Enhancers are activated by p300/CBP activity-dependent PIC assembly,RNAPII recruitment,and pause release

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pH-Driven Polymorphism of Insulin Amyloid-Like Fibrils

Prions are infective proteins, which can self-assemble into different strain conformations, leading to different disease phenotypes. An increasing number of studies suggest that prion-like self-propagation may be a common feature of amyloid-like structures. Thus it is important to unravel every possible factor leading to the formation of different amyloid strains. Here we report on the formation of two types of insulin amyloid-like fibrils with distinct infrared spectroscopic features grown under slightly different pH conditions. Similar to prion strains, both insulin fibril types are able to self-propagate their conformational template under conditions, favoring spontaneous formation of different type fibrils. The low-pH-induced insulin amyloid strain is structurally very similar to previously reported strains formed either in the presence of 20% ethanol, or by modification of the amino acid sequence of insulin. A deeper analysis of literature data in the context of our current findings suggests a shift of the monomer-dimer equilibrium of insulin as a possible factor controlling the formation of different strains.     

The interplay between transient α-helix formation and side chain rotamer distributions in disordered proteins probed by methyl chemical shifts

The peptide backbones of disordered proteins are routinely characterized by NMR with respect to transient structure and dynamics. Little experimental information is, however, available about the side chain conformations and how structure in the backbone affects the side chains. Methyl chemical shifts can in principle report the conformations of aliphatic side chains in disordered proteins and in order to examine this two model systems were chosen: the acid denatured state of acyl-CoA binding protein (ACBP) and the intrinsically disordered activation domain of the activator for thyroid hormone and retinoid receptors (ACTR). We find that small differences in the methyl carbon chemical shifts due to the γ-gauche effect may provide information about the side chain rotamer distributions. However, the effects of neighboring residues on the methyl group chemical shifts obscure the direct observation of γ-gauche effect. To overcome this, we reference the chemical shifts to those in a more disordered state resulting in residue specific random coil chemical shifts. The (13)C secondary chemical shifts of the methyl groups of valine, leucine, and isoleucine show sequence specific effects, which allow a quantitative analysis of the ensemble of χ(2)-angles of especially leucine residues in disordered proteins. The changes in the rotamer distributions upon denaturation correlate to the changes upon helix induction by the co-solvent trifluoroethanol, suggesting that the side chain conformers are directly or indirectly related to formation of transient α-helices.     

Skin extracellular matrix components accelerate the regenerative potential of Lin− cells

Due to their unique properties, bone marrow-derived Lin^? cells can be used to regenerate damaged tissues, including skin. The objective of our study was to determine the influence of the skin tissue-specific microenvironment on mouse Lin^? cell proliferation and migration in vitro. Cells were analyzed for the expression of stem/progenitor surface markers by flow cytometry. Proliferation of MACS-purified cells in 3D cultures was investigated by WST-8 assay. Lin^? cell migration was evaluated by in vitro scratch assay. The results obtained show that basement membrane matrix is more effective for Lin^? cell proliferation in vitro. However, type I collagen matrix better enhances the re-epithelization process, that depends on the cell migratory properties. These studies are important for preparing cells to be used in transplantation.     

Elongation of Mouse Prion Protein Amyloid-Like Fibrils: Effect of Temperature and Denaturant Concentration

Prion protein is known to have the ability to adopt a pathogenic conformation, which seems to be the basis for protein-only infectivity. The infectivity is based on self-replication of this pathogenic prion structure. One of possible mechanisms for such replication is the elongation of amyloid-like fibrils.We measured elongation kinetics and thermodynamics of mouse prion amyloid-like fibrils at different guanidine hydrochloride (GuHCl) concentrations. Our data show that both increases in temperature and GuHCl concentration help unfold monomeric protein and thus accelerate elongation. Once the monomers are unfolded, further increases in temperature raise the rate of elongation, whereas the addition of GuHCl decreases it.We demonstrated a possible way to determine different activation energies of amyloid-like fibril elongation by using folded and unfolded protein molecules. This approach separates thermodynamic data for fibril-assisted monomer unfolding and for refolding and formation of amyloid-like structure.     

Solvation-assisted pressure tuning of insulin fibrillation: from novel aggregation pathways to biotechnological applications

Solvation-assisted pressure tuning has been employed to unravel unknown structural and kinetic aspects of the insulin aggregation and fibrillation process. Our approach, using fluorescence, Fourier transform infrared and atomic force microscopy techniques in combination with pressure and solvent perturbation, reveals new insights into the pre-aggregated regime as well as mechanistic details about two concurrent aggregation pathways and the differential stability of insulin aggregates. Pressure uniformly fosters the dissociation of native insulin oligomers, whereas the aggregation pathways at elevated temperatures are affected by pressure differently and in a cosolvent-dependent manner. Moderate pressures accelerate the amyloid pathway in the presence of EtOH (leading to essentially monomeric aggregating species) via relatively dehydrated transition states with negative activation volumes for nucleation and elongation. Alternatively, a novel, fast equilibrium pathway to distinct beta-sheet-rich oligomers with thioflavin T-binding capability is accessible to partially unfolded insulin monomers at pressures below approximately 200 bar in the absence of EtOH. These oligomers, probably off the normal fibrillation pathway, are stabilized mainly by electrostatic and hydrophobic interactions, lacking the precise packing of mature insulin fibrils, which renders them susceptible to quantitative pressure-induced dissociation. Due to a highly negative activation volume for dissociation (-70(+/-16)ml/mol), pressure dissociation is fast and technologically feasible at ambient temperatures and moderate pressures. Becoming kinetically very labile above 35 degrees C, the pressurized oligomers can re-enter the slower, ultimately irreversible, fibrillation pathway at higher temperatures. At pressures above approximately 1000 bar, the partial unfolding of insulin monomers, accompanied by a volumetric expansion, dominates the aggregation kinetics, which manifests in a progressive inhibition of the fibrillation. Unlike their precursors, the pressure-insensitivity of mature insulin fibrils demonstrates that an extensive hydrogen bonding network and optimized side-chain packing are crucial for their stability.     

Aploparaksis kornyushini n. sp. (Cestoda: Hymenolepididae), a parasite of the woodcock Scolopax rusticola (L.), and its life-cycle

Aploparaksis kornyushini n. sp. is described from a woodcock Scolopax rusticola L. from Lithuania, Russia (Tver' Region) and the Ukraine. Initially, one specimen of this tapeworm was described and figured by Kornyushin (1975) as A. scolopacis Yamaguti, 1935 together with another specimens belonging to the latter species. A. kornyushini n. sp. and A. scolopacis are morphologically very similar species. They can be distinguished by the slightly different length of the rostellar hooks and by the shape of the cirrus, which lacks basal bulbus in the new species. A. kornyushini can be readily distinguished from the remaining species of Aploparaksis Clerc, 1903 from woodcocks by the structure of its fully-developed embryophore, which has polar thickenings and two large or a few smaller lateral projection; this combination of characters is unknown for embryophores other Aploparaksis spp. (except for A. scolopacis). The life-cycle of A. kornyushini was studied under experimental conditions in Lithuania. The metacestodes were located under the chlorogogenous tissue of the intestine of Dendrobaena octaedra (Lumbricidae). The metacestode exhibits a pattern of postembryonal development typical for the cysticercoid modification termed an 'ovoid diplocyst'.