Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

APP Processing Enzymes (Secretases) as Therapeutic Targets: Insights from the Use of Transgenics (Tgs) and Transfected Cells

Secretases degrade amyloid precursor protein (APP) releasing fragments (-peptides A, A_x) that assemble to form hallmark extracellular deposits in Alzheimer's disease (AD) correlating with disease severity. As such, secretases supply targets for therapeutic intervention and form the focus of this overview. Progress in elucidating secretases and their modes of catalysis come from exploiting the use of transgenics or transfected cells. In addition to Ax, secretases also release C-terminal fragments with putative signaling properties (amyloid intracellular domain, AICD) similar in concept to those available for conversion of the Notch-r to release the nuclear transactivator NICD. The review considers lingering questions on APP fragmentation by secretase action, ancillary proteins such as presenilins (PS1/2), nicastrin, XII, or proteases (caspases), and the influence of familial mutations (mAPP, mPS) in terms of fibrillogenesis.     

Binding of Amyloid Beta-Protein to Intracellular Brain Proteins in Rat and Human

Amyloid beta-protein (A), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of A to intracellular proteins has not been studied. We have developed an overlay assay to study A binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to A 1–40 and A 1–42. No major difference was observed in the A binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of A to brain proteins resides between 12–28 amino acid sequence of A. The presence of several intracellular A-binding (AB) proteins suggests that these proteins may either protect A from its fibrillization or alternatively promote A polymerization. Identification of these proteins and their binding affinities for A are needed to assess their potential role in the pathogenesis of Alzheimer's disease.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Activation of LA-N-2 Cell Phospholipase D by Amyloid Beta Protein (25–35)

Amyloid protein is the major protein component of neuritic plaques found in the brain of Alzheimer's disease. The activation of phospholipase D by amyloid beta protein (25–35), quisqualate and phorbol 12, 13-dibutyrate was investigated in LA-N-2 cells by measuring phosphatidylethanol formation. The activation of phospholipase D by quisqualate and AP (25–35) was calcium-independent. The AP (25–35) and quisqualate activation of phospholipase D appeared to be mediated through a pertussis toxin-sensitive GTP-binding protein. Phospholipase D activation by AP (25–35), quisqualate and phorbol dibutyrate was not blunted by the protein kinase C inhibitors, staurosporine, H-7 and RO-31-8220. However, it was abolished by overnight exposure to phorbol dibutyrate. This activation of phospholipase D was prevented by the tyrosine kinase inhibitor, genistein but not by tyrophostin A. Several excitatory amino acid antagonists were tested for their ability to prevent the phospholipase D activation by quisqualate and AP (25–35). Only NBQX was effective with an IC₅₀ of 75 M for AP (25–35) and quisqualate. Activation of phospholipase D by AP or quisqualate was absent in LA-N-2 cells previously desensitized by quisqualate or AP (25–35), but the activation by phorbol dibutyrate was unaltered. The responsiveness to AP and quisqualate in previously desensitized cells reappeared subsequent to a period of resensitization. The observations with the antagonist NBQX, and the desensitization and resensitization experiments, are consistent with a receptor occupancy mediated activation of phospholipase D by quisqualate and by AP (25–35).     

The solution structure of rat Aβ-(1–28) and its interaction with zinc ion: insights into the scarcity of amyloid deposition in aged rat brain

The amyloid -peptide (A) is a major component of insoluble amyloid deposits in Alzheimers disease, and the ability of the -peptide to exist in different conformations is dependent on residues 1–28 [-(1–28)]. However, different from humans, no A amyloid deposition has been found in aged rats brains. Studying the three-dimensional solution structure of rat A-(1–28) and the binding circumstance of Zn²⁺ is beneficial to a clear understanding of the potential role of Zn²⁺ in Alzheimer-associated neuropathogenesis and to suggest why there is no amyloid deposition in aged rats brains. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of rat A-(1–28) and the binding constant of Zn²⁺ to rat A-(1–28). Our results suggest that (1) the three-dimensional solution structure of rat A-(1–28) is more stable than that of human A-(1–28) in DMSO-d₆ and that a helical region from Glu16 to Val24 exists in the rat A-(1–28); (2) the affinity of Zn²⁺ for rat A-(1–28) is lower than that for human A-(1–28) and the NMR data suggest that Arg13, His6, and His14 residues provide the primary binding sites for Zn²⁺; and (3) the proper binding of Zn²⁺ to rat A-(1–28) can induce the peptide to change to a more stable conformation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0556-xAbbreviations A amyloid -peptide - AD Alzheimers disease - hA-(1–28) human A-(1–28) - rA-(1-28) rat A-(1–28) - REM restrained energy minimization     

The protective role of DL-α-lipoic acid in the oxidative vulnerability triggered by Aβ-amyloid vaccination in mice

Recent reports indicate that -amyloid peptide (A) vaccine based therapy for Alzheimers disease (AD) may be on the horizon. There are however, concerns about the safety of this approach. Immunization with A has several disadvantages, because it crosses the blood brain barrier and cause inflammation and neurotoxicity. The present work is aimed to study the protective effective of -lipoic acid (LA) in the oxidative vulnerability of -amyloid in plasma, liver, spleen and brain, when A fibrils are given intraperitoneally in inflammation induced mice. Result shows that reactive oxygen species (ROS) in the astrocytes of inflammation induced mice along with A (IA) has shown 2.5-fold increase when compared with LA treated mice. The increased level of lipid peroxidase (LPO) (p < 0.05) and decreased antioxidant status (p < 0.05) were observed in the plasma, liver, spleen and brain of IA induced mice when compared with LA treated mice.Data shows that there were no significant changes observed between the control and LA treated mice. Our biochemical and histological results highlight that significant oxidative vulnerability was observed in IA treated mice, which was prevented by LA therapy. Our findings suggest that the antioxidant effect of LA when induced with A may serve as a potent therapeutic tool for inflammatory AD models. (Mol Cell Biochem 270: 29–37, 2005)     

The degradation of amyloid beta as a therapeutic strategy in Alzheimer's disease and cerebrovascular amyloidoses

The deposition of 4-kDa amyloid peptide in the brain is a prominent feature of several human diseases. Such process is heterogeneous in terms of causative factors, biochemical phenotype, localization and clinical manifestations. Amyloid accumulates in the neuropil or within the walls of cerebral vessels, and associates with dementia or stroke, both hereditary and sporadic. Amyloid is normally released by cells as soluble monomeric-dimeric species yet, under pathological conditions, it self-aggregates as soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Lowering amyloid levels may be achieved by inhibiting its generation from the amyloid -precursor protein or by promoting its clearence by transport or degradation. We will summarize recent findings on brain proteases capable of degrading amyloid with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence suggesting that they may participate in the proteolysis of amyloid in vivo. We will also put in perspective their possible utilization as therapeutic agents in amyloid diseases.     

Degradation of Soluble Amyloid β-Peptides 1–40, 1–42, and the Dutch Variant 1–40Q by Insulin Degrading Enzyme from Alzheimer Disease and Control Brains

Insulin degrading enzyme (IDE) is a metalloprotease that has been involved in amyloid peptide (A) degradation in the brain. We analyzed the ability of human brain soluble fraction to degrade A analogs 1–40, 1–42 and the Dutch variant 1–40Q at physiological concentrations (1 nM). The rate of synthetic ¹²⁵I-A degradation was similar among the A analogs, as demonstrated by trichloroacetic acid precipitation and SDS-PAGE. A 110 kDa protein, corresponding to the molecular mass of IDE, was affinity labeled with either ¹²⁵I-insulin, ¹²⁵I-A 1–40 or ¹²⁵I-A 1–42 and both A degradation and cross-linking were specifically inhibited by an excess of each peptide. Sensitivity to inhibitors was consistent with the reported inhibitor profile of IDE. Taken together, these results suggested that the degradation of A analogs was due to IDE or a closely related protease. The apparent Km, as determined using partially purified IDE from rat liver, were 2.2 ± 0.4, 2.0 ± 0.1 and 2.3 ± 0.3 M for A 1–40, A 1–42 and A 1–40Q, respectively. Comparison of IDE activity from seven AD brain cytosolic fractions and six age-matched controls revealed a significant decrease in A degrading activity in the first group, supporting the hypothesis that a reduced IDE activity may contribute to A accumulation in the brain.     

Alzheimer’s Disease: Soluble Oligomeric Aβ(1–40) Peptide in Membrane Mimic Environment from Solution NMR and Circular Dichroism Studies

Amyloid beta peptide (A) is a small peptide present in normal cells and aggregated A is the main constituent of the extracellular amyloid plaques found in Alzheimers disease (AD) brain. Recent studies suggest that soluble A oligomers are neurotoxic rather than amyloid fibrils found in amyloid plaques. This study using multidimensional NMR spectroscopy and circular dichroism (CD) provides the first direct evidence that alterations in membrane structure can trigger the conversion of soluble -helical monomeric A into oligomeric A in a -sheet conformation.     

Study of beta-amyloid peptide (Abeta40) insertion into phospholipid membranes using monolayer technique

-Amyloid peptide (A), a normal constituent of neuronal and non-neuronal cells, has been proved to be the major component of extracellular plaque of Alzheimer's disease (AD). The interaction of A with lipid membranes may be essential for its neurotoxicity. Our previous study revealed that membrane insertion may provide a possible pathway by which A prevents itself from aggregation and fibril formation. In the present work we studied the membrane insertion of A and the factors that affect its insertion ability using a monolayer approach. The results show that A is surface active and can insert into lipid monolayers. When a high level of cholesterol is present, A40 can insert into the phospholipid mixtures simulating physiological membrane composition. Acidic pH enhances A insertion, while the effect of ionic strength is rather complex. A insertion ability may be ultimately relative to cholesterol-rich domains in the monolayers, which indicates strong interaction between A and cholesterol.     

Alzheimer''s Amyloid-β as a Preventive Antioxidant for Brain Lipoproteins

1. Increased production of A in a form of lipoprotein antioxidant under the action of increased oxidative stress in aging with subsequent chelation of transition metal ions by A, accumulation of toxic A–metal lipoprotein complexes, production of reactive oxygen species, and neurotoxicity are reviewed and postulated to form the temporal sequence of events in the development of Alzheimer's disease (AD).2. Since (i) A binds copper stronger than iron and other transition metals, and (ii) copper is a more efficient catalyst of oxidation than other transition metals, chelation of copper by A is proposed to be a most important part of this pathway.3. Whereas this amyloid-binds-copper (ABC) model does not remove A peptide from its central place in our current thinking of AD, it places additional factors in the center of discussion.4. Most importantly, they embrace pathological mechanisms known to develop in aging (which is the most important risk factor for AD), such as increased production of reactive oxygen species by mitochondria, that can be positioned upstream relative to the generation of A.     

GM1 Inhibits Amyloid β-Protein-Induced Cytokine Release

The ganglioside GM1 is known to play a pivotal role in neuronal survival and/or regeneration. Recently it has been shown that GM1 binds tightly with membrane-bound amyloid protein (A) and prevents its conversion from a helical to a -sheet structure. To examine the potential physiological consequences of this binding, we studied the effect of GM1 on A-stimulated release of proinflammatory cytokines, such as interleukin (IL)-1, IL-6 and TNF-, using the human monocytic cell line, THP-1, as a model system. Treatment of THP-1 cells with A 1–40 or A 25–35 resulted in an increased cytokine release from these cells. However, treatment of A-activated THP-1 cells with GM1 and several other complex gangliosides, but not hematosides and neutral glycosphingolipids such as asialo-GM1 (GA1), lactosylceramide, and globoside, significantly decreased the cytokine release. In contrast, this effect was not observed for lipopolysaccharide (LPS)-activated and thrombin-activated THP-1 cells, indicating that the ganglioside effect is specific for A-induced cytokine release. A direct interaction between GM1 and A was demonstrated using the surface plasmon resonance technique. We found that GM1 ganglioside exhibited higher affinity for A 1–40 than GA1, suggesting that the sialic acid moiety of GM1 is necessary for its interaction with A. We conclude that the inhibitory effect of GM1 on A-induced cytokine release may reflect pre-existing abnormalities in membrane transport at the stage of amyloid formation and that GM1 may induce conformational changes in A, resulting in diminished fibrillogenesis and prevention of the inflammatory response of neuronal cells in Alzheimer's disease.     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Architecture of the Alzheimer’s AβP Ion Channel Pore

We have proposed that the cytotoxic action of Alzheimers amyloid beta protein might be initiated by the interaction with the neuronal cell membrane, and subsequent formation of toxic ion channels. Consequently, AP toxicity can be explained on the basis of harmful ion fluxes across AP channels. The conformation of AP in membranes is not known. However, several models suggests that a transmembrane annular polymeric structure is responsible for the ion channel properties of the membrane-bound AP. To identify that portion of the AP molecule making up the conducting pore we have hypothesized that the region of the AP sequence in the vicinity of the hypothetical pore might interact with complementary regions in the adjacent AP subunits. We have further hypothesized that an interaction by a peptide segment would block AP conductance. To test this hypothesis we synthesized peptides that encompass the histidine dyad (H-H) previously hypothesized to line the pore. We report here that peptides designed to most closely match the proposed pore are, in fact, the most effective at blocking ion currents through the membrane-incorporated AP channel. As previously shown for Zn²⁺ blockade, peptide blockade is also asymmetric. The results also provide additional evidence for the asymmetric insertion of the AP molecules into lipid membranes, and give support to the concept that rings of histidines line the entry to one side of the AP pore.     

Localization of Transforming Growth Factor β in Association with Neuromuscular Junctions in Adult Human Muscle

Transforming growth factors- 1, 2, and 3 are known for their regulatory function in embryogenesis, fibrogenesis, and tissue repair of different cell types. A trophic function of TGF- subclasses for motoneurons has been shown in vitro. TGF- 1 is a potent survival factor for cultured embryonic rat motoneurons. In addition, TGF- 1 stimulates proliferation of rat Schwann cells. Recently, TGF- 2 has been reported to be associated with the subsynaptic nuclei of mature rat neuromuscular junctions. In this study, we investigated the expression of TGF- 1, 2, and 3 at neuromuscular junctions in skeletal muscle of 11 adults without neuromuscular disease. On muscle biopsies, neuromuscular junctions were depicted by acetylcholine esterase reaction and acetylcholine receptor antibodies. TGF- 1, 2, and 3 were stained immunohistochemically with monoclonal antibodies. Some muscle fibers showed low levels of inhomogeneous immunoreactivity for both TGF- 1 and TGF- 3. Intense immunoreactivity of TGF- 1 and 3 was shown at the postsynaptic area of neuromuscular junctions. TGF- 2 was expressed in the same subcellular distribution, but less strongly. In conclusion, the colocalization of TGF- with neuromuscular junctions may suggest a significant function in neuromuscular communication.     

Adenosine 3′, 5′- Cyclic Monophosphate Increases Processing of Amyloid Precursor Protein (APP) to β-Amyloid in Neuroblastoma Cells Without Changing APP Levels or Expression of APP mRNA

-Amyloid (A), a 39–43 residue peptide generated by splicing of the amyloid precursor protein (APP), is one of the major components of senile plaques which are the hallmark of Alzheimer's disease (AD); and therefore, a role of A in neuronal degeneration has been proposed. The factors which regulate the levels of A have not been fully identified. Since an elevation of the intracellular levels of adenosine, 3, 5-cyclic monophosphate (cAMP) in neuroblastoma cells (NB) induces terminal differentiation, and since these differentiated NB cells undergo spontaneous degeneration, the role of cAMP in the regulation of A levels in these cells have been investigated. In order to determine the specificity of the effect of cAMP on nerve cells, rat glioma cells (C-6) were investigated in a similar manner. Results showed that an elevation of the levels of cAMP in NB cells enhances the intensity of A immunostaining without changing the levels of APP or APP mRNA. This suggests that the rate of processing of APP to A increases following an elevation of cAMP level in NB cells. Data also revealed that an elevation of cAMP level in glioma cells did not alter the intensity of staining with APP or A.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.