Role of prion protein aggregation in neurotoxicity

In several neurodegenerative diseases, such as Parkinson, Alzheimer's, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.     

Predicted secondary structure and membrane topology of the scrapie prion protein

The integral membrane sialoglycoprotein PrP^Sc is the only identifiablecomponent of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakobdisease in humans are transmissible, degenerative neurologicaldiseases caused by prions. Standard predictive strategies havebeen used to analyze the secondary structure of the prion proteinin conjunction with Fourier analysis of the primary sequencehydrophobicities to detect potential amphipathic regions. Severalhydrophobic segments, a proline- and glycine-rich repeat regionand putative glycosylation sites are incorporated into a modelfor the integral membrane topology of PrP. The complete aminoacid sequences of the hamster, human and mouse prion proteinsare compared and the effects of residue substitutions upon thepredicted conformation of the polypeptide chain are discussed.While PrP has a unique primary structure, its predicted secondarystructure shares some interesting features with the serum amyloidA proteins. These proteins undergo a post-translational modificationto yield amyloid A, molecules that share with PrP the abilityto polymerize into birefringent filaments. Our analyses mayexplain some experimental observations on PrP, and suggest furtherstudies on the properties of the scrapie and cellular PrP isoforms.     

Disruption of the X-loop turn of the prion protein linked to scrapie resistance

The prion diseases are a class of neurodegenerative diseases caused by the misfolding and aggregation of the prion protein (PrP(C)) into toxic and infectious oligomers (PrP(Sc)). These oligomers are critical to understanding and combating these diseases. Differences in the sequence of PrP affect disease susceptibility, likely by shifting the tolerance of the protein for adaptation to PrP(Sc) conformations and/or the recognition event between PrP(Sc) and PrP(C) prior to conversion of the PrP(C). We selected two sets of PrP(Sc)-resistant mutant sequences for solvated atomistic molecular dynamics simulation to investigate the structural basis of resistance. The first group involved mutation in the X-loop (residues 164-171) resulting from selective breeding of sheep. The second group included eight mutants in mice identified by random mutagenesis targeting helix C followed by screening in cell cultures. Multiple simulations were performed of 14 different mutant and control constructs under different pH conditions for a total of 3.6 μs of simulation time. The X-loop formed a stable turn at neutral pH in wild-type PrP from both species. PrP(Sc)-resistant mutations disrupted this turn even though only one of the mutants is in the X-loop. The X-loop is compact and buried in our previously described spiral models of PrP(Sc)-like oligomers. On the basis of the findings presented here and in the context of the spiral oligomer model, we propose that expansion of the X-loop disrupts protofibril packing, providing a structural basis for resistance.     

Novel dominant-negative prion protein mutants identified from a randomized library

Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the cellular prion protein (PrP(C)). We generated a library of PrP variants with random mutations in the helix-3 domain and screened for dominant-negative mutants (DNMs) that would inhibit replication of prions (the Rocky Mountain Laboratory strain) in infected N2a cells. Two of the identified PrP mutants, Q167R and Q218K, were already known to counteract prion replication, thereby validating the effectiveness of this approach. In addition, novel DNMs were found efficiently to antagonize PrP(Sc) propagation in cells. In contrast to Q167R and Q218K, the newly identified DNMs S221P and Y217C resided on the cell surface at a substantially lower level, suggesting that robust cell surface display of DNM might not be a general prerequisite for efficient prion antagonism. The newly identified DNMs point to useful target-selective therapeutic tools for the treatment of prion diseases.     

Risk assessment for prion protein reduction under the conditions of the biodiesel production process

Due to the increased demand for biofuels, all different feedstocks from oils and fats have to be considered for biodiesel production. Animal fats have proved to be excellent sources for biodiesel due to their high cetane number and good stability. Large amounts of fat from so‐called high‐risk material, possibly contaminated with infectious prions, are available for biodiesel production. In this paper, the grade of destruction of prions during the biodiesel production process, including pre‐esterification with conc. sulfuric acid followed by KOH‐catalyzed transesterification, was studied. The starting material of the different production steps was spiked with purified and highly infectious prion rods, and the destruction of these prions was determined by gel electrophoresis (SDS‐PAGE) and Western blot. The pre‐esterification step led to a destruction factor of at least 100, the transesterification led to a factor of at least 250, and the distillation of the final biodiesel showed a destruction factor of at least 1000. During all experiments, no traces of prions could be detected after the different reaction steps. Based on these data, a complete and unequivocal risk assessment regarding the industrial process of biodiesel production was carried out, leading to a calculated overall risk of 5.8×10^?15 ID₅₀ units/person and year, which means that a hypothetical BSE contamination from biodiesel is more than 10⁹ times lower than the background risk.     

Novel substrate specificity engineered in the arabinose binding protein

The L-arabinose binding protein (ABP) of Escherichia coli naturallybinds L-arabinose and D-galactose with very high affinity and,with reduced affinity, a variety of other sugars that differonly at the C5 position of the pyranose ring.However, thereare stringent specificity requirements at the 1, 2, 3 and 4positions. Based on the high resolution crystallographic structureof the Ugand-protein complex, remodelling of the binding pocketwas attempted to shift the specificity towards Cl-substitutedgalactosides. To create space in the vicinity of the reducingend of bound galactose, four residues, LyslO, Asp90, Thrl47and Leul45, have been mutated for residues with smaller sidechains. Forty-seven mutants containing different combinationsof these mutations were tested by fluorometry for their abilityto bind methylß-D-galactoside (met-ß-Gal)or iso-propyl-ß-D-thio-galactoside (IPTG). Two double-residuemutants carrying Ser at position 147 and Ala or Gly at position90 appeared of particular interest for being able to bind met-ß-Galor IPTG, respectively, and no longer galactose. Fluorescenceexperiments and molecular modelling indicate that the mode ofbinding of the new substrates to the mutant proteins might besimilar to that of the natural ligands to wild-type ABP.     

Similarity between the C-terminal domain of the prion protein and chimpanzee cytomegalovirus glycoprotein UL9

Prion diseases are a group of fatal neurodegenerative disordersassociated with structural conversion of a normal, mostly     

High-level expression and characterization of a glycosylated covalently linked dimer of the prion protein

There is evidence that prion protein dimers may be involvedin the formation of the scrapie prion protein, PrP^Sc, from itsnormal (cellular) form, PrP^c. Recently, the crystal structureof the human prion protein in a dimeric form was reported. Herewe report for the first time the overexpression of a human PrPdimer covalently linked by a FLAG peptide (PrP::FLAG::PrP) inthe methylotrophic yeast Pichia pastoris. FLAG-tagged humanPrP (aa1-aa253) (huPrP::FLAG) was also expressed in the samesystem. Treatment with tunicamycin and endoglycosidase H showedthat both fusion proteins are expressed as various glycoforms.Both PrP proteins were completely digested by proteinase K (PK),suggesting that the proteins do not have a PrP^Sc structure andare not infectious. Plasma membrane fractionation revealed thatboth proteins are transported to the plasma membrane of thecell. The glycosylated proteins might act as powerful toolsfor crystallization trials, PrP^c/PrP^Sc conversion studies andother applications in the life cycle of prions.     

电解铜中铜的快速测定

恒电流电解重量法测定铜及铜合金中铜含量是目前唯一经典的实验方法[1～ 4 ] ,具有干扰少、准确度高等优点 ,但分析过程冗长 ,影响分析速度 ,不易控制 ,操作稍有不慎将引入系统误差 ,不能满足日常铜的快速分析。本法采用密闭电解杯水浴加热快速溶样 ,在不断搅拌下加大电流密度 (即 3.5A/dm2 ) ,缩短电解时间 ,能在 3h中完成测定 ,提高了效率 ,满足有关质检部门的日常快速分析。1　实验1.1　方法提要试样经酸分解 ,在硝、硫混合酸介质中 ,在阴、阳电极之间加上适当电流进行电解。电解终止时 ,将沉积在铂阴极上的金属铜洗净 ,烘干后称量 ,同…     

Copper binding to the neurotoxic peptide PrP106-126: thermodynamic and structural studies

The human prion protein fragment PrP(106-126) is a highly fibrillogenic peptide, resistant to proteinases and toxic to neurons; it derives from the normal prion protein (PrP(C)), with which it can interact, thus inhibiting its superoxide dismutase-like activity. The same properties are also shown by the abnormal isoform of the prion protein (PrP(Sc)), and this similarity makes PrP(106-126) an interesting model for the neurotoxic action of PrP(Sc). A role for copper in PrP(106-126) aggregation and toxicity has recently been evidenced, and the interaction of terminal Lys, His and Met residues with the copper ion at neutral pH has been suggested. In order to shed more light on the complex-formation equilibria of PrP(106-126) with the copper ion, a thorough investigation has been carried out by means of several experimental techniques: potentiometry, solution calorimetry, VIS spectrophotometry, circular dichroism, EPR and NMR spectroscopy. A shorter and more soluble fragment-PrP(106-113), which lacks the hydrophobic C-terminal domain of PrP(106-126) but contains all the potential donor groups-has also been considered for the sake of comparison. The involvement of terminal amino, imidazolic and amido nitrogens in complex formation has been confirmed, while no evidence was found for the interaction of side chains of Met and Lys residues with the copper ion. Solution structures for the main complexes are suggested.     

铜精矿中铜含量测定的不确定度评定

通过滴定法测定铜精矿中铜含量,对其测量不确定度进行评定,详尽地分析了整个实验过程中的不确定度来源,并对各部分不确定度分量进行量化计算,得出合成标准不确定度和扩展不确定度,并以不确定度的形式给出测定结果。     

镀铜液中铜的测定

以硫脲掩蔽Ｃｕ,ＥＤＴＡ滴定共存离子,然后用Ｈ２Ｏ２破坏硫脲铜配合物,再以标准ＥＤＴＡ滴定,计算得出铜含量。     

PoPMuSiC, an algorithm for predicting protein mutant stability changes. Application to prion proteins

A novel tool for computer-aided design of single-site mutationsin proteins and peptides is presented. It proceeds by performingin silico all possible point mutations in a given protein orprotein region and estimating the stability changes with linearcombinations of database-derived potentials, whose coefficientsdepend on the solvent accessibility of the mutated residues.Upon completion, it yields a list of the most stabilizing, destabilizingor neutral mutations. This tool is applied to mouse, hamsterand human prion proteins to identify the point mutations thatare the most likely to stabilize their cellular form. The selectedmutations are essentially located in the second helix, whichpresents an intrinsic preference to form ß-structures,with the best mutations being T183     

Control of binding of protein in the structure of chemical fibres and films

The principles of control of binding of water-soluble protein compounds in the structure of fibres and films were elaborated. The possibilities of regulating the rate of diffusion of the protein from the polymeric material into the aqueous medium by altering the conditions of formation of the structure of the fibre or film and by using additional components in the spinning composition that alter the kinetic properties of the protein were demonstrated. The efficiency of using protein-containing fibre and film materials with given pharmacodynamic properties in surgery was demonstrated. Moscow State Textile Academy. Translated fromKhimicheskie Volokna, No. 4, pp. 29–32, July–August, 1999.     

Characterization of the DNA binding domain of the mouse IRF-2 protein

The DNA binding domain of the interferon regulatory factor-2protein (IRF-2) has been produced and characterized, -chymotrypsindigestion of the purified IRF-2 protein bound to a syntheticbinding site yields a peptide fragment of 14 K in molecularweight. N-terminal analysis of this peptide fragment showedthat its sequence is the same as that of the intact IRF-2. Apeptide fragment of {small tilde} 14 K, IRF-2(113), which correspondsto the N-terminal 113 amino acids of the intact IRF-2 protein,has been expressed in a functional form in Escherichia coli.The first methionine was processed during the expression andthe purified IRF-2(113) thus contains 112 amino acids. DNaseI footprinting and gel retardation assaying showed that IRF-2(113)binds to a synthetic DNA having the consensus binding site andto the upstream regulatory sequence of the IFN-ß geneas intact IRF-2 does. These results showed that this peptidefragment, IRF-2(113), may be a good material for investigationof the DNA binding domain of IRF-2 and of the DNA–proteininteraction.     

影响海绵铜中铜含量测定的因素

为了确定测量海绵铜中铜含量的最佳条件,提高工作效率,确保高价值海绵铜含量的准确性,采用碘量法,通过单因素实验研究了几种因素对测定结果的影响。结果表明:样品在放置时间上,完成滴定分析应控制在5 min以上为佳。PH控制在溶液中有铁沉淀(pH=1.9),取样量为3 g条件为宜。     

Spectroscopic properties of an engineered maltose binding protein

The maltose binding protein (MBP) has been site specifically labelled with a nitrobenzoxadiazole (NBD) group following mutation of a serine to a cysteine residue at position 337. The resulting protein shows a large ligand (maltose or beta-cyclodextrin) dependent increase in its steady-state fluorescence intensity. Analysis of the static (intensity and anisotropy) and dynamic (lifetime distributions) fluorescence of the NBD label as well as the tryptophan residues in both ligand-bound and ligand-free states of this molecule reveals complex multi-component decays that are interpreted in terms of a ligand-induced solvent shielding mechanism. In the context of the known crystal structures of the various forms of the maltose-binding protein (MBP), ligand- dependent changes in both the fluorescence parameters as well as the circular dichroism spectra of the NBD group are interpreted by a twisted intramolecular charge-transfer (TICT) mechanism, wherein ligand binding locks the NBD group into a conformation that prevents efficient relaxation of the excited state.      

铜矿石制备硫酸铜工艺研究进展

概述了目前国内外利用铜矿石生产硫酸铜的方法及特点,提出了细菌浸出－ 萃取生产硫酸铜的新方法