in the establishment of the canonical codon set.
- Published
- 2020
35. Resistance-Guided Discovery of Elfamycin Antibiotic Producers with Antigonococcal Activity
- Author
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Venkateswarlu Yarlagadda, Georgina Cox, Timothy A. Johnson, Gerard D. Wright, Maulik N. Thaker, Ricardo Medina, and Kalinka Koteva
- Subjects
0301 basic medicine ,Modern medicine ,medicine.drug_class ,030106 microbiology ,Antibiotics ,Peptide Elongation Factor Tu ,medicine.disease_cause ,Streptomyces ,Microbiology ,03 medical and health sciences ,Polyketide ,Streptomyces isolates ,Antibiotic resistance ,Drug Resistance, Bacterial ,medicine ,Humans ,Translation factor ,biology ,biology.organism_classification ,3. Good health ,Anti-Bacterial Agents ,030104 developmental biology ,Infectious Diseases ,Protein Biosynthesis ,Neisseria gonorrhoeae - Abstract
The rise of bacterial antibiotic resistance coupled with a diminished antibiotic drug pipeline underlines the importance of developing rational strategies to discover new antimicrobials. Microbially derived natural products are the basis for most of the antibiotic arsenal available to modern medicine. Here, we demonstrate a resistance-based approach to identify producers of elfamycins, an under-explored class of natural product antibiotics that target the essential translation factor EF-Tu. Antibiotic producers carry self-resistance genes to avoid suicide. These genes are often found within the same biosynthetic gene cluster (BGC) responsible for making the antibiotic, and we exploited this trait to identify members of the kirromycin class of elfamycin producers. Genome mining of Streptomyces spp. led to the identification of three isolates that harbor kirromycin-resistant EF-Tu (EF-TuKirR) within predicted natural product BGCs. Activity-guided purification on extracts of one of the Streptomyces isolates, which was not known to produce an elfamycin, identified it as a producer of phenelfamycin B, a linear polyketide. Phenelfamycin B demonstrates impressive antibacterial activity (MIC ∼ 1 μg/mL) against multidrug-resistant Neisseria gonorrhoeae, a clinically important Gram negative pathogen. The antigonococcal activity of phenelfamycin was shown to be the result of inhibition of protein biosynthesis by binding to EF-Tu. These results indicate that a resistance-based approach of identifying elfamycin producers is translatable to other antibiotic classes that can identify new and overlooked antibiotics necessary to address the antibiotic crisis.
- Published
- 2020
36. Altered tumor suppressor genes expression in Egyptian pesticide applicators exposed to organophosphate insecticides
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Eman A Salem, Ibrahim A. Elhalafawy, Faten E Younis, Mofrih Hegazy, Ola A Swellim, and Moustafa A. Sakr
- Subjects
Adult ,Male ,Insecticides ,Adolescent ,Genotype ,Health, Toxicology and Mutagenesis ,Cell Cycle Proteins ,Biology ,Peptide Elongation Factor Tu ,Toxicology ,law.invention ,Mitochondrial Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Young Adult ,0302 clinical medicine ,Organophosphorus Compounds ,law ,Occupational Exposure ,Biomarkers, Tumor ,Humans ,Genes, Tumor Suppressor ,RNA, Messenger ,Pesticides ,Gene ,Organophosphate insecticides ,Carcinogen ,030304 developmental biology ,0303 health sciences ,Organophosphate ,Public Health, Environmental and Occupational Health ,Proto-Oncogene Proteins c-mdm2 ,Pesticide ,Middle Aged ,Genes, p53 ,Cross-Sectional Studies ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,Acetylcholinesterase ,Mdm2 ,Suppressor ,Egypt ,Occupational exposure ,DNA Damage - Abstract
Occupational exposure in spraying and application of non-arsenical insecticides has been classified as a probable human carcinogen. The fundamental molecular mechanisms involved the tumor-related genes. This study aimed to investigate the carcinogenesis effects related to chronic exposure to organophosphate (OP) pesticides in pesticide applicators. This was a cross-sectional study conducted on 27 pesticide applicators and 24 matched controls through the period from June to December 2018. The level of acetylcholinesterase (AChE) was determined and the effects of OPs exposure on messenger RNA (mRNA) expression of the DNA-damage responsive genes P53, P21, GADD45a, and MDM2 were determined using real-time quantitative polymerase chain reaction. A significant reduction of serum AChE enzyme activities was observed in chronically exposed subjects in comparison with the control group ( p = 0.001). The expression of P53, P21 mRNA was significantly downregulated in the exposed group compared with the healthy nonexposed control group ( p < 0.05). Conversely, the expression of MDM2 and GADD45a did not significantly differ between the exposed subjects and the control group ( p > 0.05). No significant differences were noted between the exposed and control groups regarding the genotype or allele distributions of P53 Arg72Pro polymorphism. These results suggested that chronic exposure to OP insecticides may have mitogenic and carcinogenicity activity for the exposed cases due to downregulation of P53 and P21 but did not demonstrate any DNA damage properties for the exposed cases, and finally, a regular follow-up of the exposed cases for tumor markers is recommended.
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- 2020
37. Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the
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Yanyu, Zhu, James C, Weisshaar, and Mainak, Mustafi
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Cytoplasm ,Escherichia coli Proteins ,Protein Biosynthesis ,Escherichia coli ,Ribosome Subunits, Small, Bacterial ,Ribosome Subunits, Large, Bacterial ,Peptide Elongation Factor Tu ,Molecular Biophysics ,Antimicrobial Cationic Peptides - Abstract
Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro. Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.
- Published
- 2020
38. Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection
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Hans-Joachim Wieden, Dylan Girodat, Scott C. Blanchard, Justin C. Morse, Karissa Y. Sanbonmatsu, Mikael Holm, Roger B. Altman, and Benjamin J. Burnett
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Peptidyl transferase ,GTP' ,protein synthesis ,translation ,Peptide Elongation Factor Tu ,RNA, Transfer, Amino Acyl ,Ribosome ,single-molecule FRET ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,RNA, Transfer ,Large ribosomal subunit ,Escherichia coli ,Fluorescence Resonance Energy Transfer ,aminoacyl-tRNA ,030304 developmental biology ,0303 health sciences ,Aminoacyl-tRNA ,Multidisciplinary ,biology ,Chemistry ,Escherichia coli Proteins ,Translation (biology) ,Biological Sciences ,elongation factor Tu ,Kinetics ,Biophysics and Computational Biology ,PNAS Plus ,Protein Biosynthesis ,Transfer RNA ,Biophysics ,biology.protein ,Guanosine Triphosphate ,Ribosome Subunits, Large ,Ribosomes ,030217 neurology & neurosurgery ,EF-Tu - Abstract
Significance Elongation factor Tu (EF-Tu) facilitates rapid and accurate selection of aminoacyl-tRNA (aa-tRNA) by the bacterial ribosome during protein synthesis. We show that EF-Tu dissociates from the ribosome as aa-tRNA navigates the accommodation corridor en route to peptide bond formation. We find that EF-Tu’s release from the ribosome during aa-tRNA selection can be reversible. We also demonstrate that new ternary complex formation, accompanied by futile cycles of GTP hydrolysis, can occur on aa-tRNA bound within the ribosome. These findings inform on the decoding mechanism, the contributions of EF-Tu to the fidelity of translation, and the potential consequences of reduced rates of peptide bond formation on cellular physiology., The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.
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- 2020
39. Substrate recognition mechanism of tRNA-targeting ribonuclease, colicin D, and an insight into tRNA cleavage-mediated translation impairment
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Toshihiro Aono, Makoto Hidaka, Haruhiko Masaki, Kazutoshi Takahashi, Tohru Terada, Wataru Ishida, and Tetsuhiro Ogawa
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Protein Conformation, alpha-Helical ,Thiouridine ,Colicins ,RNA, Transfer, Arg ,Peptide Elongation Factor Tu ,medicine.disease_cause ,Cleavage (embryo) ,Substrate Specificity ,03 medical and health sciences ,0302 clinical medicine ,Bacteriocins ,Anticodon ,Escherichia coli ,medicine ,Protein Interaction Domains and Motifs ,Nucleotide ,Ribonuclease ,Base Pairing ,Uridine ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,biology ,Gene Expression Regulation, Bacterial ,Cell Biology ,Ribosomal RNA ,In vitro ,Cell biology ,Molecular Docking Simulation ,RNA, Bacterial ,chemistry ,Protein Biosynthesis ,030220 oncology & carcinogenesis ,Colicin ,Transfer RNA ,biology.protein ,Nucleic Acid Conformation ,bacteria ,Protein Conformation, beta-Strand ,Ribosomes ,Plasmids ,Protein Binding ,Research Paper - Abstract
Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNAArg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNAArgs; the cleavage occurs at the 3′ end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNAArgs from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNAArgICG, the most abundant species of E. coli tRNAArgs, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNAArgICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNAArgICG dominant-negatively impairs translation in vitro. Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNAArgICG, which is consistent with our structural model. Finally, elevation of cleaved tRNAArgICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNAArgICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops. Abbreviations: mnm5U: 5-methylaminomethyluridine; mcm5s2U: 5-methoxycarbonylmethyl-2-thiouridine
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- 2020
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40. Disorder guides domain rearrangement in elongation factor Tu
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Paul C. Whitford, Jonathan Perrier, and Huan Yang
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Models, Molecular ,0301 basic medicine ,Conformational change ,Protein Conformation ,Static Electricity ,Protein domain ,Plasma protein binding ,Peptide Elongation Factor Tu ,RNA, Transfer, Amino Acyl ,Intrinsically disordered proteins ,Biochemistry ,Ribosome ,03 medical and health sciences ,Protein structure ,Protein Domains ,Structural Biology ,Static electricity ,Molecular Biology ,Escherichia coli K12 ,030102 biochemistry & molecular biology ,Chemistry ,Escherichia coli Proteins ,Intrinsically Disordered Proteins ,030104 developmental biology ,Biophysics ,Ribosomes ,EF-Tu ,Protein Binding - Abstract
Elongation factor Tu (EF-Tu) is a three-domain protein that is responsible for delivering aminoacyl-tRNA (aa-tRNA) molecules to the ribosome. During the delivery process, EF-Tu undergoes a large-scale (~50Å) conformational transition that results in rearrangement of domain I, relative to the II/III superdomain. Despite the central role of EF-Tu during protein synthesis, little is known about the structural and energetic properties of this reordering process. To study the physical-chemical properties of domain motion, we constructed a multi-basin structure-based (i.e., Gō-like) model, with which we have simulated hundreds of spontaneous conformational rearrangements. By analyzing the statistical properties of these events, we show that EF-Tu is likely to adopt a disordered intermediate ensemble during this transition. We further show that this disordered intermediate will favor a specific sequence of conformational substeps when bound to the ribosome, and the disordered ensemble can influence the kinetics of the incoming aa-tRNA molecule. Overall, this study highlights the dynamic nature of EF-Tu by revealing a relationship between conformational disorder and biological function.
- Published
- 2018
41. Analysis of heat-induced protein aggregation in human mitochondria
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Anne Wilkening, Cornelia Rüb, Marc Sylvester, and Wolfgang Voos
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0301 basic medicine ,Hot Temperature ,Mitochondrial translation ,Endogeny ,Peptide Elongation Factor Tu ,Protein aggregation ,Mitochondrion ,Biochemistry ,Mitochondrial Proteins ,Protein Aggregates ,03 medical and health sciences ,Adenosine Triphosphate ,Protein biosynthesis ,Humans ,Molecular Biology ,Loss function ,Membrane Potential, Mitochondrial ,Chemistry ,Translation (biology) ,Cell Biology ,Mitochondria ,Cell biology ,Elongation factor ,Cytosol ,030104 developmental biology ,Proteotoxicity ,EF-Tu ,Function (biology) ,Heat-Shock Response ,HeLa Cells - Abstract
Proteins in mammalian cells exhibit optimal stability at physiological temperatures, and even small temperature variations may cause unfolding and nonspecific aggregation. Because this process leads to a loss of function of the affected polypeptides and to cytotoxic stress, formation of protein aggregates has been recognized as a major pathogenic factor in human diseases. In this study, we determined the impact of physiological heat stress on mitochondria isolated from HeLa cells. We found that the heat-stressed mitochondria had lower membrane potential and ATP level and exhibited a decreased production of reactive oxygen species. An analysis of the mitochondrial proteome by 2D PAGE showed that the overall solubility of endogenous proteins was only marginally affected by elevated temperatures. However, a small subset of polypeptides exhibited an high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat-stress conditions. Moreover, mitochondrial translation and the import of cytosolic proteins were defective in the heat-stressed mitochondria. Both types of nascent polypeptides, produced by translation or imported into the mitochondria, exhibited a strong tendency to aggregate in the heat-exposed mitochondria. We propose that a fast and specific inactivation of elongation factors may prevent the accumulation of misfolded nascent polypeptides and may thereby attenuate proteotoxicity under heat stress.
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- 2018
42. Engineered EF-Tu and tRNA-Based FRET Screening Assay to Find Inhibitors of Protein Synthesis in Bacteria
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Rachana Bhatt, Maxim Chudaev, Wlodek Mandecki, and Emanuel Goldman
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0301 basic medicine ,Microbial Sensitivity Tests ,Peptide Elongation Factor Tu ,Protein Engineering ,medicine.disease_cause ,01 natural sciences ,03 medical and health sciences ,fluids and secretions ,RNA, Transfer ,Drug Discovery ,Escherichia coli ,Fluorescence Resonance Energy Transfer ,medicine ,Protein biosynthesis ,Ternary complex ,Protein synthesis inhibitor ,biology ,010405 organic chemistry ,Chemistry ,Escherichia coli Proteins ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,biology.organism_classification ,Anti-Bacterial Agents ,0104 chemical sciences ,030104 developmental biology ,Förster resonance energy transfer ,Biochemistry ,Staphylococcus aureus ,Protein Biosynthesis ,Transfer RNA ,bacteria ,Molecular Medicine ,Bacteria ,EF-Tu - Abstract
Antibiotic-resistant infections that do not respond to available drugs are becoming more common. Methicillin-resistant Staphylococcus aureus, carbapenem-resistant enterobacteria ("superbugs"), and many others pose a continuous threat to public health. To provide tools to combat such deadly infections, we present in this study a homogeneous assay focused on an insufficiently addressed molecular interaction linked to ribosomal translation. We show that a fluorescence resonance energy transfer (FRET) based screening assay can identify antibiotic molecules that inhibit ternary complex (EF-Tu:tRNA:GTP complex) formation, and therefore, protein synthesis in bacteria. Specifically engineered Escherichia coli EF-Tu and tRNA
- Published
- 2018
43. Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-TuH84A
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Chandra Sekhar Mandava, Joachim Frank, Zuben P. Brown, Suparna Sanyal, Marcus Fislage, Jingji Zhang, Måns Ehrenberg, Department of Bio-engineering Sciences, and Structural Biology Brussels
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Models, Molecular ,0301 basic medicine ,GTP' ,Guanosine Triphosphate/chemistry ,GTPase ,Peptide Elongation Factor Tu ,RNA, Transfer, Amino Acyl ,Biology ,Ribosome ,03 medical and health sciences ,Structural Biology ,RNA, Ribosomal, 16S ,Genetics ,RNA, Messenger ,Codon ,RNA, Ribosomal, 16S/chemistry ,Ternary complex ,Ribosomes/chemistry ,Cryoelectron Microscopy ,Biochemistry and Molecular Biology ,RNA ,Ribosomal RNA ,Peptide Elongation Factor Tu/chemistry ,A-site ,030104 developmental biology ,Transfer RNA ,Biophysics ,RNA, Messenger/chemistry ,Guanosine Triphosphate ,RNA, Transfer, Amino Acyl/chemistry ,mutation ,Ribosomes ,Biokemi och molekylärbiologi - Abstract
The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of the monitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.
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- 2018
44. Immunogenicity and protective capacity of EF-Tu and FtsZ of Streptococcus suis serotype 2 against lethal infection
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Weicheng Bei, Jinquan Li, Chao Yuan, Wen Mei, Xiaohong Wang, Stephan P. Willias, Niu Xiaona, Li Weitian, Yuan Liu, and Liping Feng
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0301 basic medicine ,Serotype ,Streptococcus suis ,030106 microbiology ,Peptide Elongation Factor Tu ,law.invention ,DNA vaccination ,Microbiology ,03 medical and health sciences ,Th2 Cells ,Bacterial Proteins ,Antigen ,law ,Streptococcal Infections ,Vaccines, DNA ,Animals ,FtsZ ,Mice, Inbred BALB C ,Streptococcus suis serotype 2 ,General Veterinary ,General Immunology and Microbiology ,biology ,Immunogenicity ,Public Health, Environmental and Occupational Health ,Th1 Cells ,biology.organism_classification ,Cytoskeletal Proteins ,Disease Models, Animal ,030104 developmental biology ,Infectious Diseases ,Bacterial Vaccines ,biology.protein ,Recombinant DNA ,bacteria ,Molecular Medicine ,Female - Abstract
Vaccine development efforts against Streptococcus suis serotype 2 (S. suis 2) are often constrained by strain/serotype antigen variability. Bioinformatics analyses revealed two highly conserved S. suis 2 factors, EF-Tu and FtsZ. Murine immunization with recombinant proteins emulsified in white oil adjuvant or eukaryotic DNA vaccine vectors provided significant protection against lethal S. suis 2 challenge. Immune responses elicited by recombinant protein immunization revealed the robust generation of humoral immune responses, with a mixed induction of Th1-type and Th2-type responses. Furthermore, the antiserum from mice immunized with recombinant proteins significantly inhibited the growth of S. suis 2 in healthy pig whole blood, suggesting the triggering of a strong opsonizing response. Histological examination found that immunizing mice with purified recombinant proteins reduced neutrophil and macrophage accumulation in brain and lung tissues after challenge with virulent S. suis. Taken together, these findings reveal that EF-Tu and FtsZ may be promising targets for subunit and DNA vaccine candidates against S. suis 2 infection.
- Published
- 2018
45. Challenges of site-specific selenocysteine incorporation into proteins byEscherichia coli
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Dieter Söll, Xian Fu, and Anastasia Sevostyanova
- Subjects
Models, Molecular ,0301 basic medicine ,Review ,Peptide Elongation Factor Tu ,Biology ,01 natural sciences ,Amino Acyl-tRNA Synthetases ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,RNA, Transfer ,Escherichia coli ,Molecular Biology ,SECIS element ,chemistry.chemical_classification ,Selenocysteine ,010405 organic chemistry ,Cell Biology ,Protein engineering ,Stop codon ,0104 chemical sciences ,Amino acid ,030104 developmental biology ,chemistry ,Biochemistry ,Genetic Code ,Protein Biosynthesis ,Transfer RNA ,Codon, Terminator ,Nucleic Acid Conformation ,Selenocysteine incorporation ,Genetic Engineering ,Ribosomes ,EF-Tu - Abstract
Selenocysteine (Sec), a rare genetically encoded amino acid with unusual chemical properties, is of great interest for protein engineering. Sec is synthesized on its cognate tRNA (tRNA(Sec)) by the concerted action of several enzymes. While all other aminoacyl-tRNAs are delivered to the ribosome by the elongation factor Tu (EF-Tu), Sec-tRNA(Sec) requires a dedicated factor, SelB. Incorporation of Sec into protein requires recoding of the stop codon UGA aided by a specific mRNA structure, the SECIS element. This unusual biogenesis restricts the use of Sec in recombinant proteins, limiting our ability to study the properties of selenoproteins. Several methods are currently available for the synthesis selenoproteins. Here we focus on strategies for in vivo Sec insertion at any position(s) within a recombinant protein in a SECIS-independent manner: (i) engineering of tRNA(Sec) for use by EF-Tu without the SECIS requirement, and (ii) design of a SECIS-independent SelB route.
- Published
- 2018
46. Gallibacterium elongation factor-Tu possesses amyloid-like protein characteristics, participates in cell adhesion, and is present in biofilms
- Author
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Jaqueline López-Ochoa, Sergio Vaca, Victor M. Pérez-Márquez, Candelario Vázquez, Patrick J. Blackall, Patricia Sánchez-Alonso, J. Fernando Montes-García, and Erasmo Negrete-Abascal
- Subjects
0301 basic medicine ,Amyloid ,Virulence Factors ,Amyloidogenic Proteins ,Peptide Elongation Factor Tu ,Biology ,Applied Microbiology and Biotechnology ,Microbiology ,Bacterial Adhesion ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Protein Domains ,Animals ,Computer Simulation ,Poultry Diseases ,Anatis ,Biofilm ,Congo Red ,General Medicine ,biology.organism_classification ,Enterobacteriaceae ,Congo red ,Bacterial adhesin ,030104 developmental biology ,chemistry ,Biochemistry ,Polyclonal antibodies ,Biofilms ,biology.protein ,Pasteurellaceae ,Pasteurellaceae Infections ,Bacterial outer membrane ,Chickens ,Protein Binding - Abstract
Gallibacterium, which is a bacterial pathogen in chickens, can form biofilms. Amyloid proteins present in biofilms bind Congo red dye. The aim of this study was to characterize the cell-surface amyloid-like protein expressed in biofilms formed by Gallibacterium strains and determine the relationship between this protein and curli, which is an amyloid protein that is commonly expressed by members of the Enterobacteriaceae family. The presence of amyloid-like proteins in outer membrane protein samples from three strains of G. anatis and one strain of Gallibacterium genomospecies 2 was evaluated. A protein identified as elongation factor-Tu (EF-Tu) by mass spectrometric analysis and in silico analysis was obtained from the G. anatis strain F149T. This protein bound Congo red dye, cross-reacted with anti-curli polyclonal serum, exhibited polymerizing properties and was present in biofilms. This protein also reacted with pooled serum from chickens that were experimentally infected with G. anatis, indicating the in vivo immunogenicity of this protein. The recombinant EF-Tu purified protein, which was prepared from G. anatis 12656-12, polymerizes under in vitro conditions, forms filaments and interacts with fibronectin and fibrinogen, all of which suggest that this protein functions as an adhesin. In summary, EF-Tu from G. anatis presents amyloid characteristics, is present in biofilms and could be relevant for the pathogenesis of G. anatis.
- Published
- 2017
47. Identification of a new diagnostic antigen for glanders using immunoproteome analysis
- Author
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Sudhir K. Dohre, Aayushi Kamthan, Syed Imteyaz Alam, Sandeep Singh, and Subodh Kumar
- Subjects
Proteomics ,0301 basic medicine ,Immunoproteins ,Hydrolases ,Immunoblotting ,030106 microbiology ,Immunology ,Enzyme-Linked Immunosorbent Assay ,Peptide Elongation Factor Tu ,Burkholderia mallei ,Sensitivity and Specificity ,Microbiology ,law.invention ,03 medical and health sciences ,Affinity chromatography ,Antigen ,Malate Dehydrogenase ,law ,Escherichia coli ,medicine ,Animals ,Immunology and Allergy ,Serologic Tests ,Horses ,Arginine deiminase ,GroEL Protein ,Antigens, Bacterial ,General Veterinary ,biology ,Glanders ,Chaperonin 60 ,General Medicine ,Peptide Elongation Factors ,biology.organism_classification ,medicine.disease ,Antibodies, Bacterial ,GroEL ,Molecular biology ,Recombinant Proteins ,030104 developmental biology ,Infectious Diseases ,Recombinant DNA ,Horse Diseases - Abstract
Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.
- Published
- 2017
48. Novel mutation in mitochondrial Elongation Factor EF-Tu associated to dysplastic leukoencephalopathy and defective mitochondrial DNA translation
- Author
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Daria Diodato, Rosalba Carrozzo, Enrico Bertini, Marzia Bianchi, Carlo Dionisi-Vici, Arianna Montanari, Teresa Rizza, Massimo Zeviani, Erika Fernandez-Vizarra, Michela Di Nottia, Romina Oliva, Alessandra Torraco, Michela Catteruccia, Daniela Verrigni, and Silvia Francisci
- Subjects
0301 basic medicine ,Male ,Mitochondrial DNA ,Mitochondrial translation ,Mutant ,Peptide Chain Elongation, Translational ,Saccharomyces cerevisiae ,Biology ,Leukodystrophy ,OXPHOS defects ,TUFM ,DNA, Mitochondrial ,Female ,Humans ,Leukoencephalopathies ,Mitochondria ,Mitochondrial Proteins ,Peptide Elongation Factor Tu ,Ribosomes ,Base Sequence ,Sequence Deletion ,Article ,03 medical and health sciences ,Mitochondrial ribosome ,Gene ,Molecular Biology ,Peptide Chain Elongation ,Molecular Medicine ,Translational ,Translation (biology) ,DNA ,Molecular biology ,Mitochondrial ,Elongation factor ,030104 developmental biology ,EF-Tu - Abstract
The mitochondrial Elongation Factor Tu (EF-Tu), encoded by the TUFM gene, is a highly conserved GTPase, which is part of the mitochondrial protein translation machinery. In its activated form it delivers the aminoacyl-tRNAs to the A site of the mitochondrial ribosome. We report here on a baby girl with severe infantile macrocystic leukodystrophy with micropolygyria and a combined defect of complexes I and IV in muscle biopsy, caused by a novel mutation identified in TUFM. Using human mutant cells and the yeast model, we demonstrate the pathological role of the novel variant. Moreover, results of a molecular modeling study suggest that the mutant is inactive in mitochondrial polypeptide chain elongation, probably as a consequence of its reduced ability to bind mitochondrial aa-tRNAs. Four patients have so far been described with mutations in TUFM, and, following the first description of the disease in a single patient, we describe similar clinical and neuroradiological features in an additional patient., Highlights • Using a custom-targeted panel, a novel TUFM mutation was found. • The patient shows a severe infantile macrocystic leukodystrophy with micropolygyria. • In Saccharomyces cerevisiae the mutation impairs mitochondrial respiration. • The 3D model supports that the mutation destabilizes the protein structure.
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- 2017
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49. Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in E. coli and isolated by metal affinity chromatography
- Author
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M. Trawkina, Sergei A. Usanov, Ya. V. Dzichenka, G. V. Sergeev, M. A. Shapiro, Andrey Gilep, A. V. Ivanchik, A. V. Yantsevich, and T. V. Shkel
- Subjects
Hemeproteins ,Proteomics ,Ribosomal Proteins ,0301 basic medicine ,Genetic Vectors ,Gene Expression ,Peptide Elongation Factor Tu ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Biochemistry ,Chromatography, Affinity ,Copurification ,Chaperonin ,03 medical and health sciences ,Affinity chromatography ,FLAG-tag ,Ribosomal protein ,Escherichia coli ,medicine ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Heat-Shock Proteins ,Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) ,Chromatography ,Flavoproteins ,030102 biochemistry & molecular biology ,Chemistry ,Escherichia coli Proteins ,Sepharose ,Peptidylprolyl Isomerase ,Catalase ,Recombinant Proteins ,030104 developmental biology ,Proteome ,Myc-tag - Abstract
Contaminating proteins have been identified by “shotgun” proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.
- Published
- 2017
50. Expression and immunological evaluation of elongation factor Tu of Streptococcus suis serotype 2
- Author
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S G Li, Z Q Shen, L Wang, Xiaojing Xia, L K Cheng, and J L Wang
- Subjects
0301 basic medicine ,Streptococcus suis ,Peptide Elongation Factor Tu ,Biology ,Serogroup ,medicine.disease_cause ,Microbiology ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,Plasmid ,Bacterial Proteins ,Antigen ,law ,Streptococcal Infections ,medicine ,Animals ,030212 general & internal medicine ,Cloning, Molecular ,Escherichia coli ,Pathogen ,Antiserum ,Streptococcus suis serotype 2 ,General Veterinary ,Gene Expression Regulation, Bacterial ,General Medicine ,biology.organism_classification ,Antibodies, Bacterial ,Virology ,030104 developmental biology ,Bacterial Vaccines ,Recombinant DNA ,Rabbits - Abstract
Streptococcus suis serotype 2 (SS2) is considered as a major pathogen that causes sepsis and meningitis in piglets and humans, but knowledge of its antigenic proteins remains limited so far. The surface-related proteins of pathogens often play significant roles in bacterium-host interactions and infection. Here, we obtained the elongation factor Tu (EF-Tu) gene of Streptococcus suis and constructed the recombinant expression plasmid successfully. The target recombinant plasmid was then expressed in Escherichia coli and the immuno-protection of the recombinant protein was subsequently evaluated as well. The EF-Tu gene of Streptococcus suis is 1197 bp in length, encodes 398 amino acids. The target recombinant EF-Tu (rEF-Tu) protein can recognize the antiserum of Streptococcus suis and can provoke obvious humoral immune responses in rabbits and conferred protection to rabbits against Streptococcus suis ear-vein challenge, implying that the EF-Tu may be used as an attractive candidate antigen for a component of subunit vaccine.
- Published
- 2017
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