Posttranslational Modifications of the Histone 3 Tail and Their Impact on the Activity of Histone Lysine Demethylases In Vitro

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k _cat/K _m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K _m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.     

Cross-Limb Interference during Motor Learning

It is well known that following skill learning, improvements in motor performance may transfer to the untrained contralateral limb. It is also well known that retention of a newly learned task A can be degraded when learning a competing task B that takes place directly after learning A. Here we investigate if this interference effect can also be observed in the limb contralateral to the trained one. Therefore, five different groups practiced a ballistic finger flexion task followed by an interfering visuomotor accuracy task with the same limb. Performance in the ballistic task was tested before the training, after the training and in an immediate retention test after the practice of the interference task for both the trained and the untrained hand. After training, subjects showed not only significant learning and interference effects for the trained limb but also for the contralateral untrained limb. Importantly, the interference effect in the untrained limb was dependent on the level of skill acquisition in the interfering motor task. These behavioural results of the untrained limb were accompanied by training specific changes in corticospinal excitability, which increased for the hemisphere ipsilateral to the trained hand following ballistic training and decreased during accuracy training of the ipsilateral hand. The results demonstrate that contralateral interference effects may occur, and that interference depends on the level of skill acquisition in the interfering motor task. This finding might be particularly relevant for rehabilitation.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

The Solution Structure of a Locked Nucleic Acid (LNA) Hybridized to DNA

Abstract

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2′-0, 4′-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional'H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32Å. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3Å. The average twist over the sequence are 35.9° ± 0.3°. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by ¹H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5′-CT^LAG-3′ site than in the unmodified 5′-CT^LAG-3′ site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

Impacts of eriophyoid gall mites on arctic willow in a rapidly changing Arctic

Arctic plants and herbivores are subject to ongoing climatic changes that are more rapid and extreme than elsewhere on the planet, and thus it is pivotal to understand the arctic plant-herbivore interactions in a global change context. We examined how infestation by an eriophyoid gall mite affects the circumpolar shrub Salix arctica, and how the effects vary across vegetation types. Specifically, we compared multiple leaf characteristics (leaf area, biomass, nutrient levels, δ¹⁵N and δ¹³C, and stress and performance of the photosynthetic apparatus) of infested leaves to those of un-infested leaves. Furthermore, we examined how altered environmental conditions, here experimentally manipulated levels of temperature, water and nutrients, shading, and UV-B radiation, affect the prevalence, density, and intensity of gall mite infestation and its impacts on S. arctica. Infested leaves were smaller in area and biomass and had lower nitrogen and carbon pools. However, their carbon concentration was higher, possibly because the galls acted as carbon sinks. The smaller photosynthetic area and lower nutrient content caused increased stress on the photosynthetic apparatus in infested leaves. The remaining leaf tissue responded with a higher photosynthetic performance, although there were indications of a general reduction in photosynthesis. Female leaves were more affected than male leaves. The experimental manipulations of environmental conditions did not affect the gall prevalence, density, or intensity on S. arctica leaves. Rather, plants responded positively to the treatments, reducing the effects of the galls to in-significance. This suggests a higher tolerance and defense against gall mites under future climate conditions.     

Structural,Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare

Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 Å resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the −7 to −4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo-initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.     

Fluctuating water levels control water chemistry and metabolism of a charophyte‐dominated pond

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