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PubMed2002

CD36, a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta-amyloid fibrils.

A pathological hallmark of Alzheimer's disease is the senile plaque, composed of beta-amyloid fibrils, microglia, astrocytes, and dystrophic neurites. We reported previously that class A scavenger receptors mediate adhesion of microglia and macrophages to beta-amyloid fibrils and oxidized low-density lipoprotein (oxLDL)-coated surfaces. We also showed that CD36, a class B scavenger receptor and an oxLDL receptor, promotes H(2)O(2) secretion by macrophages adherent to oxLDL-coated surfaces. Whether CD36 is expressed on microglia, and whether it plays a role in secretion of H(2)O(2) by microglia interacting with fibrillar beta-amyloid is not known. Using fluorescence-activated cell sorting analysis and immunohistochemistry, we found that CD36 is expressed on human fetal microglia, and N9-immortalized mouse microglia. We also found that CD36 is expressed on microglia and on vascular endothelial cells in the brains of Alzheimer's disease patients. Bowes human melanoma cells, which normally do not express CD36, gained the ability to specifically bind to surfaces coated with fibrillar beta-amyloid when transfected with a cDNA encoding human CD36, suggesting that CD36 is a receptor for fibrillar beta-amyloid. Furthermore, two different monoclonal antibodies to CD36 inhibited H(2)O(2) production by N9 microglia and human macrophages adherent to fibrillar beta-amyloid by approximately 50%. Our data identify a role for CD36 in fibrillar beta-amyloid-induced H(2)O(2) production by microglia, and imply that CD36 can mediate binding to fibrillar beta-amyloid. We propose that similar to their role in the interaction of macrophages with oxLDL, class A scavenger receptors and CD36 play complimentary roles in the interactions of microglia with fibrillar beta-amyloid.

Alzheimer Disease↗
PubMed1997

In vitro aggregation facilities beta-amyloid peptide-(25-35)-induced amnesia in the rat.

The beta-amyloid peptide-(25-35) fragment, but not beta-amyloid peptide-(1-28), shares with beta-amyloid protein-(1-42) the ability to self-aggregate and to induce neurotoxicity in vitro. This study examined the induction of amnesia in rats given intracerebroventricularly soluble or aggregated beta-amyloid peptide-(25-35) (5-45 nmol), or beta-amyloid peptide-(1-28) (15 nmol). Memory deficit in the water-maze test, examined 14 days after aggregated beta-amyloid peptide-(25-35) injection, was more pronounced than with soluble beta-amyloid peptide-(25-35). beta-Amyloid peptide-(1-28) only affected retention. These results confirm the direct amnesic properties of beta-amyloid peptides in the rat brain and showed that prior peptide aggregation markedly facilitates the appearance of amnesia.

Amnesia↗
PubMed1997

Transthyretin quaternary and tertiary structural changes facilitate misassembly into amyloid.

Human transthyretin (TTR) can be transformed into amyloid fibrils by partial acid denaturation to yield a monomeric amyloidogenic intermediate that self-associates into amyloid through quaternary structural intermediates, which are identified by sedimentation velocity methods. The monomeric amyloidogenic intermediate has substantial beta-sheet structure with a nonnative but intact tertiary structure as discerned from spectroscopic methods. Proteolysis sensitivity studies suggest that the C-strand-loop-D-strand portion of TTR becomes disordered and moves away from the core of the beta-sandwich fold upon formation of the monomeric amyloidogenic intermediate over the pH range 5.1-3.9. The single site mutations that are associated with early onset amyloid disease [familial amyloid polyneuropathy (FAP)] function by destabilizing tetrameric TTR. Under mild denaturing conditions, the FAP variants populate the monomeric amyloidogenic intermediate conformation, which assembles into amyloid, whereas wild-type TTR remains tetrameric and nonamyloidogenic. The FAP mutations do not significantly alter the native folded structure; instead, they appear to act by making the thermodynamics and perhaps the kinetics more favorable for formation of the amyloidogenic intermediate. Suppressor mutations have also been characterized that strongly stabilize tetrameric TTR and disfavor the formation of the monomeric amyloidogenic intermediate, thus inhibiting amyloid formation. The mechanistic details characterizing transthyretin amyloid fibril formation available from the biophysical studies outlined within have been utilized to develop a new therapeutic strategy for intervention in human amyloid disease. This approach features small molecules that bind with high affinity to the normal fold of transthyretin, inhibiting the quaternary and tertiary structural changes associated with the formation of the monomeric amyloidogenic intermediate that self-assembles into amyloid. Ligand binding to TTR stabilizes the native tetrameric fold, which is nonamyloidogenic.

Amyloid↗
PubMed1995

Image analysis of beta-amyloid load in Alzheimer's disease and relation to dementia severity.

The protein beta-amyloid is said to be central to the disease process of Alzheimer's disease (AD). Several groups have developed transgenic models that overexpress the amyloid precursor protein or beta-amyloid and then develop AD-like neuropathology. Another report suggests that beta-amyloid accumulation in old dogs correlates with cognitive impairment. However, many other researchers argue that beta-amyloid deposition in senile plaques is a secondary event because plaque numbers in man do not correlate well with cognition. We set out to analyse this conumdrum in man. We selected 16 mild to severely demented AD cases on the basis of mini-mental state exam scores (MMSE; n = 16). We also included 4 controls who represented the upper range of cognitive ability. We used a computer-based image analysis of cross-sectional area of the brain occupied by beta-amyloid immunopositive deposition. We used this technique in preference to conventional methods of manual plaque counts and found a strong relation between beta-amyloid load in entorhinal cortex and cognition measured on various scales (r = -0.93 versus the Blessed IMC). Our study suggests that the size of cortical area affected by beta-amyloid deposition is an important factor in the clinical manifestation of dementia, and lends support to the possibility that beta-amyloid is central to the aetiology of AD.

Aged↗
PubMedDate not supplied

The inflammation-induced pathological chaperones ACT and apo-E are necessary catalysts of Alzheimer amyloid formation.

Biochemical, genetic, and epidemiological evidence indicates that inflammation is an essential part of the pathogenesis of Alzheimer's disease. Over the last decade, we and others have focused on the mechanism by which specific inflammatory molecules contribute to the Alzheimer pathogenic pathway. In particular, we have learned that several acute phase/inflammatory molecules, specifically alpha(1)-antichymotrypsin (ACT) and apolipoprotein E (apoE) that are overproduced in the AD brain can promote the formation of, and are associated with, the neurotoxic amyloid deposits that are a key pathological hallmark of the disease. Because both of these proteins bind to the Abeta peptide and catalyze its polymerization into amyloid filaments, they have been termed "pathological chaperones".ACT, and, to a lesser extent, apoE are greatly overproduced only in areas of the AD brain that are prone to amyloid formation. This restriction suggests a local inflammatory reaction may underlie the regional specificity of amyloid deposition by inducing the production of pathological chaperones. The data that will be discussed indicate that ACT over-expression is caused by the activation of ACT mRNA synthesis in astrocytes in response to increased production of the inflammatory cytokine IL-1. IL-1 is released from microglia that become activated by pre-amyloid seeds of Abeta. Recently, this inflammatory cascade has been extended to include the amyloid precursor protein (APP), for IL-1 also upregulates the production of APP in astrocytes, but at the translational rather that the transcriptional level. Thus many of the key elements of the Alzheimer's disease pathogenic pathway are products of a local inflammatory reaction in the brain. Further support for a mechanistic role of inflammation in the Alzheimer's disease pathogenic pathway has been provided by genetic studies, which have associated an increased risk of developing AD with specific polymorphisms in the apoE, ACT, and the IL-1 genes. Most recently, transgenic mouse models of AD have demonstrated that ACT and apoE are amyloid promoters/pathological chaperones in vivo whose contribution is necessary for both amyloid formation and for amyloid-associated cognitive decline and memory loss. The importance of these findings is that they help to place inflammation at the center of the pathogenic pathway to Alzheimer's disease and identify specific steps in the pathway that may be amenable to therapeutic intervention.

Alzheimer Disease↗
PubMed2003

Immunocytochemical evidence that amyloid beta (1-42) impairs endogenous antioxidant systems in vivo.

Amyloid beta, the major constituent of the senile plaques in the brains of patients with Alzheimer's disease, is cytotoxic to neurons and has a central role in the pathogenesis of the disease. We have previously demonstrated that potent antioxidants idebenone and alpha-tocopherol prevent learning and memory impairment in rats which received a continuous intracerebroventricular infusion of amyloid beta, suggesting a role for oxidative stress in amyloid beta-induced learning and memory impairment. To test the hypothesis, in the present study, we investigated alterations in the immunoreactivity of endogenous antioxidant systems such as mitochondrial Mn-superoxide dismutase, glutathione, glutathione peroxidase and glutathione-S-transferase following the continuous intracerebroventricular infusion of amyloid beta for 2 weeks. The infusion of amyloid beta (1-42) resulted in a significant reduction of the immunoreactivity of these antioxidant substances in such brain areas as the hippocampus, parietal cortex, piriform cortex, substantia nigra and thalamus although the same treatment with amyloid beta (40-1) had little effect. The alterations induced by amyloid beta (1-42) were not uniform, but rather specific for each immunoreactive substance in a brain region-dependent manner. These results demonstrate a cytological effect of oxidative stress induced by amyloid beta (1-42) infusion. Furthermore, our findings may indicate a heterogeneous susceptibility to the oxidative stress produced by amyloid beta.

Amyloid beta-Peptides↗
PubMed1999

No hippocampal neuron or synaptic bouton loss in learning-impaired aged beta-amyloid precursor protein-null mice.

Aged beta-amyloid precursor protein-null mice were used to investigate the relationship between beta-amyloid precursor protein, hippocampal neuron and synaptic bouton number, and cognitive function. Learning and memory performance of aged beta-amyloid precursor protein-null mice and age-matched controls were assessed in the Morris water maze. Beta-amyloid precursor protein-null mice demonstrated impaired task acquisition as measured by significantly longer swim path lengths, a higher percentage of failed trials, and more frequent thigmotaxis behavior than controls. In a subsequent probe trial, beta-amyloid precursor protein-null mice spent significantly less time in the old goal quadrant, and made fewer crossings over the old platform location than did controls. No differences in motor or visual skills were observed which could account for the performance differences. In light of these findings and previous evidence for a role of beta-amyloid precursor protein in neuronal maintenance and synaptogenesis, we pursued the hypothesis that the learning impairment of beta-amyloid precursor protein-null mice may be a reflection of differences in neuron or synaptophysin-positive presynaptic bouton number. Thus, unbiased stereological analysis was used to estimate neuron and synaptic bouton number in dentate gyrus and hippocampal CA1 of the behaviorally characterized mice. No difference in neuron or synaptophysin-positive presynaptic bouton number was found between the beta-amyloid precursor protein-null mice and age-matched controls. Our results suggest that the learning impairment of beta-amyloid precursor protein-null mice is not mediated by a loss of hippocampal neurons or synaptic boutons.

Aging↗
PubMed1999

Transgenic murine cortical neurons expressing human Bcl-2 exhibit increased resistance to amyloid beta-peptide neurotoxicity.

The generation of reactive oxygen species has been implicated in the neurotoxicity of amyloid beta-peptide, the main constituent of the senile plaques that accumulates in the brain of Alzheimer's disease victims. In this study, we have compared the toxicity of amyloid beta-peptide on cultured cortical neurons from control mice and transgenic mice expressing either human copper-zinc superoxide dismutase or human Bcl-2, two proteins that protect cells against oxidative damage. Copper-zinc superoxide dismutase overexpression failed to protect cortical neurons against the toxicity of amyloid beta-peptide(25-35) [the minimal cytotoxic fragment of amyloid beta-peptide(1-42)] as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and an enzyme-linked immunoabsorbent assay using an antibody directed against microtubule-associated protein-2 (a specific neuronal protein), ruling out a role for superoxide anion and peroxynitrite in amyloid beta-peptide-evoked neurotoxicity. On the contrary, cortical neurons expressing human copper-zinc superoxide dismutase exhibited increased apoptotic nuclei in both untreated and amyloid beta-peptide(25-35)-exposed neurons. Transgenic neurons expressing human Bcl-2 were partially protected against amyloid beta-peptide-induced neuronal death. This neuroprotection appears to be related to the complete inhibition of apoptosis induced by both amyloid beta-peptide(25-35) and amyloid beta-peptide(1-42). This study may be relevant for developing neuroprotective gene therapy to inhibit neuronal apoptosis in Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1997

Protocol for quantitative analysis of paired helical filament solubilization: a method applicable to insoluble amyloids and inclusion bodies.

Biochemical studies of amyloidoses have been plagued by the sparing solubility of most amyloids in denaturant solvents. Consequently often only a subclass of amyloid protein is analyzed, a fact that is omitted in most studies. This means that there is often no evaluation of the chemical basis for amyloid insolubility, a factor that may provide valuable information concerning amyloid pathogenesis. We have devised a protocol to quantitatively evaluate the solubilization of insoluble amyloid proteins. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents that dissolve all protein. Here we describe the application of this protocol to quantitatively establish complete solubilization of the paired helical filaments (PHFs) from Alzheimer disease. PHFs are distinct from the other amyloid that defines Alzheimer disease (AD), i.e., extracellular amyloid-beta deposits of senile plaques, nonetheless, PHFs share all the properties of, and are defined as, an amyloid, i.e., binding Congo red; beta-pleated sheet conformation and, most significantly, sparing solubility. PHFs of neurofibrillary tangles are the most striking intraneuronal change seen within the brains of patients with AD. Despite intense efforts to understand the molecular composition of this amyloid, quantitative biochemical analyses have been severely hampered by the extreme insolubility of PHF and by difficulties obtaining a homogeneous PHF fraction. Therefore, to date, all of the published studies on the biochemical composition of insoluble PHFs (SDS-insoluble) are qualitative and have provided little or no quantitative data on the proportion of material assayed. Using the solubilization protocol described herein, we found that only high pH was effective in solubilizing PHF while a variety of denaturants and chaotropes resulted in only partial release of component protein. Significantly, the approach is analytical because it allows direct assessment of the significance of two posttranslational modifications in mediating PHF insolubility, i.e., phosphorylation and glycation. Further this protocol provides solubilized protein that can be readily characterized. For example, coupling the method to immunoblotting, ELISA, microsequencing or other analytical techniques would identify components as well as provide a quantitative measure.

Alzheimer Disease↗
PubMedDate not supplied

Noninvasive imaging of peripherally injected Alzheimer's disease type synthetic A beta amyloid in vivo.

The pathological hallmark of Alzheimer's disease (AD) is accumulation in the brain of amyloid composed of the 40-mer peptide A beta. Many fundamental questions about the biology of (AD) remain unanswered because there is currently no method of quantifying A beta amyloid in vivo. A noninvasive method of detecting and quantifying A beta amyloid in vivo would have wide application for the premortem diagnosis of AD and the efficient evaluation of candidate therapeutics aimed at inhibiting the formation and growth of A beta amyloid. Taking advantage of the extraordinarily high affinity of A beta for itself, we have synthesized an N'-terminal diethylenetriaminepentaacetic acid (DTPA) derivative of A beta possessing the kinetic activity and specificity for A beta amyloid desired of a probe to be used for noninvasive imaging. DTPA-A beta(3-40) is readily labeled with (111)InOAc(3) to yield a stable probe with exquisite specificity for naturally occurring and synthetic A beta amyloid in vitro. Moreover, (111)In-DTPA-A beta(3-40), administered intravascularly can specifically deposit onto and label previously injected synthetic A beta amyloid and be imaged in vivo with a gamma camera. The present results demonstrate the design, synthesis, and use of an A beta amyloid-specific probe and methods for its use as a noninvasive imaging agent. In vivo imaging of A beta amyloid represents an important step toward the development of biochemically based objective tools for the assessment of progression of AD and efficacy of potential therapeutics.

Alzheimer Disease↗
PubMed2005

Critical balance of electrostatic and hydrophobic interactions is required for beta 2-microglobulin amyloid fibril growth and stability.

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.

Acids↗
PubMed2005

Oxidation of low-density lipoproteins induces amyloid-like structures that are recognized by macrophages.

The macrophage scavenger receptor CD36 plays a key role in the initiation of atherosclerosis through its ability to bind to and internalize oxidized low-density lipoproteins (oxLDL). Prompted by recent findings that the CD36 receptor also recognizes amyloid fibrils formed by beta-amyloid and apolipoprotein C-II, we investigated whether the oxidation of low-density lipoproteins (LDL) generates characteristic amyloid-like structures and whether these structures serve as CD36 ligands. Our studies demonstrate that LDL oxidized by copper ions, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), or ozone react with the diagnostic amyloid dyes thioflavin T and Congo Red and bind to serum amyloid P component (SAP), a universal constituent of physiological amyloid deposits. X-ray powder diffraction patterns for native LDL show a diffuse powder diffraction ring with maximum intensity corresponding to an atomic spacing of approximately 4.7 A, consistent with the spacing between beta-strands in a beta-sheet. Ozone treatment of LDL generates an additional diffuse powder diffraction ring with maximum intensity indicating a spacing of approximately 9.8 A. This distance is consistent with the presence of cross-beta-structure, a defining characteristic of amyloid. Evidence that these cross-beta-amyloid structures in oxLDL are recognized by macrophages is provided by the observation that SAP strongly inhibits the association and internalization of (125)I-labeled copper-oxidized LDL by peritoneal macrophages. The ability of SAP to bind to amyloid-like structures in oxLDL and prevent lipid uptake by macrophages highlights the potential importance of these structures and suggests an important preventative role for SAP in foam cell formation and early-stage atherosclerosis.

Amyloid↗
PubMed1997

Ultrastructural organization of hemodialysis-associated beta 2-microglobulin amyloid fibrils.

Fibrils of hemodialysis-associated beta 2-microglobulin amyloid were examined by high resolution electron microscopy and immunohistochemical labeling. The amyloid containing tissues obtained through autopsy were prepared for thin section observations. In contrast to other forms of amyloid, the most conspicuous feature of these fibrils were their curved conformations. The fibril core showed ultrastructural and immunohistochemical features in common with the core of connective tissue microfibrils and of previously observed fibrils of experimental murine AA amyloidosis and familial amyloid polyneuropathy (FAP). The core was wrapped in a layer of 3 nm wide ribbon-like "double tracked" structures identified as chondroitin sulfate proteoglycan (CSPG) with immunogold labeling as well as from the results of previous in vitro experiments. Finally, the outer surface of the fibril was associated with a loose assembly of 1 nm wide filaments immunohistochemically identified as beta 2-microglobulin. This is similar to the manner in which AA protein and transthyretin filaments are associated with their respective fibrils. The results of this study provide an additional example for the concept that amyloid fibrils in general are microfibril-like structures externally associated with amyloid protein filaments. An unusual feature of the fibrils of hemodialysis-associated amyloid, however, is the presence of a peripheral layer composed of CSPG rather than of heparan sulfate proteoglycan (HSPG) as in the case of the other two amyloids above. These chondroitin sulfate chains in the outer CSPG layer may be less effective in providing rigidity to the fibril core, thus allowing for the curved conformations of beta 2-microglobulin amyloid fibrils.

Aged↗
PubMed1998

Detection of single amyloid beta-protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy.

Alzheimer's disease is associated with the intraparenchymal growth of plaque-like amyloid deposits. Amyloid plaques are formed by the progressive deposition and transformation of soluble amyloid beta-protein monomers into insoluble and fibrillar aggregates that contain amyloid beta-protein in a beta-pleated sheet conformation. This process is described as 'seeded polymerization' of the monomers with slow-nucleation and fast-growth kinetics. Soluble amyloid beta-protein monomers are present in the cortical extracellular space and in the cerebrospinal fluid, whereas insoluble aggregates so far can be found only by the examination of brain tissue by biopsy or autopsy. Here we present a biophysical method that uses the principle of seeded polymerization in combination with fluorescence correlation spectroscopy, which allowed us to detect single amyloid beta-peptide aggregates in the cerebrospinal fluid samples from Alzheimer's patients. All of 15 Alzheimer's samples but none of the 19 age-matched control samples produced large peaks with fluorescence correlation spectroscopy indicating the rapid aggregation of the fluorescent labelled synthetic amyloid beta-protein probe onto the amyloid beta-protein 'seeds' present in the cerebrospinal fluid. Our method could enable easy in vivo detection of the cerebral amyloid beta-protein pathology of Alzheimer's disease and might be of potential value to facilitate its routine diagnosis.

Aged↗
PubMed1998

Beta 2-microglobulin amyloid deposition in hip revision arthroplasty tissues.

AIMS: Hip joint disease associated with progressive amyloid deposition in uraemic patients receiving chronic haemodialysis treatment often requires treatment by joint arthroplasty. The aim of this study was to determine whether beta 2-microglobulin amyloid deposition occurred in the periprosthetic tissues of arthroplasties that had undergone aseptic loosening and required a revision procedure. METHODS AND RESULTS: Sections of the pseudocapsule, acetabular and femoral pseudomembrane surrounding failed total hip replacements of five uraemic patients known to have beta 2-microglobulin amyloid deposits at the time of primary joint replacement were examined for the presence of beta 2-microglobulin amyloid deposition by Congo red staining and immunohistochemical staining for beta 2-microglobulin and other amyloid proteins. Clinical and radiological features of each case, including postoperative history and extent of osteolysis, were also noted. In all cases evidence of beta 2-microglobulin amyloid deposits were found in one or more of the above periprosthetic tissues. In three of these cases amyloid deposition was extensive. CONCLUSIONS: This study shows that beta 2-microglobulin amyloid deposition occurs in revision arthroplasty tissues. Accelerated loosening of the prosthesis is known to occur in uraemic patients and it is possible that beta 2-microglobulin amyloid deposition may contribute to early arthroplasty failure in uraemic patients who remain on haemodialysis treatment.

Adult↗
PubMed2002

BIGH3 (TGFBI) Arg124 mutations influence the amyloid conversion of related peptides in vitro.

Amyloid deposits with Arg124 mutated TGFBI protein have been identified in autosomal dominant blinding corneal dystrophies. We assessed in vitro the mechanisms determining TGFBI protein amyloid transformation involving mutations of Arg124. Eight peptides synthesized following the TGFBI protein sequence, centered on codon Arg124 holding the previously reported amyloidogenic mutations and the respective controls were studied. Cys124 and His124 mutated peptide preparations contained significantly higher amounts of amyloid than the native peptide. Blocking the SH group of Cys124 and deleting the first four NH2-terminal amino acids including Val112-Val113 resulted in a decrease in amyloid fibril formation while deletion of the nine CONH2-terminal residues increased amyloid fibril concentration. Fourrier transformed-infrared spectroscopy analysis of the different peptide solutions showed an increase in beta-pleated sheet structures in those with enhanced amyloid yielding. We designed a peptide (BB1) likely to counteract the role of Val112-Val113 in amyloid fibril formation. Incubation of Cys124 peptide with BB1 indeed resulted in a 35% inhibition of amyloid fibril formation. Our results are in keeping with the clinical observations of Arg124 mutation-linked amyloidosis and show the importance of Val112-Val113, disulfide and hydrogen bonding in increasing the beta-pleated conformation and amyloid formation. These findings shed new light on the molecular mechanisms of TGFBI protein amyloidogenesis and encourage further research on the use of specifically designed peptides as putative therapeutic agents for these disabling diseases.

Amino Acid Sequence↗
PubMed2001

Oxidative and hydrolytic properties of beta-amyloid.

beta-Amyloid protein is the major component of senile plaques found in the brains of Alzheimer's patients. Previously, a new biochemical property of amyloid, its ability to disrupt ester and peptide bonds, was described [Elbaum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. Commun. 267, 733-738]. In the present work we compare the ability of beta-amyloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofluorescein [dichlorodihydrofluorescein (H2DCF) or dichlorofluorescein diacetate (DCF-DA), respectively] to the same final product (dichlorofluorescein). Although there is accumulating evidence of oxidative properties of beta-amyloid, little is known about its hydrolytic abilities. Chemical modification studies revealed that hydrolytic properties are related to a His, Ser and Asp/Glu triad, while residues of His, Tyr and Met are involved in the oxidative activity of amyloid. Studies with the rat homologue of human beta-amyloid (1-40), containing three amino-acid substitutions (Arg5-->Gly, Tyr10-->Phe and His13-->Arg) confirmed a role of His in the studied processes. Reduction of the hydrolysis product caused by inhibitors of Ser esterases (phenylmethylsulphonyl fluoride and eserine) suggests that beta-amyloid-mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators that reduced H2DCF oxidation did not change or increase DCF-DA hydrolysis. Solvent isotope effects suggest the involvement of hydrogen bonds in the hydrolysis reaction. Hydrolysis was inhibited by redox-active metal ions and was practically oxygen independent while the oxidation process was redox-active-metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Product formation was significantly inhibited by catalase and superoxide dismutase as well as benzoquinone, a specific superoxide anion radical scavenger. Increase of fluorescence by oxidation was strongly inhibited by azide and His and enhanced in samples prepared with deuterated phosphate buffer, suggesting singlet oxygen intermediacy. Our data are consistent with superoxide-mediated singlet oxygen intermediate in this Fenton mechanism-driven reaction. These results indicate that hydrolytic and oxidative properties of beta-amyloid are distinct features of this peptide and probably require different mechanisms to occur, but both of them may contribute to beta-amyloid toxicity.

Amino Acid Sequence↗
PubMed2003

A comparison of amyloid fibrillogenesis using the novel fluorescent compound K114.

Proteinaceous inclusions with amyloidogenic properties are a common link between many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Histological and in vitro studies of amyloid fibrils have advanced the understanding of protein aggregation, and provided important insights into pathogenic mechanisms of these neurodegenerative brain amyloidoses. The classical amyloid dyes Congo Red (CR) and thioflavin T and S, have been used extensively to detect amyloid inclusions in situ. These dyes have also been utilized to monitor the maturation of amyloid fibrils assembled from monomer subunits in vitro. Recently, the compound (trans,trans)-1-bromo-2,5-bis-(3- hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB), derived from the structure of CR, was shown to bind to a wide range of amyloid inclusions in situ. More importantly it was also used to label brain amyloids in live animals. Herein, we show that an analogue of BSB, (trans,trans)-1-bromo-2,5-bis-(4-hydroxy)styrylbenzene (K114), recognizes amyloid lesions, and has distinctive properties which allowed the quantitative monitoring of the formation of amyloid fibrils assembled from the amyloid-beta peptide, alpha-synuclein, and tau.

Amyloid↗