Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Computational study on the allosteric mechanism of Leishmania major IF4E-1 by 4E-interacting protein-1: Unravelling the determinants of m⁷GTP cap recognition.

ABSTRACT: During their life cycle, Leishmania parasites display a fine-tuned regulation of the mRNA translation through the differential expression of isoforms of eukaryotic translation initiation factor 4E (LeishIF4Es). The interaction between allosteric modulators such as 4E-interacting proteins (4E-IPs) and LeishIF4E affects the affinity of this initiation factor for the mRNA cap. Here, several computational approaches were employed to elucidate the molecular bases of the previously-reported allosteric modulation in L. major exerted by 4E-IP1 (Lm4E-IP1) on eukaryotic translation initiation factor 4E 1 (LmIF4E-1). Molecular dynamics (MD) simulations and accurate binding free energy calculations (ΔG_bind ) were combined with network-based modeling of residue-residue correlations. We also describe the differences in internal motions of LmIF4E-1 apo form, cap-bound, and Lm4E-IP1-bound systems. Through community network calculations, the differences in the allosteric pathways of allosterically-inhibited and active forms of LmIF4E-1 were revealed. The ΔG_bind values show significant differences between the active and inhibited systems, which are in agreement with the available experimental data. Our study thoroughly describes the dynamical perturbations of LmIF4E-1 cap-binding site triggered by Lm4E-IP1. These findings are not only essential for the understanding of a critical process of trypanosomatids' gene expression but also for gaining insight into the allostery of eukaryotic IF4Es, which could be useful for structure-based design of drugs against this protein family.

SUBMITTER: Hernandez-Alvarez L

PROVIDER: S-EPMC8085901 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

1H, 13C, and 15N backbone chemical shift assignments of m7GTP cap-bound Leishmania initiation factor 4E-1.

Project description:Most of the translational control of gene expression in higher eukaryotes occurs during the initiation step of protein synthesis. While this process is well characterized in mammalian cells, it is less defined in parasites, including the ones that cause human Leishmaniasis. The Leishmania cap-binding isoform 1 (LeishIF4E-1) is the only isoform that binds the specific trypanosomatids-specific hypermethylated 5' cap, called cap-4, in the human stage of the parasite life cycle. We report here the extensive NMR resonance assignment of LeishIF4E-1 bound to a cap analog, m7GTP. The chemical shift data constitute a prerequisite to understanding specific translation initiation mechanisms used in Leishmania parasites and to developing antiparasitic drugs targeting their translation initiation factors.

| S-EPMC7462832 | biostudies-literature

A novel 4E-interacting protein in Leishmania is involved in stage-specific translation pathways.

Project description:In eukaryotes, exposure to stress conditions causes a shift from cap-dependent to cap-independent translation. In trypanosomatids, environmental switches are the driving force of a developmental program of gene expression, but it is yet unclear how their translation machinery copes with their constantly changing environment. Trypanosomatids have a unique cap structure (cap-4) and encode four highly diverged paralogs of the cap-binding protein, eIF4E; none were found to genetically complement a yeast mutant failing to express eIF4E. Here we show that in promastigotes, a typical cap-binding complex is anchored through LeishIF4E-4, which associates with components of the cap-binding pre-initiation complex. In axenic amastigotes, expression of LeishIF4E-4 decreases and the protein does not bind the cap, whereas LeishIF4E-1 maintains its expression level and associates with the cap structure and with translation initiation factors. However, LeishIF4E-1 does not interact with eIF4G-like proteins in both life stages, excluding its involvement in cap-dependent translation. Using pull-down assays and mass-spectrometry, we identified a novel, non-conserved 4E-Interacting Protein (Leish4E-IP), which binds to LeishIF4E-1 in promastigotes, but not in amastigotes. Yeast two-hybrid and NMR spectroscopy confirmed the specificity of this interaction. We propose that Leish4E-IP is a translation regulator that is involved in switching between cap-dependent and alternative translation pathways.

| S-EPMC3201875 | biostudies-other

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability.

Project description:Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length determines microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.

| S-EPMC7018511 | biostudies-literature

A newly identified Leishmania IF4E-interacting protein, Leish4E-IP2, modulates the activity of cap-binding protein paralogs.

Project description:Translation of most cellular mRNAs in eukaryotes proceeds through a cap-dependent pathway, whereby the cap-binding complex, eIF4F, anchors the preinitiation complex at the 5' end of mRNAs and regulates translation initiation. The requirement of Leishmania to survive in changing environments can explain why they encode multiple eIF4E (LeishIF4Es) and eIF4G (LeishIF4Gs) paralogs, as each could be assigned a discrete role during their life cycle. Here we show that the expression and activity of different LeishIF4Es change during the growth of cultured promastigotes, urging a search for regulatory proteins. We describe a novel LeishIF4E-interacting protein, Leish4E-IP2, which contains a conserved Y(X)4L? IF4E-binding-motif. Despite its capacity to bind several LeishIF4Es, Leish4E-IP2 was not detected in m7GTP-eluted cap-binding complexes, suggesting that it could inhibit the cap-binding activity of LeishIF4Es. Using a functional assay, we show that a recombinant form of Leish4E-IP2 inhibits the cap-binding activity of LeishIF4E-1 and LeishIF4E-3. Furthermore, we show that transgenic parasites expressing a tagged version of Leish4E-IP2 also display reduced cap-binding activities of tested LeishIF4Es, and decreased global translation. Given its ability to bind more than a single LeishIF4E, we suggest that Leish4E-IP2 could serve as a broad-range repressor of Leishmania protein synthesis.

| S-EPMC7192595 | biostudies-literature

Estimating the microtubule GTP cap size in vivo.

Project description:Microtubules (MTs) polymerize via net addition of GTP-tubulin subunits to the MT plus end, which subsequently hydrolyze to GDP-tubulin in the MT lattice. Relatively stable GTP-tubulin subunits create a "GTP cap" at the growing MT plus end that suppresses catastrophe. To understand MT assembly regulation, we need to understand GTP hydrolysis reaction kinetics and the GTP cap size. In vitro, the GTP cap has been estimated to be as small as one layer (13 subunits) or as large as 100-200 subunits. GTP cap size estimates in vivo have not yet been reported. Using EB1-EGFP as a marker for GTP-tubulin in epithelial cells, we find on average (1) 270 EB1 dimers bound to growing MT plus ends, and (2) a GTP cap size of ?750 tubulin subunits. Thus, in vivo, the GTP cap is far larger than previous estimates in vitro, and ?60-fold larger than a single layer cap. We also find that the tail of a large GTP cap promotes MT rescue and suppresses shortening. We speculate that a large GTP cap provides a locally concentrated scaffold for tip-tracking proteins and confers persistence to assembly in the face of physical barriers such as the cell cortex.

| S-EPMC3461128 | biostudies-literature

Hyperglycemia mediates a shift from cap-dependent to cap-independent translation via a 4E-BP1-dependent mechanism.

Project description:Diabetes and its associated hyperglycemia induce multiple changes in liver function, yet we know little about the role played by translational control of gene expression in mediating the responses to these conditions. Here, we evaluate the hypothesis that hyperglycemia-induced O-GlcNAcylation of the translational regulatory protein 4E-BP1 alters hepatic gene expression through a process involving the selection of mRNA for translation. In both streptozotocin (STZ)-treated mice and cells in culture exposed to hyperglycemic conditions, expression of 4E-BP1 and its interaction with the mRNA cap-binding protein eIF4E were enhanced in conjunction with downregulation of cap-dependent and concomitant upregulation of cap-independent mRNA translation, as assessed by a bicistronic luciferase reporter assay. Phlorizin treatment of STZ-treated mice lowered blood glucose concentrations and reduced activity of the cap-independent reporter. Notably, the glucose-induced shift from cap-dependent to cap-independent mRNA translation did not occur in cells lacking 4E-BP1. The extensive nature of this shift in translational control of gene expression was revealed using pulsed stable isotope labeling by amino acids in cell culture to identify proteins that undergo altered rates of synthesis in response to hyperglycemia. Taken together, these data provide evidence for a novel mechanism whereby O-GlcNAcylation of 4E-BP1 mediates translational control of hepatic gene expression.

| S-EPMC3712054 | biostudies-literature

Tau Avoids the GTP Cap at Growing Microtubule Plus-Ends.

Project description:Plus-end tracking proteins (+TIPs) associate with the growing end of microtubules and mediate important cellular functions. The majority of +TIPs are directed to the plus-end through a family of end-binding proteins (EBs), which preferentially bind the stabilizing cap of GTP-tubulin present during microtubule growth. One outstanding question is whether there may exist other microtubule-associated proteins (MAPs) that preferentially bind specific nucleotide states of tubulin. Here, we report that the neuronal MAP tau preferentially binds GDP-tubulin (K D = 0.26 μM) over GMPCPP-tubulin (K D = 1.1 μM) in vitro, as well as GTP-tubulin at the tips of growing microtubules, causing tau binding to lag behind the plus-end both in vitro and in live cells. Thus, tau is a microtubule tip avoiding protein, establishing the framework for a possible new class of tip avoiding MAPs. We speculate that disease-relevant tau mutations may exert their phenotype by their failure to properly recognize GDP-tubulin.

| S-EPMC7691178 | biostudies-literature

Opposite allosteric mechanisms in TetR and CAP.

Project description:Regulation of the DNA binding affinity of an oligomeric protein can be considered to consist of an intrinsic component, in which the affinity of an individual DNA-binding domain is modulated in response to effector binding, and an extrinsic component, in which the relative position of the protein's two DNA-binding domains are altered so that they can or cannot contact both half-site operators simultaneously. We demonstrated directly that the TetR repressor utilizes an extrinsic mechanism and CAP, the catabolite activator protein, utilizes an intrinsic mechanism.

| S-EPMC2762589 | biostudies-literature

Identification of proteins interacting with GTP cyclohydrolase I.

Project description:GTP cyclohydrolase I (GCH-1) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, an essential cofactor for nitric oxide synthase and aromatic amino acid hydroxylase. To explore the interactome of GCH-1, we established a HEK 293 cell line stably expressing tetracycline-inducible FLAG-GCH-1. FLAG-GCH-1 and associated proteins were immunoprecipitated and analyzed by liquid chromatography/tandem mass spectrometry. Twenty-nine proteins, derived from different subcellular components such as cytosol, membranes, nucleus and mitochondria were identified to interact with GCH-1. Cell fractionation studies also showed that GCH-1 was present in the cytosol, membranes and nucleus. Gene ontology analysis revealed that GCH-1 interactome was involved in a variety of biological processes such as signal transduction, apoptosis, metabolism, transport and cell organization. To our knowledge, this study is the first to provide a comprehensive analysis of the GCH-1 interactome. Findings expand the number and diversity of proteins that are known to associate with GCH-1.

| S-EPMC2737525 | biostudies-literature

Mechanism of allosteric regulation of transglutaminase 2 by GTP.

Project description:Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the beta-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.

| S-EPMC1750866 | biostudies-literature