Transmissible amyloid.

There are around 30 human diseases associated with protein misfolding and amyloid formation, each one caused by a certain protein or peptide. Many of these diseases are lethal and together they pose an enormous burden to society. The prion protein has attracted particular interest as being shown to be the pathogenic agent in transmissible diseases such as kuru, Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Whether similar transmission could occur also in other amyloidoses such as Alzheimer's disease, Parkinson's disease and serum amyloid A amyloidosis is a matter of intense research and debate. Furthermore, it has been suggested that novel biomaterials such as artificial spider silk are potentially amyloidogenic. Here, we provide a brief introduction to amyloid, prions and other proteins involved in amyloid disease and review recent evidence for their potential transmission. We discuss the similarities and differences between amyloid and silk, as well as the potential hazards associated with protein-based biomaterials.

Proteolytic generation and aggregation of peptides from transmembrane regions: lung surfactant protein C and amyloid beta-peptide.

The formation of amyloid fibrils is associated with several devastating diseases in humans and animals, including e.g. Alzheimer's disease (AD) and the spongiform encephalopathies. Here, we review and discuss the current knowledge on two amyloid peptides: lung surfactant protein C (SP-C) and the amyloid beta-peptide (Abeta), implicated in human lung disease and in AD, respectively. Both these hydrophobic peptides are derived from the transmembrane region of their precursor protein, and can transit from a monomeric alpha-helical state to a beta-sheet fibril. The alpha helices of SP-C and Abeta are composed of amino acid residues with inherently higher propensities for beta strand than helix conformation. Their helical states are stabilized by a membrane environment, and loss of membrane association thus promotes structural conversion and fibril formation. We speculate that the loss of structural context for sequences with a high propensity for formation of beta sheets may be a common feature of amyloid formation in general.

Controlling polymerization of beta-amyloid and prion-derived peptides with synthetic small molecule ligands.

The Alzheimer beta-amyloid peptide (Abeta) and a fragment of the prion protein have the capacity of forming amyloid-like fibrils when incubated under physiological conditions in vitro. Here we show that a small amyloid ligand, RO-47-1816/001, enhances this process severalfold by binding to amyloid molecules and apparently promote formation of the peptide-to-peptide bonds that join the monomers of the amyloid fibrils. This effect could be antagonized by other ligands, including analogues of RO-47-1816/001, as well as the structurally unrelated ligand Congo red. Analogues of RO-47-1816/001 with low affinity for amyloid did not display any antagonistic effect. In conclusion, these data suggest that synthetic molecules, and possibly also small natural substances present in the brain, may act in a chaperone-like fashion, promoting Abeta polymerization and growth of amyloid fibrils in vitro and possibly also in vivo. Furthermore, we demonstrate that small organic molecules can be used to inhibit the action of amyloid-enhancing compounds.

Medin: an integral fragment of aortic smooth muscle cell-produced lactadherin forms the most common human amyloid.

Aortic medial amyloid is a form of localized amyloid that occurs in virtually all individuals older than 60 years. The importance and impact of the amyloid deposits are unknown. In this study we have purified a 5.5-kDa aortic medial amyloid component, by size-exclusion chromatography and RP-HPLC, from three individuals, and we have shown by amino acid sequence analysis that the amyloid is derived from an integral proteolytic fragment of lactadherin. Lactadherin is a 364-aa glycoprotein, previously known to be expressed by mammary epithelial cells as a cell surface protein and secreted as part of the milk fat globule membrane. The multidomain protein has a C-terminal domain showing homology to blood coagulation factors V and VIII. We found that the main constituent of aortic medial amyloid is a 50-aa-long peptide, here called medin, that is positioned within the coagulation factor-like domain of lactadherin. Our result is supported by the specific labeling of aortic medial amyloid in light and electron microscopy with two rabbit antisera raised against two synthetic peptides corresponding to different parts of medin. By using in situ hybridization we have shown that lactadherin is expressed by aortic medial smooth muscle cells. Furthermore, one of the synthetic peptides forms amyloid-like fibrils in vitro. Lactadherin was not previously known to be an amyloid precursor protein or to be expressed in aortic tissue. The structure of lactadherin may implicate an important regulatory function in the aorta.

Binding of amyloid beta-peptide to mitochondrial hydroxyacyl-CoA dehydrogenase (ERAB): regulation of an SDR enzyme activity with implications for apoptosis in Alzheimer's disease.

The intracellular amyloid beta-peptide (A beta) binding protein, ERAB, a member of the short-chain dehydrogenase/reductase (SDR) family, is known to mediate apoptosis in different cell lines and to be a class II hydroxyacyl-CoA dehydrogenase. The A beta peptide inhibits the enzymatic reaction in a mixed type fashion with a Ki of 1.2 micromol/l and a KiES of 0.3 micromol/l, using 3-hydroxybutyryl-CoA. The peptide region necessary for inhibition comprises residues 12-24 of A beta1-40, covering the 16-20 fragment, which is the minimum sequence for the blockade of A beta polymerization, but that minimal fragment is not sufficient for more than marginal inhibition. The localization of ERAB to the endoplasmic reticulum and mitochondria suggests a complex interaction with components of the programmed cell death machinery. The interaction of A beta with ERAB further links oxidoreductase activity with both apoptosis and amyloid toxicity.

Endogenous proteins controlling amyloid beta-peptide polymerization. Possible implications for beta-amyloid formation in the central nervous system and in peripheral tissues.

We report that certain plasma proteins, at physiological concentrations, are potent inhibitors of amyloid beta-peptide (Abeta) polymerization. These proteins are also present in cerebrospinal fluid, but at low concentrations having little or no effect on Abeta. Thirteen proteins representing more than 90% of the protein content in plasma and cerebrospinal fluid were studied. Quantitatively, albumin was the most important protein, representing 60% of the total amyloid inhibitory activity, followed by alpha1-antitrypsin and immunoglobulins A and G. Albumin suppressed amyloid formation by binding to the oligomeric or polymeric Abeta, blocking a further addition of peptide. This effect was also observed when the incorporation of labeled Abeta into genuine beta-amyloid in tissue section was studied. The Abeta and the anti-diabetic drug tolbutamide apparently bind to the same site on albumin. Tolbutamide displaces Abeta from albumin, increasing its free concentration and enhancing amyloid formation. The present results suggest that several endogenous proteins are negative regulators of amyloid formation. Plasma contains at least 300 times more amyloid inhibitory activity than cerebrospinal fluid. These findings may provide one explanation as to why beta-amyloid deposits are not found in peripheral tissues but are only found in the central nervous system. Moreover, the data suggest that some drugs that display an affinity for albumin may enhance beta-amyloid formation and promote the development of Alzheimer's disease.

A molecular model of Alzheimer amyloid beta-peptide fibril formation.

Polymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta-fragments containing the Abeta16-20 sequence, previously shown essential for Abeta-Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta14-23 oligomers in an antiparallel beta-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy.

BACKGROUND: The accumulation of fibrillar deposits of amyloid beta-peptide (Abeta) in brain parenchyma and cerebromeningeal blood vessels is a key step in the pathogenesis of Alzheimer's disease. In this report, polymerization of Abeta was studied using fluorescence correlation spectroscopy (FCS), a technique capable of detecting small molecules and large aggregates simultaneously in solution. RESULTS: The polymerization of Abeta dissolved in Tris-buffered saline, pH 7.4, occurred above a critical concentration of 50 microM and proceeded from monomers/dimers into two discrete populations of large aggregates, without any detectable amount of oligomers. The aggregation showed very high cooperativity and reached a maximum after 40 min, followed by an increase in the amount of monomers/dimers and a decrease in the size of the large aggregates. Electron micrographs of samples prepared at the time for maximum aggregation showed a mixture of an amorphous network and short diffuse fibrils, whereas only mature amyloid fibrils were detected after one day of incubation. The aggregation was reduced when Abeta was incubated in the presence of Abeta ligands, oligopeptides previously shown to inhibit fibril formation, and aggregates were partly dissociated after the addition of the ligands. CONCLUSIONS: The polymerization of Abeta is a highly cooperative process in which the formation of very large aggregates precedes the formation of fibrils. The entire process can be inhibited and, at least in early stages, partly reversed by Abeta ligands.

Controlling amyloid beta-peptide fibril formation with protease-stable ligands.

We have previously shown that short peptides incorporating the sequence KLVFF can bind to the approximately 40amino acid residue Alzheimer amyloid beta-peptide (Abeta) and disrupt amyloid fibril formation (Tjernberg, L. O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) J. Biol. Chem. 271, 8545-8548). Here, it is shown that KLVFF binds stereospecifically to the homologous sequence in Abeta (i.e. Abeta16-20). Molecular modeling suggests that association of the two homologous sequences leads to the formation of an atypical anti-parallel beta-sheet structure stabilized primarily by interaction between the Lys, Leu, and COOH-terminal Phe. By screening combinatorial pentapeptide libraries exclusively composed of D-amino acids, several ligands with a general motif containing phenylalanine in the second position and leucine in the third position were identified. Ligands composed of D-amino acids were not only capable of binding Abeta but also prevented formation of amyloid-like fibrils. These ligands are protease-resistant and may thus be useful as experimental agents against amyloid fibril formation in vivo.

Generation of Alzheimer amyloid beta peptide through nonspecific proteolysis.

Polymerization of Alzheimer amyloid beta peptide (Abeta) into amyloid fibrils is associated with resistance to proteolysis and tissue deposition. Here, it was investigated whether Abeta might be generated as a protease-resistant core from a polymerized precursor. A 100-amino acid C-terminal fragment of the Alzheimer beta-amyloid precursor protein (C100), containing the Abeta and cytoplasmic domains, polymerized both when inserted into membranes and after purification. When subjected to digestion using the nonspecific enzyme proteinase K, the cytoplasmic domain of C100 was degraded, whereas the Abeta domain remained intact. In contrast, dissociated C100 polymers were almost completely degraded by proteinase K. Mammalian cells transfected with the human Alzheimer beta-amyloid precursor gene contained a fragment corresponding to C100, which needed similar harsh conditions to be dissolved, as did polymers formed by purified C100. Hence, it was concluded that C100 polymers are formed in mammalian cells. These results suggest that the C terminus of Abeta can be generated by nonspecific proteases, acting on a polymerized substrate, rather than a specific gamma-secretase. This offers an explanation of how the Abeta peptide can be formed in organelles containing proteases capable of cleaving most peptide bonds.

High-resolution separation of amyloid beta-peptides: structural variants present in Alzheimer's disease amyloid.

In Alzheimer's disease (AD), one of the cardinal neuropathological signs is deposition of amyloid, primarily consisting of the amyloid beta-peptide (Abeta). Structural variants of AD-associated Abeta peptides have been difficult to purify by high-resolution chromatographic techniques. We therefore developed a novel chromatographic protocol, enabling high-resolution reverse-phase liquid chromatography (RPLC) purification of Abeta variants displaying very small structural differences. By using a combination of size-exclusion chromatography and the novel RPLC protocol, Abeta peptides extracted from AD amyloid were purified and subsequently characterized. Structural analysis by microsequencing and electrospray-ionization mass spectrometry revealed that the RPLC system resolved a complex mixture of Abeta variants terminating at either residue 40 or 42. Abeta variants differing by as little as one amino acid residue could be purified rapidly to apparent homogeneity. The resolution of the system was further illustrated by its ability to separate the structural isomers of Abeta1-40. The present chromatography system might provide further insight into the role of N-terminally and posttranslationally modified Abeta variants, because each variant can now be studied individually.

Arrest of beta-amyloid fibril formation by a pentapeptide ligand.

Polymerization of amyloid beta-peptide (Abeta) into amyloid fibrils is a critical step in the pathogenesis of Alzheimer's disease. Here, we show that peptides incorporating a short Abeta fragment (KLVFF; Abeta16-20) can bind full-length Abeta and prevent its assembly into amyloid fibrils. Through alanine substitution, it was demonstrated that amino acids Lys16, Leu17, and Phe20 are critical for binding to Abeta and inhibition of Abeta fibril formation. A mutant Abeta molecule, in which these residues had been substituted, had a markedly reduced capability of forming amyloid fibrils. The present data suggest that residues Abeta16-20 serve as a binding sequence duringA beta polymerization and fibril formation. Moreover, the present KLVFF peptide may serve as a lead compound for the development of peptide and non-peptide agents aimed at inhibiting Abeta amyloidogenesis in vivo.

Characterization of stable complexes involving apolipoprotein E and the amyloid beta peptide in Alzheimer's disease brain.

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer's disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.

The Alzheimer A beta peptide develops protease resistance in association with its polymerization into fibrils.

An intriguing property of the polypeptide constituents of amyloid is that they apparently can escape the proteolytic mechanisms that normally catalyze turnover and prevent abnormal tissue accumulation of polypeptides. Here, we demonstrate that the A beta peptide, the principal component of cerebrovascular amyloid deposits in Alzheimer's disease, becomes resistant to an array of proteases as a result of structural changes associated with its polymerization into amyloid fibrils. It is further demonstrated that fibril formation per se does not lead to protease resistance but probably structural changes associated with polymerization. The results suggest that higher order structural changes, regulated by the primary structure, enable amyloidogenic polypeptides to escape proteolytic degradation and accumulate in tissues.

The metabolic pathway generating p3, an A beta-peptide fragment, is probably non-amyloidogenic.

The Alzheimer a beta amyloid precursor protein is metabolized by at least two secretory pathways. One generates the A beta peptide and the other a N-terminally truncated A beta fragment termed p3 that is considered non-amyloidogenic. However, direct evidence is missing. We have undertaken to synthesize and purify p3. Pure p3 polymerizes in vitro, forming a lattice with an ultrastructure distinct from the linear fibrils of A beta. In contrast to amyloid, polymerized p3 does not bind thioflavine T. It is therefore concluded that amino acids in the N-terminal part of the A beta molecule are required for formation of typical amyloid fibrils and that the metabolic pathway generating p3 probably is non-amyloidogenic

Relative abundance of Alzheimer A beta amyloid peptide variants in Alzheimer disease and normal aging.

The Alzheimer A beta amyloid peptide (A beta) is the principal proteinaceous component of amyloid associated with Alzheimer disease (AD). We have determined the relative abundance of A beta structural variants present in amyloid from brains of 10 individuals with sporadic AD, 2 individuals with familial AD carrying specific mutations in the Alzheimer amyloid precursor protein gene, and 5 nondemented elderly controls. A procedure of isolation based on the extreme insolubility of A beta amyloid was used. The purified, nondigested A beta was analyzed by N-terminal sequencing and electrospray-ionization mass spectrometry. Three principal A beta variants were detected--A beta-(1-40), A beta-(1-42), and A beta-(11-42)--in all brains analyzed. The predominant variant in sporadic AD was A beta-(1-40), whereas the principal A beta variant in nondemented elderly controls was A beta-(1-42). The ratio A beta-(1-40)/A beta-(1-42) differed by 10-fold between brains from nondemented controls and those with sporadic AD.