Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer's disease. Analysis of circular dichroism spectra.

The A4 or beta-peptide (39 to 43 amino acid residues) is the principal proteinaceous component of amyloid deposits in Alzheimer's disease. Using circular dichroism (c.d.), we have studied the secondary structures and aggregational properties in solution of 4 synthetic amyloid beta-peptides: beta-(1-28), beta-(1-39), beta-(1-42) and beta-(29-42). The natural components of cerebrovascular deposits and extracellular amyloid plaques are beta-(1-39) and beta-(1-42), while beta-(1-28) and beta-(29-42) are unnatural fragments. The beta-(1-28), beta-(1-39) and beta-(1-42) peptides adopt mixtures of beta-sheet, alpha-helix and random coil structures, with the relative proportions of each secondary structure being strongly dependent upon the solution conditions. In aqueous solution, beta-sheet structure is favored for the beta-(1-39) and beta-(1-42) peptides, while in aqueous solution containing trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), alpha-helical structure is favored for all 3 peptides. The alpha-helical structure unfolds with increasing temperature and is favored at pH 1 to 4 and pH 7 to 10; the beta-sheet conformation is temperature insensitive and is favored at pH 4 to 7. Peptide concentration studies showed that the beta-sheet conformation is oligomeric (intermolecular), whereas the alpha-helical conformation is monomeric (intramolecular). The rate of aggregation to the oligomeric beta-sheet structure (alpha-helix----random coil----beta-sheet) is also dependent upon the solution conditions such as the pH and peptide concentration; maximum beta-sheet formation occurs at pH 5.4. These results suggest that beta-peptide is not an intrinsically insoluble peptide. Thus, solution abnormalities, together with localized high peptide concentrations, which may occur in Alzheimer's disease, may contribute to the formation of amyloid plaques. The hydrophobic beta-(29-42) peptide adopts exclusively an intermolecular beta-sheet conformation in aqueous solution despite changes in temperature or pH. Therefore, this segment may be the first region of the beta-peptide to aggregate and may direct the folding of the complete beta-peptide to produce the beta-pleated sheet structure found in amyloid deposits. Differences between the solution conformations of the beta-(1-39) and beta-(1-42) peptides suggests that the last 3 C-terminal amino acids are crucial to amyloid deposition.     

Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1

alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.     

Interaction of cyclodextrins with fluorescent probes and its application to kinetic studies of amylase.

It was found that 6-p-toluidinylnaphthalene-2-sulfonate (TNS) showed pronounced fluorescence enhancement when it was added to alpha-, beta-, and gamma-cyclodextrin solutions. 2. The following results were obtained by quantitative study of the interactions of three kinds of cyclodextrins with TNS by following TNS fluorescence at pH5.3. and 25 degrees. i) alpha-Cyclodextrin forms a l : l complex with TNS. ii) beta- and gamma-Cyclodextrins form 1 : 1 and also 2 : 1 complexes; in the latter two cyclodextrin molecules bind to one TNS molecule. iii) The dissociation constants of cyclodextrin-TNS complexes were determined to be 54.9 mM for alpha-cyclodextrin, 0.65 mM for beta-cyclodextrin and 0.66 mM for gamma-cyclodextrin in the 1 : 1 complex, and the secondary dissociation constants in the 2 : 1 complex were 71.4 mM for beta-cyclodextrin in the 1 : 1 complex, and the secondary dissociation constants in the 2 : 1 complex were 71.4 mM for beta-cyclodextrin and 32.6 mM for gamma-cyclodextrin. iv)...     

NMR and CD studies on the interaction of Alzheimer beta-amyloid peptide (12-28) with beta-cyclodextrin

Polymerization of the amyloid beta-peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease (AD). Inhibition of the formation of these toxic polymers of Abeta has emerged as an approach for developing therapeutics for AD. NMR and CD spectra were used to investigate the interaction between cyclodextrin and Abeta(12-28) peptide, which was reported to be an important region for forming amyloid fibrils. CD spectral analyses show that of the alpha-, beta- and gamma-cyclodextrins only beta-cyclodextrin inhibits the aggregation of Abeta(12-28) at pH 5.0. Analysis of the one-dimensional proton NMR spectra of Abeta(12-28) and the mixture of Abeta(12-28) with beta-cyclodextrin clearly indicates that there are chemical shift changes in the aromatic ring of Phe19 and the methyl groups of Val18 in the peptide. The NOESY spectra show cross-peaks between H-3 and H-5 of beta-cyclodextrin and the aromatic protons of Phe19 and Phe20. These chemical shift differences and NOEs demonstrate that there is an interaction between Abeta(12-28) and beta-cyclodextrin. Analysis of the cross-peak intensity in the NOESY spectra reveals that the aromatic rings of Phe19 and 20 are generally inserted into beta-cyclodextrin at the broad side and are oriented toward the narrow side of the cavity.     

Prion protein helix1 promotes aggregation but is not converted into beta-sheet

Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).     

Prevention of Alzheimer's disease-associated Abeta aggregation by rationally designed nonpeptidic beta-sheet ligands

A new concept is introduced for the rational design of beta-sheet ligands, which prevent protein aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor (DAD) hydrogen bond pattern complementary to that of a beta-sheet efficiently block the solvent-exposed beta-sheet portions in Abeta-(1-40) and thereby prevent formation of insoluble protein aggregates. Density gradient centrifugation revealed that in the initial phase, the size of Abeta aggregates was efficiently kept between the trimeric and 15-meric state, whereas after 5 days an additional high molecular weight fraction appeared. With fluorescence correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of nine similar aminopyrazole ligands were identified as efficient aggregation retardants whose minimum energy conformations showed a perfect complementarity to a beta-sheet. The concentration dependence of the inhibitory effect of a trimeric aminopyrazole derivative allowed an estimation of the dissociation constant in the range of 10(-5) m. Finally, electrospray ionization mass spectrometry (ESI-MS) was used to determine the aggregation kinetics of Abeta-(1-40) in the absence and in the presence of the ligands. From the comparable decrease in Abeta monomer concentration, we conclude that these beta-sheet ligands do not prevent the initial oligomerization of monomeric Abeta but rather block further aggregation of spontaneously formed small oligomers. Together with the results from density gradient centrifugation and fluorescence correlation spectroscopy it is now possible to restrict the approximate size of soluble Abeta aggregates formed in the presence of both inhibitors from 3- to 15-mers.     

Circular-dichroism studies on two murine serum amyloid A proteins.

C.d. studies have shown that mouse SAA2 (serum amyloid A2) protein has about one-half of the alpha-helix content of the SAA1 (serum amyloid A1) analogue (15 as against 32%), although secondary-structure prediction analyses based on sequence data do not suggest such a large difference between the forms. The decreased helical content may be a reflection or indication of a stronger propensity to aggregation of the SAA2 form compared with SAA1. The main elements of secondary structure in both proteins are beta-sheets/turns. Interactions with Ca2+ are accompanied by small losses in alpha-helix content, whereas binding to chondroitin-6-sulphate in the presence of millimolar Ca2+ also decreases the amount of secondary structure. However, SAA2 binding to heparan sulphate increases its beta-sheet structure, whereas with SAA1 secondary structure is not apparently altered by its interaction with heparan sulphate. Computer-generated surface profiles show slight differences in accessibility, hydrophilicity and flexibility between the proteins. Understanding these differences may help to explain why SAA2 is found in amyloid fibrils whereas SAA1 is not. In particular, a stronger tendency to aggregation might be the reason why SAA2 is deposited exclusively in these fibrils.     

Kinetic partitioning between aggregation and vesicle permeabilization by modified ADan

The neurodegenerative illness Familial Danish Dementia (FDD) is linked to formation and aggregation of the 34-residue ADan peptide, whose cytotoxicity may be mediated by membrane interactions. Here we characterize the derived peptide SerADan, in which the two cysteines found in ADan have been changed to serines to emulate the reduced peptide. SerADan aggregates rapidly at pH 5.0 and 7.5 in a series of conformational transitions to form beta-sheet rich fibril-like structures, which nevertheless do not bind amyloid-specific dyes, probably due to the absence of organized beta-sheet contacts. Aggregation is prevented at neutral/acidic pH and low ionic strength by anionic lipid vesicles. These vesicles are permeabilized by monomeric SerADan assembling on the membrane to form stable beta-sheet structures which are different from the solution aggregates. In contrast, solution ageing of SerADan first reduces and then abolishes permeabilization properties. The competition between lipid binding and aggregation may reflect bifurcating pathways for the ADan peptide in vivo between accumulation of inert aggregates and formation of cytotoxic permeabilizing species. Our work demonstrates that non-fibrillar aggregates can assemble in a series of steps to form a hierarchy of higher-order assemblies, where rapid formation of stable local beta-sheet structure may prevent rearrangement to amyloid proper.     

Structural transformation and aggregation of human alpha-synuclein in trifluoroethanol: non-amyloid component sequence is essential and beta-sheet formation is prerequisite to aggregation

Amyloid-like aggregation of alpha-synuclein and deposit in Lewy bodies are thought to be the major cause of Parkinson's disease. Here we describe the secondary structural transformation and aggregation of human alpha-synuclein and its C-terminus truncated fragments in trifluoroethanol. Proteins containing the NAC (non-amyloid component) segment undergo a three-state transition: from native random coil to beta-sheet and to alpha-helical structure, while the NAC deficient fragment and gamma-synuclein undergo a typical two-state coil-to-alpha transition. The beta-sheet form is highly hydrophobic that strongly binds to 1-anilinonaphthalene-8-sulfonic acid (ANS) and is prone to self-aggregation. The results suggest that the NAC sequence is essential to beta-sheet formation and the aggregation originates from the beta-sheet intermediate, which may be implicated in the pathogenesis of Parkinson's disease.     

An infrared spectroscopic study of the secondary structure of protein kinase C alpha and its thermal denaturation

The secondary structure of protein kinase C alpha (PKC alpha) has been studied using infrared spectroscopy in the presence of both H(2)O and D(2)O buffers. In the absence of ligands at 20 degrees C, it was shown that beta-sheet is the major component, representing about 44% of the total structure, whereas the alpha-helix amounts to 22%. The addition of Ca(2+) produced only small changes in the secondary structure at 20 degrees C with the beta-sheet increasing up to 48%. On the other hand, the other ligands, such as phorbol 12-myristate 13-acetate (PMA), ATP, and phospholipids, did not produce any significant change. When the thermal unfolding of PKC alpha was studied after heating to 75 degrees C, the presence of the ligands affected the unfolding process. PKC alpha was better preserved from thermal denaturation in the presence of Ca(2+), the aggregated beta-sheet at 1618 cm(-1) decreasing from 19% in the absence of this ligand to 13% in its presence. Protection was also afforded by the presence of PMA or phospholipids. A two-dimensional correlation study of the denaturation of PKC alpha in the presence of these different ligands also showed differences among them. Synchronous 2D-IR correlation showed that the main change occurred at 1616-1619 cm(-1), this component being assigned to the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in the alpha-helix component in all cases except in the presence of a mixture of ligands including Ca(2+), ATP, PMA, and phospholipids, when the intermolecular aggregation of beta-sheet was correlated with the change in the beta-sheet component. In addition, the asynchronous 2D-IR correlation study of PKC alpha showed that the aggregated beta-sheet increased after changes in other components. It was interesting that alpha-helix changed before the beta-sheet in the control experiment and in the presence of Ca(2+), while the order of change was reversed when PMA was added.     

Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability

Genetic evidence predicts a causative role for amyloid-beta (A beta) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A beta are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A beta-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta-(1-42)-induced inhibition significant at 10 nm. Under A beta-(1-42) oligomer- and fibril-forming conditions, A beta-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A beta-(1-42). We applied the aggregation protocols developed for wild type A beta-(1-42) to A beta-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A beta-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A beta-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A beta-(1-42) are useful in distinguishing the structural and functional differences between A beta-(1-42) and A beta-(1-40) and genetic mutations of A beta-(1-42).     

Enantioselective preparative HPLC separation of the HBCD-Stereoisomers from the technical product and their absolute structure elucidation using X-ray crystallography

1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a widely used flame retardant, which tends to persist in the environment and accumulates in biota. The six stereoisomers (three racemates named alpha-, beta-, and gamma-HBCD) of the technical mixture were isolated with high-performance liquid chromatography (HPLC). Direct separations were performed on a chiral stationary phase containing permethylated beta-cyclodextrin (NUCLEODEX beta-PM column) and the pure enantiomers of alpha-, beta-, and gamma-HBCD were physically characterized for the first time. The absolute configurations of all six isomers were determined by anomalous dispersion using single crystal X-ray crystallography. Optical rotations alphaD in tetrahydrofuran were +4.2/-4.0 (alpha-HBCD), +26.1/-27.5 (beta-HBCD), and +68.0/-66.3 (gamma-HBCD). The sense of rotation could be correlated with the absolute configurations of alpha-, beta-, and gamma-HBCD enantiomers and their order of elution on a chiral permethylated beta-cyclodextrin-bonded stationary phase. The diastereomersalpha-, beta-, and gamma-HBCD displayed distinctly different melting points as well as (1)H-, (13)C NMR, and IR spectra.     

Entrapping intermediates of thermal aggregation in alpha-helical proteins with low concentration of guanidine hydrochloride

Aggregation of proteins is a problem with serious medical implications and economic importance. To develop strategies for preventing aggregation, the mechanism(s) and pathways by which proteins aggregate must be characterized. In this study, the thermally induced aggregation processes of three alpha-helix proteins (myoglobin, cytochrome c, and lysozyme) in the presence and absence of 1.0 m guanidine hydrochloride (GdnHCl) were investigated by means of infrared spectroscopy. In the absence of GdnHCl, intensities of the alpha-helix bands (approximately 1656 cm(-1)) decrease as a function of temperature at above 50 degrees C. With myoglobin and cytochrome c, the loss of helix bands was accompanied by the appearance of two new bands at 1694 and 1623 cm(-1), indicative of the formation of intermolecular beta-sheet aggregates. For lysozyme, bands indicative of intermolecular beta-sheet aggregates did not appear in any significant intensity. In the presence of 1.0 m GdnHCl, two major intermediate states rich in 3(10)-helix (represented by the band at 1663 cm(-1)) and beta-turn structure (represented by the band at 1667 cm(-1)), respectively, were observed. These findings demonstrated that IR spectroscopic studies of protein aggregation using a combination of thermal and chemical denaturing factors could provide a means to populate and characterize aggregation intermediates.     

Aggregation and neurotoxicity of mutant amyloid beta (A beta) peptides with proline replacement: importance of turn formation at positions 22 and 23

Aggregation of the amyloid beta peptides (A beta 1-42 and A beta 1-40) plays a pivotal role in pathogenesis of Alzheimer's disease. Although it is widely accepted that the aggregates of A betas mainly consist of beta-sheet structure, the precise aggregation mechanism remains unclear. To identify amino acid residues that are important for the beta-sheet formation, a series of proline-substituted mutants of A beta 1-42 peptides at positions 19-26 was synthesized in a highly pure form and their aggregation ability and neurotoxicity on PC12 cells were investigated. All proline-substituted A beta 1-42 mutants except for 22P- and 23P-A beta 1-42 were hard to aggregate and showed weaker cytotoxicity than wild-type A beta 1-42, suggesting that the residues at positions 19-21 and 24-26 are important for the beta-sheet formation. In contrast, 22P-A beta 1-42 extensively aggregated with stronger cytotoxicity than wild-type A beta 1-42. Since proline has a propensity for beta-turn structure as a Pro-X corner, these data implicate that beta-turn formation at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of A beta peptides.     

Pressure effects on the heat-induced aggregation of equine serum albumin by FT-IR spectroscopic study: Secondary structure, kinetic and thermodynamic properties

Pressure can restrain the heat-induced aggregation and dissociate the heat-induced aggregates. We investigated the aggregation-preventing pressure effect and the aggregates-dissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by Fourier transform infrared spectroscopy. The results suggest that the alpha-helical structure collapses at the beginning of heat-induced aggregation, then the rearrangement of structure from partially unfolded structure to the intermolecular beta-sheet takes place through the activated state. We determined the activation volume for the heat-induced aggregation (DeltaV( not equal)=+92+/-8 ml mol(-1)) and the partial molar volume difference between native state and heat-induced aggregates (DeltaV(N-->HA)=+32 ml mol(-1)). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular beta-sheet is unfavorable under high pressure. We also determined the free energy profile of ESA. This energy profile explains the restriction of the formation of heat-induced aggregates by pressure. These results explain the structural differences between heat-induced aggregates with intermolecular beta-sheet and pressure-induced aggregates without intermolecular beta-sheet.     

Angiotensin-converting enzyme degrades Alzheimer amyloid beta-peptide (A beta ); retards A beta aggregation, deposition, fibril formation; and inhibits cytotoxicity

We have demonstrated that the angiotensin-converting enzyme (ACE) genotype is associated with Alzheimer's disease (AD) in the Japanese population (). To determine why ACE affects susceptibility to AD, we examined the effect of purified ACE on aggregation of the amyloid beta-peptide (A beta) in vitro. Surprisingly, ACE was found to significantly inhibit A beta aggregation in a dose response manner. The inhibition of aggregation was specifically blocked by preincubation of ACE with an ACE inhibitor, lisinopril. ACE was confirmed to retard A beta fibril formation with electron microscopy. ACE inhibited A beta deposits on a synthaloid plate, which was used to monitor A beta deposition on autopsied brain tissue. ACE also significantly inhibited A beta cytotoxicity on PC12 h. The most striking fact was that ACE degraded A beta by cleaving A beta-(1-40) at the site Asp(7)-Ser(8). This was proven with reverse-phase HPLC, amino acid sequence analysis, and MALDI-TOF/MS. Compared with A beta-(1-40), aggregation and cytotoxic effects of the degradation products A beta-(1-7) and A beta-(8-40) peptides were reduced or virtually absent. These findings led to the hypothesis that ACE may affect susceptibility to AD by degrading A beta and preventing the accumulation of amyloid plaques in vivo.     

Selective binding of chiral molecules of cinchona alkaloid by beta- and gamma-cyclodextrins and organoselenium-bridged bis(beta-cyclodextrin)s

The inclusion complexation behavior of chiral members of cinchona alkaloid with beta- and gamma-cyclodextrins (1 and 2) and 6,6(')-trimethylenediseleno-bridged bis(beta-cyclodextrin) (3) was assessed by means of fluorescence and 2D-NMR spectroscopy. The spectrofluorometric titrations have been performed in aqueous buffer solution (pH 7.20) at 25.0 degrees C to determine the stability constants of the inclusion complexation of 1-3 with guest molecules (i.e., cinchonine, cinchonidine, quinine, and quinidine) in order to quantitatively investigate the molecular selective binding ability. The stability constants of the resulting complexes of 2 with guest molecules are larger than that of 1. As a result of cooperative binding, the stability constants of inclusion complexation of dimeric beta-cyclodextrin 3 with cinchonidine and cinchonine are higher than that of parent 1 by factor of 4.5 and 2.4, respectively. These results are discussed from the viewpoint of the size-fit and geometric complementary relationship between the host and guest.     

Conformational similarity indices between different residues in proteins and alpha-helix propensities

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS(XX'), between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS(AX') indicates the extent of similarity in the main-chain and side-chain conformations (phi,psi and chi1) of different residues (X) with Ala (A) and is found to have strong correlation with alpha-helix propensities. However, no subset of CS(XX') provides any linear relationship with beta-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an alpha-helix is different from the one favouring the beta-sheet. Conformationally similar residues (close CS(AX) values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS(AX) values.     

A toxic monomeric conformer of the polyglutamine protein

Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a beta-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into beta-sheet-rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble beta-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic beta-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.     

Metallothionein mRNA levels are influenced by dietary cyclodextrins in rats

The relationship between metallothionein mRNA levels and the amounts of copper and zinc in liver, kidney and small intestine by feeding dietary cyclodextrin was examined in growing Wistar rats. alpha-, beta- or gamma-cyclodextrin was fed at 50 g/kg of diet for a 7-days period (ad libitum). After feeding, the liver zinc of rats fed beta-cyclodextrin was greater than those of rats fed the other three diets. Copper accumulated in kidney of rats fed alpha- or beta-cyclodextrin. Copper content in the small intestine did not show any alterations among rats fed all kinds of diets. The cyclodextrin-supplemented diets were ineffective in zinc content in every organ. There was the greatest level of copper in serum of rats fed beta-cyclodextrin, whereas the highest level of serum zinc was observed in rats fed gamma-cyclodextrin diet. Northern blot analysis demonstrated that dietary beta- and gamma-cyclodextrins, but not alpha-cyclodextrin markedly increased the metallothionein mRNA in the liver, whereas small intestinal metallothionein mRNA levels were markedly decreased. Kidney metallothionien mRNA levels were raised appreciably by all dietary cyclodextrin intakes. Metallothionein gene expressions in liver, kidney and small intestine were not proportional to liver and serum copper or zinc levels in those tissues. These results suggest that regulation of the metallothionein mRNA levels may at least partly involved with the accumulation of metals as copper in liver and kidney of rats fed cyclodextrins.