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© 2015 American Chemical Society. A variety of species express the amyloid β-protein (Aβ (the term A refers both to Aβ40 and Aβ42, whereas Aβ40 and Aβ42 refer to each isoform specifically). Those species expressing Aβ with primary structure identical to that expressed in humans have been found to develop amyloid deposits and Alzheimer's disease-like neuropathology. In contrast, the Aβ sequence in mice and rats contains three amino acid substitutions, Arg5Gly, His13Arg, and Tyr10Phe, which apparently prevent the development of AD-like neuropathology. Interestingly, the brush-tailed rat, Octodon degus, expresses Aβ containing only one of these substitutions, His13Arg, and does develop AD-like pathology. We investigate here the biophysical and biological properties of Aβ peptides from humans, mice (Mus musculus), and rats (Octodon degus). We find that each peptide displays statistical coil β-sheet secondary structure transitions, transitory formation of hydrophobic surfaces, oligomerization, formation of annuli, protofibrils, and fibrils, and an inverse correlation between rate of aggregation and aggregate size (faster aggregation produced smaller aggregates). The rank order of assembly rate was mouse > rat > Aβ42. The rank order of neurotoxicity of assemblies formed by each peptide immediately after preparation was Aβ42 > mouse rat. These data do not support long-standing hypotheses that the primary factor controlling development of AD-like neuropathology in rodents is Aβ sequence. Instead, the data support a hypothesis that assembly quaternary structure and organismal responses to toxic peptide assemblies mediate neuropathogenetic effects. The implication of this hypothesis is that a valid understanding of disease causation within a given system (organism, tissue, etc.) requires the coevaluation of both biophysical and cell biological properties of that system.

Amyloid β-protein (Aβ) assembly is a seminal process in Alzheimer's disease. Elucidating the mechanistic features of this process is thought to be vital for the design and targeting of therapeutic agents. Computational studies of the most pathologic form of Aβ, the 42-residue Aβ42 peptide, have suggested that hydrogen bonding involving Ser26 may be particularly important in organizing a monomer folding nucleus and in subsequent peptide assembly. To study this question, we experimentally determined structure-activity relationships among Aβ42 peptides in which Ser26 was replaced with Gly, Ala, α-aminobutryic acid (Abu), or Cys. We observed that aliphatic substitutions (Ala and Abu) produced substantially increased rates of formation of β-sheet, hydrophobic surface, and fibrils, and higher levels of cellular toxicity. Replacement of the Ser hydroxyl group with a sulfhydryl moiety (Cys) did not have these effects. Instead, this peptide behaved like native Aβ42, even though the hydropathy of Cys was similar to that of Abu and very different from that of Ser. We conclude that H bonding of Ser26 is the factor most important in its contribution to Aβ42 conformation, assembly, and subsequent toxicity.

Amyloid β-protein (Aβ) assembly is hypothesized to be a seminal neuropathologic event in Alzheimer’s disease (AD). We used an unbiased D-amino acid substitution strategy to determine structure-assembly relationships of 76 different Aβ40 and Aβ42 peptides. We determined the effects of the substitutions on peptide oligomerization, secondary structure dynamics, fibril assembly dynamics, and fibril morphology. Our experiments revealed that the assembly of Aβ42 was more sensitive to chiral substitutions than was Aβ40 assembly. Substitutions at identical positions in the two peptides often, but not always, produced the same effects on assembly. Sites causing substantial effects in both Aβ40 and Aβ42 include His14, Gln15, Ala30, Ile31, Met35, and Val36. Sites whose effects were unique to Aβ40 include Lys16, Leu17, and Asn 27, whereas sites unique to Aβ42 include Phe20 and Ala21. These sites may be appropriate targets for therapeutic agents that inhibit or potentiate, respectively, these effects.

Mutations in the amyloid beta-protein (Abeta) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid beta-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the Abeta40 and Abeta42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil --alpha/beta --beta and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide oligomers using in situ chemical cross-linking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial beta-strand (H6R) or alpha/beta (D7N) structure, in contrast to the predominately statistical coil structure of wild type Abeta oligomers. Mutant oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter Abeta assembly at its earliest stages, monomer folding and oligomerization, and produce oligomers that are more toxic to cultured neuronal cells than are wild type oligomers.

The structure of the 21-30 fragment of the amyloid β-protein (Aβ) was investigated by ion mobility mass spectrometry and replica exchangdynamics simulations. Mutations associated with familial Alzheimer's disease (E22G, E22Q, E22K, and D23N) of Aβ(21-30) were also studied, in order to understand any structural changes that might occur with these substitutions. The structure of the peptide shows a bend and a perpendicular turn in the backbone which is maintained by a network of D23 hydrogen bonding. Results for the mutants show that substitutions at E22 do little to alter the overall structure of the fragment. A substitution at D23 resulted in a change of structure for Aβ(21-30). A comparison of these gas-phase studies to previous solution-phase studies reveals that the peptide can fold in the absence of solvent to a structure also seen in solution, highlighting the important rolof the D23 hydrogen bonding network in stabilizing the fragment's folded structure. © 2009 American Chemical Society.

Epidemiological evidence suggests that moderate consumption of red wine reduces the incidence of Alzheimer disease (AD). To study the protective effects of red wine, experiments recently were executed in the Tg2576 mouse model of AD. These studies showed that a commercially available grape seed polyphenolic extract, MegaNatural-AZ (MN), significantly attenuated AD-type cognitive deterioration and reduced cerebral amyloid deposition (Wang, J., Ho, L., Zhao, W., Ono, K., Rosensweig, C., Chen, L., Humala, N., Teplow, D. B., and Pasinetti, G. M. (2008) J. Neurosci. 28, 6388-6392). To elucidate the mechanistic bases for these observations, here we used CD spectroscopy, photo-induced cross-linking of unmodified proteins, thioflavin T fluorescence, size exclusion chromatography, and electron microscopy to examine the effects of MN on the assembly of the two predominant disease-related amyloid beta-protein alloforms, Abeta40 and Abeta42. We also examined the effects of MN on Abeta-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism and lactate dehydrogenase activity in Abeta-treated, differentiated pheochromocytoma (PC12) cells. Initial studies revealed that MN blocked Abeta fibril formation. Subsequent evaluation of the assembly stage specificity of the effect showed that MN was able to inhibit protofibril formation, pre-protofibrillar oligomerization, and initial coil --alpha-helix/beta-sheet secondary structure transitions. Importantly, MN had protective effects in assays of cytotoxicity in which MN was mixed with Abeta prior to peptide assembly or following assembly and just prior to peptide addition to cells. These data suggest that MN is worthy of consideration as a therapeutic agent for AD.

More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the γ-secretase complex, cause aberrant amyloid β-peptide (Aβ) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. γ-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor α and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of β-amyloid precursor protein (βAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by γ-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Aβ40 production, whereas Aβ42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Aβ40. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Colletotrichum dematium is an endophytic fungus recovered from a Pteromischum sp. growing in a tropical forest in Costa Rica. This fungus makes a novel peptide antimycotic, colutellin A, with a MIC of 3.6 microg ml(-1) (48 h) against Botrytis cinerea and Sclerotinia sclerotiorum. Collutellin A has a mass of 1127.7 Da and contains residues of Ile, Val, Ser, N-methyl-Val and beta-aminoisobutryic acid in nominal molar ratios of 3 : 2 : 1 : 1 : 1, respectively. Independent lines of evidence suggest that the peptide is cyclic and sequences of Val-Ile-Ser-Ile and Ile-Pro-Val have been deduced by MS/MS as well as Edman degradation methods. Colutellin A inhibited CD4(+) T-cell activation of interleukin 2 (IL-2) production with an IC(50) of 167.3+/-0.38 nM, whereas cyclosporin A in the same test yielded a value of 61.8 nM. Inhibition of IL-2 production by collutellin A at such a low concentration indicates the potential immunosuppressive activity of this compound. In repeated experiments, cyclosporin A at or above 8 microg ml(-1) exhibited high levels of cytotoxicity on human peripheral blood mononuclear cells, whereas collutellin A or DMSO (carrier) alone, after 24 and 48 h of culture, exhibited no toxicity. Because of these properties collutellin A has potential as a novel immunosuppressive drug.

Some biotechnological inventions involve expensive, sophisticated machines. Others are relatively simple innovations that nevertheless address, and solve difficult problems. Synthesis and purification of highly hydrophobic peptides can be a difficult and challenging task, particularly when these peptides have low solubility in both aqueous and organic solvents. Here we describe the synthesis and purification of a series of peptides derived from the hydrophobic Cterminus of the 42-residue form of amyloid β-protein (Aβ42), a peptide believed to be the primary cause for Alzheimer’s disease (AD). The series of C-terminal fragments (CTFs) had the general formula Aβ(x-42), x=28-39, which potentially can be used as inhibitors of Aβ42 assembly and neurotoxicity. Synthesis and purification of peptides containing 8-residues or less were straightforward. However, HPLC purification of longer peptides was problematic and provided

Streptomyces NRRL 30562 was originally isolated as an endophyte from Kennedia nigriscans, snakevine, in the Northern Territory of Australia. This plant has been used for centuries by Aboriginal peoples to treat open bleeding wounds to prevent sepsis. A solvent extract of the crude fluid from cultures of this endophyte possesses wide-spectrum antibiotic activity. Some of the bioactivity is associated with the appearance of actinomycins X2, D, and Xobeta, the first two of which had been previously designated munumbicins A and B, respectively. Other novel compounds bearing wide-spectrum antibiotic activity are also produced by Streptomyces NRRL 30562, and these are designated munumbicins E-4 and E-5. Mass spectrometric analyses of these peptide antibiotics show that they have identical masses (1445.00) but different retention times on HPLC. Both compounds showed activity against gram-positive and gram-negative bacteria. The plant pathogenic fungus, Pythium ultimum is sensitive to both munumbicins at 5.0 microg mL(-1) The malarial parasite, Plasmodium falciparum has IC50 values of 0.50+/-0.08 and 0.87+/-0.0.26 microg mL(-1) for E-4 and E-5, respectively. It appears that other bioactive compounds, related to E-4 and E-5, are also produced making it the most biologically active endophytic Streptomyces spp. on record.

Formation of toxic oligomeric and fibrillar structures by the amyloid beta-protein (Abeta) is linked to Alzheimer's disease (AD). To facilitate the targeting and design of assembly inhibitors, intrinsic fluorescence was used to probe assembly-dependent changes in Abeta conformation. To do so, Tyr was substituted in Abeta40 or Abeta42 at position 1, 10 (wild type), 20, 30, 40, or 42. Fluorescence then was monitored periodically during peptide monomer folding and assembly. Electron microscopy revealed that all peptides assembled readily into amyloid fibrils. Conformational differences between Abeta40 and Abeta42 were observed in the central hydrophobic cluster (CHC) region, Leu17-Ala21. Tyr20 was partially quenched in unassembled Abeta40 but displayed a significant and rapid increase in intensity coincident with the maturation of an oligomeric, alpha-helix-containing intermediate into amyloid fibrils. This process was not observed during Abeta42 assembly, during which small decreases in fluorescence intensity were observed in the CHC. These data suggest that the structure of the CHC in Abeta42 is relatively constant within unassembled peptide and during the self-association process. Solvent accessibility of the Tyr ring was studied using a mixed solvent (dimethyl sulfoxide/water) system. [Tyr40]Abeta40, [Tyr30]Abeta42, and [Tyr42]Abeta42 all were relatively shielded from solvent. Analysis of the assembly dependence of the site-specific intrinsic fluorescence data suggests that the CHC is particularly important in controlling Abeta40 assembly, whereas the C-terminus plays the more significant role in Abeta42 assembly.

Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) forms neurotoxic oligomers. The predominant in vivo Abeta alloforms, Abeta40 and Abeta42, have distinct oligomerization pathways. Abeta42 monomers oligomerize into pentamer/hexamer units (paranuclei) which self-associate to form larger oligomers. Abeta40 does not form these paranuclei, a fact which may explain the particularly strong linkage of Abeta42 with AD. Here, we sought to determine the structural elements controlling paranucleus formation as a first step toward the development of strategies for treating AD. Because oxidation of Met(35) is associated with altered Abeta assembly, we examined the role of Met(35) in controlling Abeta oligomerization. Oxidation of Met(35) in Abeta42 blocked paranucleus formation and produced oligomers indistinguishable in size and morphology from those produced by Abeta40. Systematic structural alterations of the C(gamma)(35)-substituent group revealed that its electronic nature, rather than its size (van der Waals volume), was the factor controlling oligomerization pathway choice. Preventing assembly of toxic Abeta42 paranuclei through selective oxidation of Met(35) thus represents a potential therapeutic approach for AD.

Proteases that degrade the amyloid beta-protein (Abeta) are important regulators of brain Abeta levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Abeta degradation assays based on the novel substrate, fluorescein-Abeta-(1-40)-Lys-biotin (FAbetaB). Liquid chromatography/mass spectrometric analysis shows that FAbetaB is hydrolyzed at closely similar sites as wild-type Abeta by neprilysin and insulin-degrading enzyme, the two most widely studied Abeta-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAbetaB degradation by several Abeta-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Abeta-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.

Munumbicins A, B, C and D are newly described antibiotics with a wide spectrum of activity against many human as well as plant pathogenic fungi and bacteria, and a Plasmodium sp. These compounds were obtained from Streptomyces NRRL 3052, which is endophytic in the medicinal plant snakevine (Kennedia nigriscans), native to the Northern Territory of Australia. This endophyte was cultured, the broth was extracted with an organic solvent and the contents of the residue were purified by bioassay-guided HPLC. The major components were four functionalized peptides with masses of 1269.6, 1298.5, 1312.5 and 1326.5 Da. Numerous other related compounds possessing bioactivity, with differing masses, were also present in the culture broth extract in lower quantities. With few exceptions, the peptide portion of each component contained only the common amino acids threonine, aspartic acid (or asparagine), glutamic acid (or glutamine), valine and proline, in varying ratios. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had an MIC of 2.5 microg x ml(-1) against a methicillin-resistant strain of Staphylococcus aureus, whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as Bacillus anthracis and multidrug-resistant Mycobacterium tuberculosis. However, the most impressive biological activity of any of the munumbicins was that of munumbicin D against the malarial parasite Plasmodium falciparum, having an IC(50) of 4.5+/-0.07 ng x ml(-1). This report also describes the potential of the munumbicins in medicine and agriculture.

beta-Site amyloid precursor protein cleavage enzyme (BACE)-1 and BACE-2 are members of a novel family of membrane-bound aspartyl proteases. While BACE-1 is known to cleave beta-amyloid precursor protein (betaAPP) at the beta-secretase site and to be required for the generation of amyloid beta-peptide (Abeta), the role of its homologue BACE-2 in amyloidogenesis is less clear. We now demonstrate that BACE-1 and BACE-2 have distinct specificities in cleavage of betaAPP in cultured cells. Radiosequencing of the membrane-bound C-terminal cleavage product revealed that BACE-2 cleaves betaAPP in the middle of the Abeta domain between phenylalanines 19 and 20, resulting in increased secretion of APPs-alpha- and p3-like products and reduced production of Abeta species. This cleavage can occur in the Golgi and later secretory compartments. We also demonstrate that BACE-1-mediated cleavage of betaAPP at Asp1 of the Abeta domain can occur as early as in the endoplasmic reticulum, while cleavage at Glu11 occurs in later compartments. These data indicate that the distinct specificities of BACE-1 and BACE-2 in their cleavage of betaAPP differentially affect the generation of Abeta.

One of the earliest events in amyloid β-protein (Aβ) self-Association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create Aβ42 from 26-O-Acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized Nα-Acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O → N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy-mass spectrometry) were quite consistent. A rank order of Ac-iAβ42 > iAβ42 > Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into "pure" Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies. © 2014 Elsevier Ltd.

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved. However, the significance of this intermediate has been a matter of debate. We report here that the formation of an oligomeric, alpha-helix-containing assembly is a key step in Abeta fibrillogenesis. The generality of this phenomenon was supported by conformational studies of 18 different Abeta peptides, including wild-type Abeta(1-40) and Abeta(1-42), biologically relevant truncated and chemically modified Abeta peptides, and Abeta peptides causing familial forms of cerebral amyloid angiopathy. Without exception, fibrillogenesis of these peptides involved an oligomeric alpha-helix-containing intermediate and the kinetics of formation of the intermediate and of fibrils was temporally correlated. The kinetics varied depending on amino acid sequence and the extent of peptide N- and C-terminal truncation. The pH dependence of helix formation suggested that Asp and His exerted significant control over this process and over fibrillogenesis in general. Consistent with this idea, Abeta peptides containing Asp-->Asn or His-->Gln substitutions showed altered fibrillogenesis kinetics. These data emphasize the importance of the dynamic interplay between Abeta monomer conformation and oligomerization state in controlling fibrillogenesis kinetics.

Several pathogenic Alzheimer's disease (AD) mutations have been described, all of which cause increased amyloid beta-protein (Abeta) levels. Here we present studies of a pathogenic amyloid precursor protein (APP) mutation, located within the Abeta sequence at codon 693 (E693G), that causes AD in a Swedish family. Carriers of this 'Arctic' mutation showed decreased Abeta42 and Abeta40 levels in plasma. Additionally, low levels of Abeta42 were detected in conditioned media from cells transfected with APPE693G. Fibrillization studies demonstrated no difference in fibrillization rate, but Abeta with the Arctic mutation formed protofibrils at a much higher rate and in larger quantities than wild-type (wt) Abeta. The finding of increased protofibril formation and decreased Abeta plasma levels in the Arctic AD may reflect an alternative pathogenic mechanism for AD involving rapid Abeta protofibril formation leading to accelerated buildup of insoluble Abeta intra- and/or extracellularly.

Synthetic amyloid beta-protein (A beta) is used widely to study fibril formation and the physiologic effects of low molecular weight and fibrillar forms of the peptide on cells in culture or in experimental animals. Not infrequently, conflicting results have arisen in these studies, in part due to variation in the starting conformation and assembly state of A beta. To avoid these problems, we sought a simple, reliable means of preparing A beta for experimental use. We found that solvation of synthetic peptide with sodium hydroxide (A beta x NaOH), followed by lyophilization, produced stocks with superior solubility and fibrillogenesis characteristics. Solubilization of the pretreated material with neutral buffers resulted in a pH transition from approximately 10.5 to neutral, avoiding the isoelectric point of A beta (pI approximately 5.5), at which A beta precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (A beta x TFA) or hydrochloric acid (A beta x HCl) salts of A beta, yields of "low molecular weight A beta" (monomers and/or dimers) were improved significantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that A beta x NaOH formed fibrils more readily than did the other A beta preparations and that these fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for the preparation of A beta for biophysical and physiologic studies.

Alzheimer's disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid beta-protein (Abeta). Fibrils, both in vitro and in vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control Abeta production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of Abeta fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364-22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight Abeta (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer's disease and for our understanding of fibril assembly are discussed.

A novel family of peptide antimycotics, termed ecomycins, is described from Pseudomonas viridiflava, a plant-associated bacterium. Ecomycins B and C have molecular masses of 1153 and 1181. They contain equimolar amounts of a beta hydroxyaspartic acid, homoserine, threonine, serine, alanine, glycine and one unknown amino acid. Fatty acids were detectable after hydrolysis, methylation and gas chromatography and mass spectroscopy. The ecomycins have significant bioactivities against a wide range of human and plant pathogenic fungi. The minimum inhibitory concentration values for ecomycin B were 4.0 micrograms ml-1 against Cryptococcus neoformans and 31 micrograms ml-1 against Candida albicans. Pseudomonas viridiflava also produces what appears to be syringotoxin, an antifungal lipopeptide previously described from Ps. syringae.

γ-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor α (TNFα) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases. © 2006 Nature Publishing Group.

Fibrillogenesis of the amyloid β-protein (Aβ) is a seminal pathogenetic event in Alzheimer’s disease. Inhibiting fibrillogenesis is thus one approach toward disease therapy. Rational design of fibrillogenesis inhibitors requires elucidation of the stages and kinetics of Aβ fibrillogenesis. We report results of studies designed to examine the initial stages of Aβ oligomerization. Size exclusion chromatography, quasielastic light scattering spectroscopy, and electron microscopy were used to characterize fibrillogenesis intermediates. After dissolution in 0.1 m Tris-HCl, pH 7.4, and removal of pre-existent seeds, Aβ chromatographed almost exclusively as a single peak. The molecules composing the peak had average hydrodynamic radii of 1.8 ± 0.2 nm, consistent with the predicted size of dimeric Aβ. Over time, an additional peak, with a molecular weight >100,000, appeared. This peak contained predominantly curved fibrils, 6–8 nm in diameter and

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β−protein (Aβ). The two additional C−terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single−molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C−termini, the N−terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N−terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N−terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.

Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer's disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu(2+). Cu(2+) cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu(2+) regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu(2+) cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu(2+) cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu(2+) cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu(2+) cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu(2+) cations also stabilize the dimers. No effects of Cu(2+) cations on Aβ-Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu(2+) cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu(2+) cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu(2+) cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu(2+) cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu(2+).

Alzheimer's disease (AD) is linked to the aberrant assembly of the amyloid β-protein (Aβ). The21AEDVGSNKGA30segment, Aβ(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aβ(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development. © 2012 American Chemical Society.

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson’s disease and Alzheimer’s disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimer’s disease.

Oligomers of the amyloid beta-protein (Abeta) play an important role in Alzheimer's disease (AD). We hypothesized that AD patients have a central nervous system environment that promotes Abeta oligomerization. We investigated the effect of cerebrospinal fluid (CSF) from 33 patients with AD and 33 age-matched, non-demented controls on oligomerization of Abeta1-40 and Abeta1-42 using the technique of photo-induced cross-linking of unmodified proteins. CSF inhibited oligomerization of both Abeta1-40 and Abeta1-42. This inhibitory effect was significantly weaker in AD patients than in non-demented controls. Our results indicate that AD patients have a CSF environment favorable for Abeta oligomerization.

Understanding the structural and assembly dynamics of the amyloid β-protein (Aβ) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the Aβ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform "scanning PICUP." Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for Aβ40) or 42 (for Aβ42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil →α/β→β transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr1-substituted homologues of Aβ40 and Aβ42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr1]Aβ40 fibrils. Substitution of Aβ40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both Aβ40 and Aβ42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr1) to alter Aβ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that Aβ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

The C-terminus of amyloid beta-protein (Abeta) 42 plays an important role in this protein's oligomerization and may therefore be a good therapeutic target for the treatment of Alzheimer's disease. Certain C-terminal fragments (CTFs) of Abeta42 have been shown to disrupt oligomerization and to strongly inhibit Abeta42-induced neurotoxicity. Here we study the structures of selected CTFs [Abeta(x-42); x=29-31, 39] using replica exchange molecular dynamics simulations and ion mobility mass spectrometry. Our simulations in explicit solvent reveal that the CTFs adopt a metastable beta-structure: beta-hairpin for Abeta(x-42) (x=29-31) and extended beta-strand for Abeta(39-42). The beta-hairpin of Abeta(30-42) is converted into a turn-coil conformation when the last two hydrophobic residues are removed, suggesting that I41 and A42 are critical in stabilizing the beta-hairpin in Abeta42-derived CTFs. The importance of solvent in determining the structure of the CTFs is further highlighted in ion mobility mass spectrometry experiments and solvent-free replica exchange molecular dynamics simulations. A comparison between structures with solvent and structures without solvent reveals that hydrophobic interactions are critical for the formation of beta-hairpin. The possible role played by the CTFs in disrupting oligomerization is discussed.

Alzheimer's disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of Aβ42 are believed to be the main cause of AD; therefore, disruption of Aβ oligomerization is a promising approach for developing therapeutics for AD. Formation of Aβ42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of Aβ42 may get incorporated into oligomers of Aβ42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using Aβ fragments with the general formula Aβ( x −42) ( x = 28–39). A cell viability screen identified Aβ(31–42) as the most potent inhibitor. In addition, the shortest peptide, Aβ(39–42), also had high activity. Both Aβ(31–42) and Aβ(39–42) inhibited Aβ-induced cell death and rescued disruption of synaptic activity by Aβ42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of Aβ42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with Aβ42 to form heterooligomers. Thus, Aβ(31–42) and Aβ(39–42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure–activity approach.

Amyloid β-protein (Aβ) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). Recently, to elucidate the oligomerization pathway, we studied Aβ monomer folding and identified a decapeptide segment of Aβ, 21 Ala– 22 Glu– 23 Asp– 24 Val– 25 Gly– 26 Ser– 27 Asn– 28 Lys– 29 Gly– 30 Ala, within which turn formation appears to nucleate monomer folding. The turn is stabilized by hydrophobic interactions between Val-24 and Lys-28 and by long-range electrostatic interactions between Lys-28 and either Glu-22 or Asp-23. We hypothesized that turn destabilization might explain the effects of amino acid substitutions at Glu-22 and Asp-23 that cause familial forms of AD and cerebral amyloid angiopathy. To test this hypothesis, limited proteolysis, mass spectrometry, and solution-state NMR spectroscopy were used here to determine and compare the structure and stability of the Aβ(21–30) turn within wild-type Aβ and seven clinically relevant homologues. In addition, we determined the relative differences in folding free energies (ΔΔ G f ) among the mutant peptides. We observed that all of the disease-associated amino acid substitutions at Glu-22 or Asp-23 destabilized the turn and that the magnitude of the destabilization correlated with oligomerization propensity. The Ala21Gly (Flemish) substitution, outside the turn proper (Glu-22–Lys-28), displayed a stability similar to that of the wild-type peptide. The implications of these findings for understanding Aβ monomer folding and disease causation are discussed.

A subset of Alzheimer disease cases is caused by autosomal dominant mutations in genes encoding the amyloid beta-protein precursor or presenilins. Whereas some amyloid beta-protein precursor mutations alter its metabolism through effects on Abeta production, the pathogenic effects of those that alter amino acid residues within the Abeta sequence are not fully understood. Here we examined the biophysical effects of two recently described intra-Abeta mutations linked to early-onset familial Alzheimer disease, the D7N Tottori-Japanese and H6R English mutations. Although these mutations do not affect Abeta production, synthetic Abeta(1-42) peptides carrying D7N or H6R substitutions show enhanced fibril formation. In vitro analysis using Abeta(1-40)-based mutant peptides reveal that D7N or H6R mutations do not accelerate the nucleation phase but selectively promote the elongation phase of amyloid fibril formation. Notably, the levels of protofibrils generated from D7N or H6R Abeta were markedly inhibited despite enhanced fibril formation. These N-terminal Abeta mutations may accelerate amyloid fibril formation by a unique mechanism causing structural changes of Abeta peptides, specifically promoting the elongation process of amyloid fibrils without increasing metastable intermediates.

Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer's disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu2+. Cu2+ cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu2+ regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu2+ cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu2+ cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu2+ cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu2+ cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu2+ cations also stabilize the dimers. No effects of Cu2+ cations on Aβ-Aβ interactions were observed at pH4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu2+ cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu2+ cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu2+ cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu2+ cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu2+.

beta-Site amyloid precursor protein cleavage enzyme (BACE)-1 and BACE-2 are members of a novel family of membrane-bound aspartyl proteases. While BACE-1 is known to cleave beta-amyloid precursor protein (betaAPP) at the beta-secretase site and to be required for the generation of amyloid beta-peptide (Abeta), the role of its homologue BACE-2 in amyloidogenesis is less clear. We now demonstrate that BACE-1 and BACE-2 have distinct specificities in cleavage of betaAPP in cultured cells. Radiosequencing of the membrane-bound C-terminal cleavage product revealed that BACE-2 cleaves betaAPP in the middle of the Abeta domain between phenylalanines 19 and 20, resulting in increased secretion of APPs-alpha- and p3-like products and reduced production of Abeta species. This cleavage can occur in the Golgi and later secretory compartments. We also demonstrate that BACE-1-mediated cleavage of betaAPP at Asp1 of the Abeta domain can occur as early as in the endoplasmic reticulum, while cleavage at Glu11 occurs in later compartments. These data indicate that the distinct specificities of BACE-1 and BACE-2 in their cleavage of betaAPP differentially affect the generation of Abeta.

The presenilin (PS)-dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP) results in the secretion of amyloid beta-peptide (Abeta). However, little is known about the corresponding C-terminal cleavage product (CTFgamma). We have now identified CTFgamma in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFgamma is facilitated by PSs, since a dominant-negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, gamma-secretase inhibitors, including one that is known to bind to PS, also block CTFgamma generation. Sequence analysis revealed that CTFgamma is produced by a novel gamma-secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.

In a Flemish kindred, an Ala(692)-->Gly amino acid substitution in the amyloid beta-protein precursor (AbetaPP) causes a form of early-onset Alzheimer's disease (AD) which displays prominent amyloid angiopathy and unusually large senile plaque cores. The mechanistic basis of this Flemish form of AD is unknown. Previous in vitro studies of amyloid beta-protein (Abeta) production in HEK-293 cells transfected with cDNA encoding Flemish AbetaPP have shown that full-length [Abeta(1-40)] and truncated [Abeta(5-40) and Abeta(11-40)] forms of Abeta are produced. In an effort to determine how these peptides might contribute to the pathogenesis of the Flemish disease, comparative biophysical and neurotoxicity studies were performed on wild-type and Flemish Abeta(1-40), Abeta(5-40) and Abeta(11-40). The results revealed that the Flemish amino acid substitution increased the solubility of each form of peptide, decreased the rate of formation of thioflavin-T-positive assemblies, and increased the SDS-stability of peptide oligomers. Although the kinetics of peptide assembly were altered by the Ala(21)-->Gly substitution, all three Flemish variants formed fibrils, as did the wild-type peptides. Importantly, toxicity studies using cultured primary rat cortical cells showed that the Flemish assemblies were as potent a neurotoxin as were the wild-type assemblies. Our results are consistent with a pathogenetic process in which conformational changes in Abeta induced by the Ala(21)-->Gly substitution would facilitate peptide adherence to the vascular endothelium, creating nidi for amyloid growth. Increased peptide solubility and assembly stability would favour formation of larger deposits and inhibit their elimination. In addition, increased concentrations of neurotoxic assemblies would accelerate neuronal injury and death.

A)40 and A)42 are peptides that adopt similar random-coil structures in solution. A)42, however, is significantly more neurotoxic than A)40 and forms amyloid fibrils much more rapidly than A)40. Here, mass spectrometry and ion mobility spectrometry are used to investigate a mixture of A)40 and A)42. The mass spectrum for the mixed solution shows the presence of a heterooligomer composed of equal parts of A)40 and A)42. Ion mobility results indicate that this mixed species comprises an oligomer distribution extending to tetramers. A)40 alone produces such a distribution, whereas A)42 alone produces oligomers as large as dodecamers. This indicates that A)40 inhibits A)42 oligomerization. Copyright © 2009 American Chemical Society.

A unique lipopeptide antimycotic, termed cryptocandin, is described from Cryptosporiopsis cf. quercina, an endophytic fungus. Cryptocandin, with a molecular mass of 1079 Da, contains equimolar amounts of 3,4-dihydroxyhomotyrosine, 4-hydroxyproline, threonine, glutamine, 3-hydroxy-4-hydroxymethylproline, 4,5-dihydroxyornithine and palmitic acid. Cryptocandin is chemically related to well-known antimycotics, the echinocandins and pneumocandins, which are produced by such fungi as Zalerion arboricola, Pezicula spp. and Aspergillus spp. Cryptocandin has minimal inhibitory concentration values of 0.03-0.07 microgram ml-1 against isolates of Candida albicans, Trichophyton mentagrophytes and Trichophyton rubrum. Cryptocandin is also active against a number of plant-pathogenic fungi including Sclerotinia sclerotiorum and Botrytis cinerea.

Background The current model for the development of Alzheimer’s (AD), Parkinson’s, Huntington’s, prion, and other neurodegenerative diseases involves protein misfolding as the early step followed by spontaneous aggregation, with specific proteins identified as the primary initiators for disease development. Therefore, elucidating the properties of the disease-prone misfolded states, understanding the mechanism of their formation, and identification of their most toxic forms will open prospects for the development of early diagnostics and specific therapeutics for these diseases.