The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2β

Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons.     

Role of mitochondria in apoptosis induced by the 2‐5A system and mechanisms involved

The 2‐5A system (2-5OAS/RNaseL) is composed of the 2′,5′oligoadenylate synthetase 1 (2-5OAS1) and 2-5A-dependent RNase (RNaseL), enzymes that play a key role in antiviral defence mechanisms. Activation of the 2-5A system by double stranded RNA (dsRNA) induces degradation of ribosomal RNAs and apoptosis in mammalian cells. To obtain further information into the molecular mechanisms by which RNaseL induces apoptosis, we expressed human RNaseL and 2-5OAS in HeLa cells using recombinant vaccinia viruses as vectors and we analysed in detail different biochemical markers of apoptosis. In this expression virus-cell system the activation of RNaseL, as index of rRNA degradation, is an upstream event of apoptosis induction. RNaseL induces apoptosis in a caspase-dependent manner (caspases 8, 9 and 2). At the beginning of apoptosis RNaseL and 2-5OAS are localized in the mitochondria and cytosol fractions, while at the onset of apoptosis both enzymes are largely in mitochondria. The 2-5A system induces the release of Cytochrome c from mitochondria to cytosol in a caspase dependent manner. The onset of apoptosis elicits the disruption of mitochondrial membrane potential (ΔΦm), as well as the generation of reactive oxygen species (ROS). Moreover, the activation of RNaseL induces morphological alterations in the mitochondria. Apoptosis induced by the 2-5A system involves mitochondrial proteins, such as the human anti-apoptotic protein Bcl-2, which blocks both the apoptosis and the change of ΔΦm induced by the activation of RNaseL. These findings provide new insights into the molecular mechanisms of apoptosis induction by the 2-5A system, demonstrating the importance of mitochondria in 2-5OAS/RNaseL-induced apoptosis.     

Structural basis of γH2AX recognition by human PTIP BRCT5-BRCT6 domains in the DNA damage response pathway

Human Pax2 transactivation domain-interacting protein (hPTIP), containing six BRCT domains, is an essential protein required for the IR induced DDR process with an unclear role. Here we report that the tandem BRCT5–BRCT6 domain of hPTIP recognizes the γH2AX tail, and this interaction depends on the phosphorylation of H2AX Ser139 and binding with the carboxyl ending peptide to the aminoacyl ending peptide. The 2.15 Å crystal structure of hPTIP BRCT5/6–γH2AX complex and mutation analysis provide molecular evidence for direct interactions between PTIP and γH2AX. This interaction proffers a new clue to identify the role of PTIP in DDR pathways.

Structured summary of protein interactions

PTIP and gamma H2AXbind by fluorescence polarization spectroscopy (View Interaction: 1, 2, 3, 4, 5, 6).PTIP and gamma H2AXbind by X-ray crystallography (View interaction).     

A modified rinsing method for the determination of the S,W–S and D + U fraction of protein and starch in feedstuff within the in situ technique

A modified rinsing method for the in situ technique was developed to separate, isolate and characterise the soluble (S), the insoluble washout (W–S) and the non-washout fractions (D + U) within one procedure. For non-incubated bags (t = 0 h), this method was compared with the conventional, Combined Fractionation (CF) method that measures the D + U and S fractions in separate steps and subsequently calculates the W–S fraction. The modified method was based on rinsing of nylon bags in a closed vessel containing a buffer solution (pH 6.2) during 1 h, where shaking speeds of 40, 100, and 160 strokes per minutes (spm) were evaluated, and tested for six feed ingredients (faba beans, maize, oats, peas, soya beans and wheat) and four forages (two ryegrass silages and two maize silages). The average recoveries as the sum of all fractions were 0.972 ± 0.041 for N and 0.990 ± 0.050 for starch (mean ± s.d.). The mean W–S fraction increased with increasing shaking speed and varied between 0.017 (N) and 0.083 (starch) at 40 spm and 0.078 (N) and 0.303 (starch) at 160 spm, respectively. For ryegrass silages, the W–S fraction was absent at all shaking speeds, but was present in the CF method. The modified method, in particular at 40 and 100 spm, reduced the loss of small particles during rinsing, resulting in lower W–S and higher D + U fractions for N and starch compared with the CF method. For soya beans and ryegrass silage, the modified method reduced the S fraction of N compared with the CF method. The results obtained at 160 spm showed the best comparison with those from the CF method. The W–S fraction of the feedstuff obtained at 160 spm contained mainly particles smaller than 40 μm (0.908 ± 0.086). In most feedstuff, starch was the most abundant chemical component in the W–S fraction and its content (726 ± 75 g/kg DM) was higher than in the D + U fraction (405 ± 177 g/kg DM). Alkaline-soluble proteins were the dominant N-containing components in the W–S fraction of dry feed ingredients and its relative content (0.79 ± 0.18 of total N in W–S) was higher than in the D + U fraction (0.59 ± 0.07 of total N in D + U) for all feedstuff except maize. The molecular weight distribution of the alkaline-soluble proteins differed between the W–S and the D + U fractions of all dry feed ingredients, except soya beans and wheat.     

Binding of β4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry

Characterization of G protein βγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native βγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and βγ labeled with heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched βγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing β and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three β isoforms, β(1), β(2), and β(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. β(1) and γ(5) were most abundant in the enriched βγ fraction, and this βγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more β(4) and γ(5) compared to the enriched βγ fraction; also, more β(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched βγ fraction. These results suggest that preferences for particular βγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.     

Catalysis and Selectivity in Prebiotic Synthesis: Initiation of the Formation of Oligo(U)s on Montmorillonite Clay by Adenosine-5′-methylphosphate

Adenosine-5′-methylphosphate (MepA) initiates the oligomerization of the 5′-phosphorimidazolide of uridine (ImpU) in the presence of montmorillonite clay. Longer oligomers form because the 5′-phosphate is blocked with a methyl group that prevents the formation of cyclic- and pyrophosphate-containing compounds. The MepA initiates 69–84% of the 5–9 charge oligomers, respectively. The montmorillonite catalyst also provides selectivity in the oligomerization reactions so that the main reaction pathway is MepA → MepA³′pU → MepA³′pU²′pU → MepA³′pU²′pU³′pU. MepA did not enhance the oligomerization of ImpA. The relative rates of the reactions were determined from an investigation of the products in competitive reactions. Selectivity was observed in the reaction of ImpU with equimolar amounts of MepA³′pU and MepA²′pU where the relative reaction rates are 10.3:1, respectively. In the reaction of ImpA with MepA³′pA and MepA²′pA the ImpA reacts 5.2 times faster with MepA³′pA. In the competitive reaction of ImpU and ImpA with MepA³′pA and MepA³′pU the elongation proceeds on MepA³′pA 5.6 times more rapidly than with MepA³′pU. There is no correlation between the extent of binding to the montmorillonite and reaction rates in the formation of longer oligomers. The formation of more than two sequential 2′,5′-linkages in the oligomer chain proceeds more slowly than the addition to a single 2′,5′-link or a 3′,5′-link and either chain termination or elongation by a 3′,5′-linage occurs. The central role that catalysis may have had in the prebiotic formation of biopolymers is discussed. Note added in proof: There are errors in the high resolution mass spectral data given in Section 4.2.1. The high resolution mass spectrum found for the cyclic dimer of UpUp (C-UpUp) was 657.02260. C₁₈H₂₁N₄O₁₆P₂Na₂ requires 657.02232. The high resolution mass spectrum found for the cyclic dimer of ApAp (C-ApAp) was 725.05850. C₂₀H₂₂N₁₀O₁₂P₂Na₃ requires 725.05839.     

Expression of α2, α5 and α6 subunits of integrin in de-differentiated NIH3T3 cells by cell-free extract of embryonic stem cells

Generation of patient specific stem cells is among the ultimate goals in regenerative medicine. Such a cell needs to be functional when it transplants. Interaction between the matrix proteins and integrin adjust many cells' function such as adhesion, migration, cell cycle and self renewal in stem cells. In this study, NIH3T3 cells were dedifferentiated by mouse Embryonic Stem Cell (mESC) extract. The expression of pluripotency markers as well as a2, a5 and a6 integrin subunits were determined. NIH3T3 cells treated with mESC extract showed noticeable changes in cell morphology as early as day 2 post-treatment forming colonies similar to typical mESC morphology by day 8, after three passages. Alkaline phosphatase (ALP) assay and immunocytochemistry staining were performed for the induced reprogrammed cells. The results indicated that these colonies showed the ALP activity and they express Sox2 and Nanog. RT-PCR revealed that the colonies also express Oct3/4. NIH3T3 cells, ESC and reprogrammed cells expressed a2 integrin. a5 integrin expression was greatest in reprogrammed cells followed by the expression of this integrin in NIH3T3 which in turn was more than in ESC. a6A integrin was expressed in NIH3T3 cells while a6B integrin was expressed in ESC and in very low quantity was expressed in reprogrammed cells. These data provide evidence for both the generation of ES like cells from differentiated somatic cells and the expression profile of integrins after de-differentiation by mESC extract.     

Involvement of METTL3/m6Adenosine and TGFβ/Smad3 signaling on Tenon’s fibroblasts and in a rabbit model of glaucoma surgery

Journal of Molecular Histology - Glaucoma filtration surgery (GFS) is a classic operation for the treatment of glaucoma, which is the second leading cause of blindness, and scar formation caused by...     

Differential morphology and composition of inclusions in the R6/2 mouse and PC12 cell models of Huntington’s disease

The histological hallmark feature of Huntington’s disease (HD) and other polyglutamine repeat diseases is the presence of intracellular inclusions. Much work has been devoted to trying to determine the relationship between inclusion formation and neuronal injury. However, little attention has been paid to the variability and characteristics of inclusions themselves. Here, we characterize the morphological and biochemical composition of inclusions in both a transgenic mouse model (R6/2 line) and an inducible cell culture model of HD (iPC12Q74). We identified several morphologically distinct kinds of inclusions in different locations (nuclei, cytoplasm and cellular processes). Ubiquitin colocalized completely with all of these inclusions in both the iPC12Q72 and R6/2 models. In the inclusions in iPC12Q74 cells, the 20S and 11S proteasome subunits colocalized variably, and the 19S subunit did not colocalize at all. In inclusions in R6/2 mouse neurons, the 20S subunit colocalized completely, but neither the 11S nor the 19S subunits colocalized at all. While the role of inclusions in the pathogenesis of HD continues to be debated, we suggest that the content and structure of inclusions vary considerably, not only from cell to cell but even within individual cells. Their role in the pathogenesis of HD is likely to depend on their location as well as their composition.     

The structure of 5a,6–anhydrotetracycline and its Mg2+ complexes in aqueous solution

Semiempirical molecular orbital theory has been used for a systematic scan of the binding positions for a Mg²⁺ ion with 5a,6–anhydrotetracycline taking both conformational flexibility and possible different tautomeric forms into account. The magnesium ion has been calculated alone and with four or five complexed water molecules in order to simulate the experimental situation more closely. The results are analyzed by comparing the behavior of the title compound with that of tetracycline itself and possible causes for the stronger induction of the Tetracycline Receptor (TetR) by 5a,6–anhydrotetracycline than by tetracycline are considered. Energetically favored 3D -structure of the zwitteranionic 5a,6-anhydrotetracycline magnesium complex in solution Electronic Supplementary Material Supplementary material is available for this article at     

Functional importance of Ψ38 and Ψ39 in distinct tRNAs,amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U₃₈ and U₃₉ in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ₃₈ or Ψ₃₉ is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA^Gln(UUG), which normally has Ψ₃₈. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s²U moiety of mcm⁵s²U₃₄ of tRNA^Gln(UUG) and the other two cytoplasmic species with mcm⁵s²U₃₄, that the reduced s²U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s²U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ_38,39 provides evidence that Ψ₃₈ is important for function of tRNA^Gln(UUG) at permissive temperature, and indicates that Ψ₃₉ is important for the function of tRNA^Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA^Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ₃₈ and Ψ₃₉ in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.     

Regulation of S100A2 expression by TGF-β-induced MEK/ERK signalling and its role in cell migration/invasion

The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.     

Stereochemistry of 2′-5′ Linked Nucleic Acids: Crystal and Molecular Structure of Ammonium Adenylyl-2′, 5′-Adenosine Tetrahydrate: A Core Fragment of 2′-5′ Oligo A′s Produced by Interferon Induced Adenylate Synthetased

Abstract

The preponderance of 3′-5′ phosphodiester links in nucleic acids is well known. Albeit less prevalent, the 2′-5′ links are specifically utilised in the formation of ‘lariat’ in group II introns and in the msDNA-RNA junction in myxobacterium. As a sequel to our earlier study on cytidylyl-2′,5′-adenosine we have now obtained the crystal structure of adenylyl-2′,5′-adenosine (A2′p5′A) at atomic resolution. This dinucleoside monophosphate crystallises in the orthorhombic space group P2₁2₁2₁ with a= 7.956(3)Å, b = 12.212(3)Å and c = 36.654 (3) Å. CuKα intensity data were collected on a diffractometer. The structure was sloved by direct methods and refined by full matrix least squares methods to R = 10.8 %. The 2′ terminal adenine is in the commonly observed anti (χ₂ =?161°) conformation and the 5′ terminal base has a syn (χ₁ = 55°) conformation more often seen in purine nucleotides. A noteworthy feature of A2′p5′ A is the intranucleotide hydrogen bond between N3 and 05′ atoms of the 5′ adenine base. The two furanose rings in A2′ p5′ A show different conformations-C2′ endo, C3′ endo puckering for the 5′ and 2′ ends respectively. In this structure too there is a stacking of the purine base on the ribose 04′ just as in other 2′-5′ dinucleoside structures, a feature characteristically seen in the left handed ZDNA. In having syn, anti conformation about the glycosyl bonds, C2′ endo, C3′ endo mixed sugar puckering and N3–05′ intramolecular hydrogen bond A2′p5′ A resembles its 3′-5′ analogue and several other 2′-5′ dinucleoside monophosphate structures solved so far. Striking similarities between the 2′-5′ dinucleoside monophosphate structures suggest that the conformation of the 5′-end nucleoside dictates the conformation of the 2′ end nucleoside. Also, the 2′-5′ dimers do not favour formation of miniature classical double helical structures like the 3′-5′ dimers. It is conceivable, 2–5(A) could be using the stereochemical features of A2′p5′ A which accounts for its higher activity.     

Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington’s Disease

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.     

Insight into the modulation of Shaw2 Kv channels by general anesthetics: Structural and functional studies of S4–S5 linker and S6 C-terminal peptides in micelles by NMR

The modulation of the Drosophila Shaw2 Kv channel by 1-alkanols and inhaled anesthetics is correlated with the involvement of the S4–S5 linker and C-terminus of S6, and consistent with stabilization of the channel's closed state. Structural analysis of peptides from S4–S5 (L45) and S6 (S6c), by nuclear magnetic resonance and circular dichroism spectroscopy supports that an α-helical conformation was adopted by L45, while S6c was only in an unstable/dynamic partially folded α-helix in dodecylphosphocholine micelles. Solvent accessibility and paramagnetic probing of L45 revealed that L45 lies parallel to the surface of micelles with charged and polar residues pointing towards the solution while hydrophobic residues are buried inside the micelles. Chemical shift perturbation introduced by 1-butanol on residues Gln320, Thr321, Phe322 and Arg323 of L45, as well as Thr423 and Gln424 of S6c indicates possible anesthetic binding sites on these two important components in the channel activation apparatus. Diffusion measurements confirmed the association of L45, S6c and 1-butanol with micelles which suggests the capability of 1-butanol to influence a possible interaction of L45 and S6c in the micelle environment.