scholarly journals Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P

2017 ◽  
Author(s):  
Ralph Krafczyk ◽  
Jakub Macošek ◽  
Daniel Gast ◽  
Swetlana Wunder ◽  
Pravin Kumar Ankush Jagtap ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Structural Data ◽  
Enzyme Characterization ◽  
Structural Basis ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Enzymatic Mechanism ◽  
Bacterial Translation

ABSTRACTGlycosylation is a universal strategy to post-translationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann-folds, thus constituting a GT-B glycosyltransferase. While TDP-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the EF-P via its KOW-like N-domain. Based on our structural data combined with an in vitro /in vivo enzyme characterization, we propose a mechanism of inverting arginine glycosylation. As EarP is essential for pathogenicity in P. aeruginosa our study provides the basis for targeted inhibitor design.

scholarly journals Overexpression of Translation Elongation Factor 1A Affects the Organization and Function of the Actin Cytoskeleton in Yeast

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1425-1436
Author(s):  
Raj Munshi ◽  
Kimberly A Kandl ◽  
Anne Carr-Schmid ◽  
Johanna L Whitacre ◽  
Alison E M Adams ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Elongation Factor ◽  
Nucleotide Exchange ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Function

Abstract The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1Bα, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

scholarly journals Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Ralph Krafczyk ◽  
Jakub Macošek ◽  
Pravin Kumar Ankush Jagtap ◽  
Daniel Gast ◽  
Swetlana Wunder ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Structural Model ◽  
Biochemical Characterization ◽  
Elongation Factor ◽  
Structural Basis ◽  
Inhibitor Design ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Enzymatic Mechanism

ABSTRACT Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-β-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design. IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection. IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection.

scholarly journals Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A

Science ◽  
2021 ◽  
Vol 371 (6532) ◽  
pp. 926-931 ◽  
Author(s):  
Kris M. White ◽  
Romel Rosales ◽  
Soner Yildiz ◽  
Thomas Kehrer ◽  
Lisa Miorin ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Elongation Factor ◽  
Resistant Mutant ◽  
Host Protein ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Drug Resistant Mutant

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

scholarly journals Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system

2002 ◽  
Vol 365 (3) ◽  
pp. 669-676 ◽  
Author(s):  
Francisco MANSILLA ◽  
Irene FRIIS ◽  
Mandana JADIDI ◽  
Karen M. NIELSEN ◽  
Brian F.C. CLARK ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Translation Elongation Factor ◽  
Yeast Two Hybrid ◽  
Translation Elongation ◽  
Guanine Nucleotide Exchange ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1Bα, eEF1Bβ and eEF1Bγ. The first two subunits possess the nucleotide-exchange activity, whereas the role of the last remains poorly defined. In mammals, two active tissue-specific isoforms of eEF1A have been identified. The reason for this pattern of differential expression is unknown. Several models on the basis of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1Bβ and eEF1Bγ subunits in vitro. Here, the human eEF1H complex is for the first time mapped using the yeast two-hybrid system, which is a powerful in vivo technique for analysing protein—protein interactions. The following complexes were observed: eEF1A1:eEF1Bα, eEF1A1:eEF1Bβ, eEF1Bβ:eEF1Bβ, eEF1Bα:eEF1Bγ, eEF1Bβ:eEF1Bγ and eEF1Bα:eEF1Bγ:eEF1Bβ, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit is a pentamer composed of two molecules of eEF1A, each interacting with either eEF1Bα or eEF1Bβ held together by eEF1Bγ. These units can dimerize via eEF1Bβ. Our model is compared with other models, and structural as well as functional aspects of the model are discussed.

scholarly journals Comparison of the ability of mammalian eEF1A1 and its oncogenic variant eEF1A2 to interact with actin and calmodulin

2017 ◽  
Vol 398 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Oleksandra Novosylna ◽  
Annette Doyle ◽  
Dmytro Vlasenko ◽  
Mark Murphy ◽  
Boris Negrutskii ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Oncogenic Potential ◽  
Regulatory Systems ◽  
Protein Variants ◽  
Homologous Tissue ◽  
Trna Binding

Abstract The question as to why a protein exerts oncogenic properties is answered mainly by well-established ideas that these proteins interfere with cellular signaling pathways. However, the knowledge about structural and functional peculiarities of the oncoproteins causing these effects is far from comprehensive. The 97.5% homologous tissue-specific A1 and A2 isoforms of mammalian translation elongation factor eEF1A represent an interesting model to study a difference between protein variants of a family that differ in oncogenic potential. We propose that the different oncogenic impact of A1 and A2 might be explained by differences in their ability to communicate with their respective cellular partners. Here we probed this hypothesis by studying the interaction of eEF1A with two known partners – calmodulin and actin. Indeed, an inability of the A2 isoform to interact with calmodulin is shown, while calmodulin is capable of binding A1 and interferes with its tRNA-binding and actin-bundling activities in vitro. Both A1 and A2 variants revealed actin-bundling activity; however, the form of bundles formed in the presence of A1 or A2 was distinctly different. Thus, a potential inability of A2 to be controlled by Ca2+-mediated regulatory systems is revealed.

scholarly journals Translation Elongation Factor EF-Tu Modulates Filament Formation of Actin-Like MreB Protein In Vitro

2015 ◽  
Vol 427 (8) ◽  
pp. 1715-1727 ◽  
Author(s):  
Hervé Joël Defeu Soufo ◽  
Christian Reimold ◽  
Hannes Breddermann ◽  
Hans G. Mannherz ◽  
Peter L. Graumann
Keyword(s):  
Elongation Factor ◽  
Filament Formation ◽  
Translation Elongation Factor ◽  
Translation Elongation

scholarly journals In vivo characterization of the role of tissue‐specific translation elongation factor 1 A 2 in protein synthesis reveals insights into muscle atrophy

FEBS Journal ◽  
2013 ◽  
Vol 280 (24) ◽  
pp. 6528-6540 ◽  
Author(s):  
Jennifer Doig ◽  
Lowri A. Griffiths ◽  
David Peberdy ◽  
Permphan Dharmasaroja ◽  
Maria Vera ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Muscle Atrophy ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
In Vivo Characterization ◽  
Elongation Factor 1
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