Direct Observation of Oligomerization by Single Molecule Fluorescence Reveals a Multistep Aggregation Mechanism for the Yeast Prion Protein Ure2.

The self-assembly of polypeptides into amyloid structures is associated with a range of increasingly prevalent neurodegenerative diseases as well as with a select set of functional processes in biology. The phenomenon of self-assembly results in species with dramatically different sizes, from small oligomers to large fibrils; however, the kinetic relationship between these species is challenging to characterize. In the case of prion aggregates, these structures can self-replicate and act as infectious agents. Here we use single molecule spectroscopy to obtain quantitative information on the oligomer populations formed during aggregation of the yeast prion protein Ure2. Global analysis of the aggregation kinetics reveals the molecular mechanism underlying oligomer formation and depletion. Quantitative characterization indicates that the majority of Ure2 oligomers are relatively short-lived, and their rate of dissociation is much higher than their rate of conversion into growing fibrils. We identify an initial metastable oligomer, which can subsequently convert into a structurally distinct oligomer, which in turn converts into growing fibrils. We also show that fragmentation is responsible for the autocatalytic self-replication of Ure2 fibrils, but that preformed fibrils do not promote oligomer formation, indicating that secondary nucleation of the type observed for peptides and proteins associated with neurodegenerative disease does not occur at a significant rate for Ure2. These results establish a framework for elucidating the temporal and causal relationship between oligomers and larger fibrillar species in amyloid forming systems, and provide insights into why functional amyloid systems are not toxic to their host organisms.

Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation.

Selenoproteins contain the amino acid selenocysteine (Sec), co-translationally inserted at a predefined UGA opal codon by means of Sec-specific translation machineries. In Escherichia coli, this process is dependent upon binding of the Sec-dedicated elongation factor SelB to a Sec insertion sequence (SECIS) element in the selenoprotein-encoding mRNA and competes with UGA-directed translational termination. Here, we found that Sec can also be efficiently incorporated at a predefined UAG amber codon, thereby competing with RF1 rather than RF2. Subsequently, utilizing the RF1-depleted E. coli strain C321.ΔA, we could produce mammalian selenoprotein thioredoxin reductases with unsurpassed purity and yield. We also found that a SECIS element was no longer absolutely required in such a system. Human glutathione peroxidase 1 could thereby also be produced, and we could confirm a previously proposed catalytic tetrad in this selenoprotein. We believe that the versatility of this new UAG-directed production methodology should enable many further studies of diverse selenoproteins.

Construction of a novel guest biomimetic glutathione peroxidase with solvent-dependent catalytic behavior by incorporating the active center into adamantyl molecule.

A novel guest biomimetic glutathione peroxidase (3,3'-tellurobis(propane-3,1-diyl) diadamantanecarboxylate, denoted as ADA-Te-ADA) was synthesized. ADA-Te-ADA functioned to overcome the disadvantages in the construction of building block for giant supramolecular biomimetic enzyme. To reveal the catalytic property of hydrophobic ADA-Te-ADA, the catalytic mechanism was investigated using PBS (phosphate buffer (pH 7.0. 50 mM))/methanol solvent mixture as assay solution. Itindicated that ADA-Te-ADA exhibited typical enzyme catalytic behavior by saturation kinetics measurement. Importantly, ADA-Te-ADA exhibited the typical solvent-dependent catalytic behavior. And the highest catalytic rate 4.29 µM x min-1 was obtained when the volume ratio of PBs: methanol was 5 : 5. Especially, the catalytic rates obtained based on various substrates proved that ADA-Te-ADA slightly displayed special substrate selectivity, which was the ideal catalytic characterization of building block for giant supramolecular biomimetic enzyme. The successfully synthesis of ADA-Te-ADA might highlight for the understanding of the catalytic mechanism of hydrophobic guest biomimetic glutathione peroxidase. And it also might provide the basement for the construction of giant supramolecular biomimetic enzyme.

Construction of an artificial glutathione peroxidase active site on copolymer vesicles.

To construct an efficient GPx mimic, a novel method for preparing polymer-based vesicles carrying GPx-active sites was developed. A series of block copolymers loaded with recognition and catalytic sites were synthesized based on polystyrene-block-poly[tri(ethylene glycol) methyl ether acrylate]s (PS-PMEO(3) MAs). By altering the molar ratio of the functional copolymers, vesicles with GPx activity were obtained by self-assembly of these functional copolymers through blending. The optimum GPx mimic constructed by the blending process exhibited high catalytic activity and acted as a real catalyst with typical saturation kinetics behavior. The method may be of benefit for designing other enzyme mimics and may cast a light on constructing other biologically related functional nanoparticles.

Design of artificial selenoenzymes based on macromolecular scaffolds.

The selenoenzyme glutathione peroxidase has received increased attention as one of the antioxidative enzymes exerting important biological roles in living bodies. Over the past decades, much effort has been invested to mimic its catalytic behavior for understanding enzymatic catalytic mechanisms and also for developing potential medicines. A great number of artificial GPxs, ranging from small molecular compounds to macromolecular ones, have been designed and prepared by combining the concept of recognition and catalysis using chemical, biological and supramolecular strategies. In this article, we specify the development of artificial GPxs based on macromolecules as scaffolds, and discuss the power of reduced models in studying the bio-catalytic nature of selenoenzymes.

A supramolecular bifunctional artificial enzyme with superoxide dismutase and glutathione peroxidase activities.

For constructing a bifunctional antioxidative enzyme with both superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, a supramolecular artificial enzyme was successfully constructed by the self-assembly of the Mn(III)meso-tetra[1-(1-adamantyl methyl ketone)-4-pyridyl] porphyrin (MnTPyP-M-Ad) and cyclodextrin-based telluronic acid (2-CD-TeO(3)H) through host-guest interaction in aqueous solution. The self-assembly of the adamantyl moieties of Mn(III) porphyrin and the beta-CD cavities of 2-CD-TeO(3)H was demonstrated by the NMR spectra. In this supramolecular enzyme model, the Mn(III) porphyrin center acted as an efficient active site of SOD and tellurol moiety endowed GPx activity. The SOD-like activity (IC(50)) of the new catalyst was found to be 0.116 microM and equals to 2.56% of the activity of the native SOD. Besides this, supramolecular enzyme model also showed a high GPx activity, and a remarkable rate enhancement of 27-fold compared to the well-known GPx mimic ebselen was observed. More importantly, the supramolecular artificial enzyme showed good thermal stability.

A novel selenium and copper-containing peptide with both superoxide dismutase and glutathione peroxidase activities.

Superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS. In order to imitate the synergism of these enzymes, we designed and synthesized a novel 32-mer peptide (32P) on the basis of the previous 15-mer peptide with GPX activity and a 17-mer peptide with SOD activity. Upon the selenation and chelation of copper, the 32-mer peptide is converted to a new Se- and Cu-containing 32-mer peptide (Se-Cu-32P) and displays both SOD and GPX activities and its kinetics was studied. Moreover, the novel peptide was demonstrated to be able to better protect vero cells from the injury induced by xanthine oxidase (XOD)/xanthine/Fe2+ damage system than its parents. Thus, this bifunctional enzyme imitated the synergism of SOD and GPX and could be a better candidate of therapeutic medicine.

Association mechanism of S-dinitrophenyl glutathione with two glutathione peroxidase mimics: 2, 2 cent-ditelluro- and 2, 2 cent-diseleno-bridged b-cyclodextrins.

Complex formation of the glutathione peroxidase mimics 2,2 cent-ditelluro-bridged b-cyclodextrin (1) and 2,2 cent-diseleno-bridged b-cyclodextrin (2), with S-substituted dinitrophenyl glutathione (3) were determined by ultraviolet-visible (UV-Vis) absorption spectroscopy in phosphate buffer (pH 7.4) and (1)H-NMR spectroscopy. Molecular mechanics (MM2) modeling calculations were used to deduce a three-dimensional model for each complex. The dinitrophenyl (DNP) group of 3 appears to penetrate the cavity of b-cyclodextrin (b-CD) or 1, but it is located between the two secondary rims of 2. The complexes' stability constants (K(s)) from 19 to 37 degrees C, Gibbs free energy changes (DG degrees ), DH degrees and TDS degrees for 1:1 complexes of b-CD, 1 and 2 with ligand 3 as obtained from UV-Vis spectra were compared. The binding of 3 by the three cyclodextrin hosts generally decreased in the order of 1>2>b-CD. The binding ability of 3 by b-CD, 1 and 2 was discussed with regard to the size/shape-fit concept, the induced-fit interaction, and the cooperative interaction of the dual hydrophobic cavities. The binding ability of 1>2indicated that the length of linkage between two cyclodextrin units plays a crucial role in the interaction with 3.

A semisynthetic glutathione peroxidase with high catalytic efficiency. Selenoglutathione transferase.

Glutathione peroxidase (GPX) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). GPX therefore has potential therapeutic value as an antioxidant, but its pharmacological development has been limited because GPX uses a selenocysteine as its catalytic group and it is difficult to generate selenium-containing proteins with traditional recombinant DNA technology. Here, we show that naturally occurring proteins can be modified to generate GPX activity. The rat theta-class glutathione transferase T2-2 (rGST T2-2) presents an ideal scaffold for the design of a novel GPX catalyst because it already binds GSH and contains a serine close to the substrate binding site, which can be chemically modified to bind selenium. The modified Se-rGST T2-2 efficiently catalyzes the reduction of hydrogen peroxide, and the GPX activity surpasses the activities of some natural GPXs.

A novel cyclodextrin-derived tellurium compound with glutathione peroxidase activity.

A novel dicyclodextrinyl ditelluride (2-TeCD) compound was devised as a functional mimic of the glutathione peroxidase (GPX) enzymes that normally remove hydroperoxides from the cell. The GPX activity of the mimic was found to be 46.7 U microM(-1), which is 46 times as active as Ebselen, a well-known GPX mimic. A detailed steady-state kinetic study was undertaken to probe the reason for the high catalytic efficiency of 2-TeCD. This high efficiency can be explained based on both the binding of the substrate to the cyclodextrin and the catalytic mechanism of 2-TeCD, which is different from that of diselenide compounds. 2-TeCD exhibits good water solubility and is chemically and biologically stable. The biological effect of 2-TeCD was evaluated by its ability to protect mitochondria from oxidative damage. 2-TeCD exhibited excellent antioxidant capacity in comparison with Ebselen.