scholarly journals Spectroscopic Investigation of the Kinetic Mechanism Involved in the Association of Potyviral VPg with the Host Plant Translation Initiation Factor eIF4E

2020 ◽  
Vol 21 (16) ◽  
pp. 5618
Author(s):  
Jocelyne Walter ◽  
Amandine Barra ◽  
Justine Charon ◽  
Geneviève Tavert-Roudet ◽  
Thierry Michon
Keyword(s):  
Amino Acids ◽  
Translation Initiation ◽  
Plant Resistance ◽  
Kinetic Mechanism ◽  
Single Step ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Intrinsically Disordered ◽  
Resistance Breaking ◽  
Central Helix

The infectious cycle of potyviruses requires the formation of a complex between the viral genome-linked protein VPg and the host eukaryotic translation initiation factor 4E, eIF4E. Mutations associated with plant resistance to potyviruses were previously mapped at the eIF4E surface, while on the virus side, mutations leading to plant resistance breaking were identified within the VPg. In the present study, fluorescence spectroscopy was used to probe the contribution of the VPg intrinsically disordered region bearing amino acids determinant of the resistance breaking, to the VPg–eIF4E binding mechanism. Synthetic peptides encompassing the VPg88–120 central region were found to tightly bind to eIF4E. Fluorescence energy transfer experiments show that, upon binding to eIF4E, the N and C termini of the VPg88–111 fragment move closer to one another, at a distance compatible with a α-helix folding. When the VPg112–120 region, which contains amino acids associated with resistance breakdown, is appended to VPg88–111, the complex formation with eIF4E switches from a single-step to a two-step kinetic model. This study revisits a recent investigation of the VPg–eIF4E complex by specifying the contribution of the VPg central helix and its appended disordered region to VPg association with eIF4E.

scholarly journals Control of Paip1-Eukayrotic Translation Initiation Factor 3 Interaction by Amino Acids through S6 Kinase

2014 ◽  
Vol 34 (6) ◽  
pp. 1046-1053 ◽  
Author(s):  
Y. Martineau ◽  
X. Wang ◽  
T. Alain ◽  
E. Petroulakis ◽  
D. Shahbazian ◽  
...  
Keyword(s):  
Amino Acids ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
S6 Kinase ◽  
Translation Initiation Factor 3

scholarly journals Disparate Phenotypes Resulting from Mutations of a Single Histidine in Switch II of Geobacillus stearothermophilus Translation Initiation Factor IF2

2020 ◽  
Vol 21 (3) ◽  
pp. 735
Author(s):  
Jerneja Tomsic ◽  
Arianna Smorlesi ◽  
Enrico Caserta ◽  
Anna Maria Giuliodori ◽  
Cynthia L. Pon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Viability ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Geobacillus Stearothermophilus ◽  
Wild Type ◽  
Initiation Complex ◽  
Structural Alterations

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 ≥ to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.

scholarly journals The Transformation Suppressor Pdcd4 Is a Novel Eukaryotic Translation Initiation Factor 4A Binding Protein That Inhibits Translation

2003 ◽  
Vol 23 (1) ◽  
pp. 26-37 ◽  
Author(s):  
Hsin-Sheng Yang ◽  
Aaron P. Jansen ◽  
Anton A. Komar ◽  
Xiaojing Zheng ◽  
William C. Merrick ◽  
...  
Keyword(s):  
Amino Acids ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Tumor Promoter ◽  
Initiation Factor ◽  
Middle Region ◽  
Entry Site ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an ATP-dependent RNA helicase needed to unwind 5′ mRNA secondary structure. Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4D418A, a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both Pdcd4 and Pdcd4D418A bound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1.

scholarly journals Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

2008 ◽  
Vol 295 (4) ◽  
pp. E876-E883 ◽  
Author(s):  
Fiona A. Wilson ◽  
Agus Suryawan ◽  
Renán A. Orellana ◽  
Hanh V. Nguyen ◽  
Asumthia S. Jeyapalan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Translation Initiation ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Growing Pigs ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Skeletal Muscle Protein

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 μg·kg−1·day−1) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) ( P < 0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level ( P < 0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1·eIF4E complex association, and increased active eIF4E·eIF4G complex formation ( P < 0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

scholarly journals Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise

2016 ◽  
Vol 310 (11) ◽  
pp. C874-C884 ◽  
Author(s):  
Marcus Moberg ◽  
William Apró ◽  
Björn Ekblom ◽  
Gerrit van Hall ◽  
Hans-Christer Holmberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Resistance Exercise ◽  
Translation Initiation ◽  
Random Order ◽  
Essential Amino Acids ◽  
Branched Chain Amino Acids ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mtorc1 Signaling ◽  
Branched Chain

Protein synthesis is stimulated by resistance exercise and intake of amino acids, in particular leucine. Moreover, activation of mammalian target of rapamycin complex 1 (mTORC1) signaling by leucine is potentiated by the presence of other essential amino acids (EAA). However, the contribution of the branched-chain amino acids (BCAA) to this effect is yet unknown. Here we compare the stimulatory role of leucine, BCAA, and EAA ingestion on anabolic signaling following exercise. Accordingly, eight trained volunteers completed four sessions of resistance exercise during which they ingested either placebo, leucine, BCAA, or EAA (including the BCAA) in random order. Muscle biopsies were taken at rest, immediately after exercise, and following 90 and 180 min of recovery. Following 90 min of recovery the activity of S6 kinase 1 (S6K1) was greater than at rest in all four trials (Placebo<Leucine<BCAA<EAA; P < 0.05 time × supplement), with a ninefold increase in the EAA trial. At this same time point, phosphorylation of eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) at Thr37/46 was unaffected by supplementation, while that of Thr46 alone exhibited a pattern similar to that of S6K1, being 18% higher with EAA than BCAA. However, after 180 min of recovery this difference between EAA and BCAA had disappeared, although with both these supplements the increases were still higher than with leucine (40%, P < 0.05) and placebo (100%, P < 0.05). In summary, EAA ingestion appears to stimulate translation initiation more effectively than the other supplements, although the results also suggest that this effect is primarily attributable to the BCAA.

scholarly journals The role of olefin geometry in the activity of hydrocarbon stapled peptides targeting eukaryotic translation initiation factor 4E (eIF4E)

2019 ◽  
Vol 17 (26) ◽  
pp. 6414-6419 ◽  
Author(s):  
James M. Song ◽  
Erin E. Gallagher ◽  
Arya Menon ◽  
Lauren D. Mishra ◽  
Amanda L. Garner
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Ring Closing Metathesis ◽  
Stapled Peptides ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

Ring-closing metathesis of monosubstituted alkenyl amino acids leads to stapled peptides with differing olefin geometry and biological activity.

scholarly journals Hydrodynamic Behavior of the Intrinsically Disordered Potyvirus Protein VPg, of the Translation Initiation Factor eIF4E and of their Binary Complex

2019 ◽  
Vol 20 (7) ◽  
pp. 1794 ◽  
Author(s):  
Jocelyne Walter ◽  
Amandine Barra ◽  
Bénédicte Doublet ◽  
Nicolas Céré ◽  
Justine Charon ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Molten Globule ◽  
Translation Initiation Factor ◽  
Size Exclusion ◽  
Initiation Factor ◽  
Binary Complex ◽  
Hydrodynamic Behavior ◽  
Hydrodynamic Properties ◽  
Intrinsically Disordered ◽  
His Tag

Protein intrinsic disorder is involved in many biological processes and good experimental models are valuable to investigate its functions. The potyvirus genome-linked protein, VPg, displays many features of an intrinsically disordered protein. The virus cycle requires the formation of a complex between VPg and eIF4E, one of the host translation initiation factors. An in-depth characterization of the hydrodynamic properties of VPg, eIF4E, and of their binary complex VPg-eIF4E was carried out. Two complementary experimental approaches, size-exclusion chromatography and fluorescence anisotropy, which is more resolving and revealed especially suitable when protein concentration is the limiting factor, allowed to estimate monomers compaction upon complex formation. VPg possesses a high degree of hydration which is in agreement with its classification as a partially folded protein in between a molten and pre-molten globule. The natively disordered first 46 amino acids of eIF4E contribute to modulate the protein hydrodynamic properties. The addition of an N-ter His tag decreased the conformational entropy of this intrinsically disordered region. A comparative study between the two tagged and untagged proteins revealed the His tag contribution to proteins hydrodynamic behavior.

scholarly journals Central domain of a potyvirus VPg is involved in the interaction with the host translation initiation factor eIF4E and the viral protein HcPro

2007 ◽  
Vol 88 (3) ◽  
pp. 1029-1033 ◽  
Author(s):  
G. Roudet-Tavert ◽  
T. Michon ◽  
J. Walter ◽  
T. Delaunay ◽  
E. Redondo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Recombinant Proteins ◽  
Translation Initiation ◽  
Viral Protein ◽  
Binding Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Biological Functions ◽  
Central Domain

Using recombinant proteins produced in bacteria or in infected plants, interactions between the VPg and HcPro of Lettuce mosaic potyvirus (LMV) and between LMV VPg and the lettuce translation initiation factor 4E, the cap-binding protein (eIF4E), were demonstrated in vitro. Interaction with eIF4E and HcPro both involved the same VPg central domain. The structure of this domain in the VPg context was predicted to include an amphiphilic α-helix, with the amino acids related to biological functions in various potyviruses exposed at the hydrophilic side.

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