Protein-protein interactions in the assembly and subcellular trafficking of the BACE (beta-site amyloid precursor protein-cleaving enzyme) complex of Alzheimer's disease.

The correct assembly of the BACE (beta-site amyloid precursor protein-cleaving enzyme or beta-secretase) complex and its subsequent trafficking to cellular compartments where it associates with the APP (amyloid precursor protein) is essential for the production of Abeta (amyloid beta-peptide), the protein whose aggregation into senile plaques is thought to be responsible for the pathogenesis of AD (Alzheimer's disease). These processes rely upon both transient and permanent BACE-protein interactions. This review will discuss what is currently known about these BACE-protein interactions and how they may reveal novel therapeutic targets for the treatment of AD.

Regulation of the lipidation of beta-secretase by statins.

Statins inhibit the dimerization of beta-secretase [BACE (beta-site amyloid precursor protein-cleaving enzyme)] by inhibiting the lipidation of BACE and associated proteins. Our studies have demonstrated a clearly defined temporal sequence for these reactions in the assembly of the BACE complex, which may provide targets for the treatment of Alzheimer's disease.

Protein lipidation of BACE.

Our research has concentrated upon the protein lipid modification of BACE [beta-site amyloid precursor protein cleaving enzyme (beta-secretase)], of which very little is currently known. Lipidation influences the production of Abeta (amyloid beta-protein) by promoting the dimerization of BACE.

Structure and neurotoxicity of novel amyloids derived from the BRI gene.

A number of human neurodegenerative diseases involve aggregated amyloid proteins in the brain, e.g. Alzheimer's disease (beta-amyloid) and Parkinson's disease (alpha-synuclein). Other examples are rare familial dementias which involve the BRI gene. In a British family, mutation of the termination codon extends the reading frame of BRI to yield a furin-processed 34-residue peptide (Abri; British dementia peptide), 11 residues longer than the wild-type (WT). In a Danish family, a ten-base insertion also yields a 34-residue peptide (Adan; Danish dementia peptide). To explore the roles of Abri and Adan in neurodegeneration, we synthesized Abri and Adan in oxidized and reduced forms and generated transgenic mice colonies expressing the WT and mutated forms of BRI. We have generated transgenic mice colonies bearing the genes coding for WT-BRI, Adan and Abri under the control of the Thy1 promoter. Whereas WT-BRI transgenic mice express full-length WT-BRI protein in their brains, Adan protein is fully processed to small peptides.

The role of intracellular cholesterol on the processing of the beta-amyloid precursor protein.

The objectives of this article are to summarise evidence that amyloidogenesis is a causal factor in Alzheimer's disease, to outline the main pathways of amyloidogenesis in Alzheimer's disease, describe contemporary evidence showing that the processing of the amyloid precursor protein and amyloidogenesis is strongly influenced by the levels of intracellular cholesterol. Moreover, we shall suggest a mechanistic hypothesis that could explain the observed epidemiological links between the use of inhibitors of cholesterol biosynthesis in patients and the observed reduced risk of Alzheimer's disease.

Non-fibrillar oligomeric species of the amyloid ABri peptide, implicated in familial British dementia, are more potent at inducing apoptotic cell death than protofibrils or mature fibrils.

Familial British dementia (FBD) is an autosomal dominant neurodegenerative disorder, with biochemical and pathological similarities to Alzheimer's disease. FBD is associated with a point mutation in the stop codon of the BRI gene. The mutation extends the length of the wild-type protein by 11 amino acids, and following proteolytic cleavage, results in the production of a cyclic peptide (ABri) 11 amino acids longer than the wild-type (WT) peptide produced from the normal gene BRI. ABri was found to be the main component of amyloid deposits in FBD brains. However, pathological examination of FBD brains has shown the presence of ABri as non-fibrillar deposits as well as amyloid fibrils. Taken together, the genetic, pathological and biochemical data support the hypothesis that ABri deposits play a central role in the pathogenesis of FBD. Here we report that ABri, but not WT peptide, can oligomerise and form amyloid-like fibrils. We show for the first time that ABri induces apoptotic cell death, whereas WT is not toxic to cells. Moreover, we report the novel findings that non-fibrillar oligomeric species of ABri are more toxic than protofibrils and mature fibrils. These findings provide evidence that non-fibrillar oligomeric species are likely to play a critical role in the pathogenesis of FBD and suggest that a similar process may also operate in other neurodegenerative diseases.

Effect of the disulfide bridge and the C-terminal extension on the oligomerization of the amyloid peptide ABri implicated in familial British dementia.

Familial British dementia (FBD) is a rare neurodegenerative disorder and shares features with Alzheimer's disease, including amyloid plaque deposits, neurofibrillary tangles, neuronal loss, and progressive dementia. Immunohistochemical and biochemical analysis of plaques and vascular amyloid of FBD brains revealed that a 4 kDa peptide named ABri is the main component of the highly insoluble amyloid deposits. In FBD patients, the ABri peptide is produced as a result of a point mutation in the usual stop codon of the BRI gene. This mutation produces a BRI precursor protein 11 amino acids longer than the wild-type protein. Mutant and wild-type precursor proteins both undergo furin cleavage between residues 243 and 244, producing a peptide of 34 amino acids in the case of ABri and 23 amino acids in the case of the wild-type (WT) peptide. Here we demonstrate that the intramolecular disulfide bond in ABri and the C-terminal extension are required to elongate initially formed dimers to oligomers and fibrils. In contrast, the shorter WT peptide did not aggregate under the same conditions. Conformational analyses indicate that the disulfide bond and the C-terminal extension of ABri are required for the formation of beta-sheet structure. Soluble nonfibrillar ABri oligomers were observed prior to the appearance of mature fibrils. A molecular model of ABri containing three beta-strands, and two beta-hairpins annealed by a disulfide bond, has been constructed, and predicts a hydrophobic surface which is instrumental in promoting oligomerization.

Inhibition of antigen transport by expression of infected cell peptide 47 (ICP47) prevents cell surface expression of HLA in choriocarcinoma cell lines.

Cell surface expression of HLA class I (including non-classical HLA-G) in JEG3 (choriocarcinoma cell line) was blocked by stable transfection with the sequence encoding the Herpes simplex virus protein, infected cell peptide 47 (ICP47) inserted into a vector pCEP4. Intracellular expression of ICP47 protein in ICP47-transfected cells was demonstrated. The lack of HLA cell surface expression was likely to be due to blockage of peptide transport from the cytoplasm into the endoplasmic reticulum by ICP47. ICP47 is known to block the heterodimeric transporter associated with antigen processing (formed from TAP1 and TAP2). Western blotting with a polyclonal antibody to the C-terminus of TAP1 showed high expression of TAP1 in BeWo and JEG3, but not JAR cells, expression that was strongly upregulated by gamma-interferon. Gamma-interferon also upregulated the cell surface expression of HLA class I. TAP1 was strongly expressed in MC2 and MC3 extravillous cytotrophoblast cell lines immortalised with the SV40 large T antigen. The results suggest a role for non-classical HLA in the presentation of antigenic peptides to the immune system.

The use of seldi proteinchip arrays to monitor production of Alzheimer's betaamyloid in transfected cells.

beta-Amyloid (Abeta), a 39-43 residue peptide, is the principal component of senile plaques found in the brains of patients with Alzheimer's disease (AD). There are two main lines of evidence that its deposition is the cause of neurodegeneration. First, mutations found in three genes in familial Alzheimer's cases give rise to increased production of the longest, most toxic, form, Abeta 1-42. Second. aggregated Abeta is toxic to neuronal cells in culture. Inhibitors of the proteases involved in its release from the amyloid precursor protein are, therefore, of major therapeutic interest. The best candidates for the releasing proteases are both aspartyl proteases, which are integrated into the membranes of the endoplasmic reticulum and Golgi network. A sensitive assay using Ciphergen's Seldi system has been developed to measure all the variants of Abeta in culture supernatants, which will be of great value in screening inhibitors of these proteases. With this assay, it has been shown that increasing intracellular cholesterol increases the activities of both beta-secretase, and gamma-secretase 42. Moreover, changing the intracellular targeting of amyloid precursor glycoprotein (APP) yields increased alpha-secretase cleavage, and increases in the amounts of oxidized/nitrated forms of Abeta.

Oligomerization and toxicity of beta-amyloid-42 implicated in Alzheimer's disease.

beta-Amyloid protein (Abeta) is the major component of senile plaques found in the brains of Alzheimer's patients. A novel ELISA has been developed which probes the early stages of oligomerization of Abeta. Incubation of Abeta solutions at 37 degrees C and pH 7.4 produces soluble oligomers in a concentration-dependent manner. Fresh Abeta42 solutions rapidly form soluble oligomers, whereas Abeta40 solutions require prolonged incubation to produce oligomers. Fresh Abeta42 solutions are more toxic to human neuroblastoma SH-SY5Y cells than Abeta40 solutions, possibly mediated by soluble oligomers. The differences between Abeta42 and Abeta40 could explain the association of the longer form with familial early-onset Alzheimer's disease. We also report a new strategy for solid-phase synthesis of Abeta peptides which gives high yield and purity of the initial crude preparation.

Oligomerization of beta-amyloid of the Alzheimer's and the Dutch-cerebral-haemorrhage types.

A novel ELISA has been developed which detects oligomerization of beta-amyloid (A beta). Oligomerization, fibrillization and neurotoxicity of native A beta associated with Alzheimer's disease (AD) type has been compared with E22Q A beta (amyloid beta-protein containing residues 1--40 with the native Glu at residue 22 changed to Gln) implicated in Dutch cerebral haemorrhage disease. Solutions of A beta rapidly yield soluble oligomers in a concentration-dependent manner, which are detected by the ELISA, and by size-exclusion gel chromatography. Conformational changes from disordered to beta-sheet occur more slowly than oligomerization, and fibrils are produced after prolonged incubation. The E22Q A beta oligomerizes, changes conformation and fibrillizes more rapidly than the native form and produces shorter stubbier fibrils. Aged fibrillar preparations of E22Q A beta are more potent than aged fibrils of native A beta in inducing apoptotic changes and toxic responses in human neuroblastoma cell lines, whereas low-molecular-mass oligomers in briefly incubated solutions are much less potent. The differences in the rates of oligomerization of the two A beta forms, their conformational behaviour over a range of pH values, and NMR data reported elsewhere, are consistent with a molecular model of oligomerization in which strands of A beta monomers initially overcome charge repulsion to form dimers in parallel beta-sheet arrangement, stabilized by intramolecular hydrophobic interactions, with amino acids of adjacent chains in register.

The role of cholesterol in the biosynthesis of beta-amyloid.

Addition of the beta-hydroxy-beta-methylglutaryl-CoA (HmG-CoA) reductase inhibitor lovastatin to human HEK cells transfected with the amyloid precursor protein (APP) reduces intracellular cholesterol/protein ratios by 50%, and markedly inhibits beta-secretase cleavage of newly-synthesized APP. Exogenous water-solubilized cholesterol at 200 microg/ml concentration increases newly synthesized beta-amyloidogenic products four-fold. These intracellular changes are detectable by immunoprecipitation and immunofluorescent labelling. Analyses of the fragments captured from culture medium by an N-terminal anti-beta-amyloid antibody on ProteinChip arrays and detected using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry revealed that culture with cholesterol (200 microg/ml) increased secretion of beta-amyloid 1-40 by 1.8-fold, and increased secretion of beta-amyloid 1-42. Changes in APP processing by cholesterol may mediate the way in which the ApoE4 allele increases risk of developing Alzheimer's disease (AD) in western populations.

Solid-phase synthesis and cyclization of a large branched peptide from IgG Fc with affinity for Fc gammaRI.

A solid phase approach has been used to synthesize a large branched disulphide peptide from IgG Fc, Ac-F-C*-A-K-V-N-N-K-D-L-P-A-P-I-E-K(Ac-E-L-L-G-G-P-S-V-F)-C*-I-NH2. This peptide combines the lower hinge region of IgG and a proximal beta-hairpin loop, both implicated in binding to Fc gammaRI. Solid phase Tl(tfa)3 cyclization of the linear branched peptide resulted in a poor yield of cyclic hinge-loop peptide (11%) most likely due to steric hindrance caused by the branch. However, if addition of the branch was preceded by solid phase Tl(tfa)3 cyclization of the loop, the yield was excellent at 75%. Cyclic hinge-loop peptide was active in displacing IgG2a from Fc gammaRI expressed on monocyte cell lines with an IC50 of 40 microM, whereas the linear form of this peptide was inactive. The Fc hinge-loop peptide demonstrates the potential for a non-mAb high affinity, immunomodulatory ligand for Fc gammaRI.

Expression and purification of the canine 54-kDa subunit of signal recognition particle as a his-tagged protein from Escherichia coli.

The 54-kDa subunit of the signal recognition particle (SRP) binds nascent secretory polypeptides, binds the 7SL RNA (SRP RNA) component of SRP, and hydrolyzes GTP. Limited proteolysis of SRP 54-kDa suggests the protein has two domains, termed the G (GTP-binding) and M (methionine-rich) domains. The M domain is predicted to contain a number of amphiphilic helices, which provide a binding cleft for signal sequences. In order to obtain sufficient material for studies of relationships between structure and function, we have expressed the canine cDNA encoding the 54-kDa subunit in Escherichia coli using a T7 expression system. To aid purification, the protein was expressed with an amino-terminal extension encoding an initiating methionine and 10 histidine residues followed by an enterokinase cleavage site; 0.3mg of HIS-SRP 54-kDa was purified to give a single band on SDS-PAGE in 20% yield from 500 ml of cultured E. coli. Purified HIS-SRP 54-kDa was shown to be folded into the G and M domains, to inhibit the translocation of pre-prolactin into canine microsomes, and to bind mammalian SRP RNA only in the presence of the 19-kDa subunit of SRP.

Metabolites of the beta-amyloid precursor protein generated by beta-secretase localise to the trans-Golgi network and late endosome in 293 cells.

Deposition of beta-amyloid occurs in the brains of all sufferers of Alzheimer's disease. beta-amyloid is proteolytically derived from the beta-amyloid precursor protein by as yet unidentified enzymes termed secretases. We have generated and characterised antisera to the carboxy-terminal domain and beta-secretase cleavage site of the Alzheimer's amyloid precursor protein. The beta-secretase cleavage event occurs at the extreme N-terminus of the beta-amyloid peptide. Our antiserum to the N-terminus of the beta-amyloid peptide (NT beta 4) specifically recognises beta-secretase cleaved species as opposed to intact beta APP. NT beta 4 specifically immunoprecipitates a 13 kDa fragment of beta APP (p13) which is potentially amyloidogenic. We have used these antisera in confocal laser scanning immunofluorescence microscopy to localise the intracellular location of potentially amyloidogenic beta APP processing fragments such as p13. Using a number of marker antisera of known intracellular location, we have defined the major location of beta APP fragments possessing the Asp-1 N-terminus of beta-amyloid as the trans-Golgi network or late endosome on the basis of colocalisation with a monoclonal antibody to the cation-independent mannose-6-phosphate receptor. The colocalisation was further investigated using brefeldin A which demonstrated that the p13 fragment and mannose-6-phosphate receptor are trafficked by alternative pathways from the trans-Golgi network.

Substitution of fifty four homologue (Ffh) in Escherichia coli with the mammalian 54-kDa protein of signal-recognition particle.

The fifty four homologue (Ffh) of Escherichia coli promotes the translocation of a subset of periplasmic, membrane and secreted proteins across the cytoplasmic membrane. The ffh gene product is essential for cell viability and efficient protein export. Here we show that the mammalian homologue signal-recognition particle (SRP) 54 kDa is not able to suppress the translocation defect in an Ffh conditional mutant Wam 113 [Phillips, G.J. & Silhavy, T.J. (1992) Nature 359, 744-746]. The expression of SRP 54kDa, which is increased when Ffh is suppressed in the Wam 113 strain, causes a pleiotropic defect characterised by cell elongation, and increased accumulation of precursor proteins. The accumulation of precursors of outer membrane protein A (Omp A) and maltose-binding protein (MBP), See-B dependent pre-proteins, was less than the Ffh-dependent proteins ribose-binding protein (RBP) and beta-lactamase. Sec B expression was suppressed by Ffh expression. The recombinant SRP 54 kDa, which forms a ribonucleoprotein complex in E coli, was shown to bind to precursor proteins, but is unable to interact with the filamentous temperature-sensitive Y (Fts Y) membrane receptor of the translocation machinery.

The Vpr protein of human immunodeficiency virus type 1 binds to nucleocapsid protein p7 in vitro.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) is incorporated into the virion by the Gag polyprotein precursor Pr55gag. The importance of the p6gag sequence at the C-terminal end of Pr55gag has a crucial role in Vpr incorporation. To identify the Gag sequences directly involved in Vpr binding, we compared the Vpr binding affinities of the 71 amino acid nucleocapsid protein p7, the C-terminal peptide (35-71) p7C and p6gag by affinity chromatography. p7 and p7C have the strongest Vpr binding activities compared to p6gag. These results suggest that the nucleocapsid protein and its C-terminal domain may be important for the incorporation of Vpr into the mature HIV-1 virion and the subsequent localisation of viral nucleic acid to the cell nucleus by Vpr.

The gp120 envelope of HIV-1 binds peptides in a similar manner to human leukocyte antigens.

OBJECTIVE: All the conserved regions of HIV gp120 have at least some partial homology with human leukocyte antigen (HLA) class I or class II. One functional similarity is the ability of gp120 and HLA class II to bind CD4. Given the close association between HIV-induced disease and the amount of immune activation and anergy, features closely associated with chronic allogenic stimulation, we asked whether gp120 shared any other properties of HLA, in this case the ability to bind peptides. DESIGN: T-cell epitope peptides known to bind to soluble HLA class I or class II were photolabelled and made radioactive. Cross-linking of modified peptides to soluble HLA class I, II and gp120 was activated by ultraviolet light and analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis. RESULTS: A signal peptide binding to HLA class I and a haemagglutinin peptide that binds to HLA class II were found to bind soluble gp120 specifically; binding and cross-linking could be competed out with excess of the unmodified peptides but not unrelated control peptides. Molecular modelling of gp120 suggests shared anchor sites for peptides binding to both HLA and gp120 soluble molecules. CONCLUSIONS: The ability to bind these two peptides suggests that gp120 has a peptide-binding site of broad specificity, which if functional in vivo, could compete with normal peptide loading of major histocompatibility complex (MHC) class I and/or class II peptides, as well as aberrantly stimulate the T-cell receptor (by virtue of its potential to be mistaken for an allogenic MHC/peptide complex), resulting in immune activation, anergy and apoptosis in susceptible hosts.