Dimerization of beta-site beta-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.     

Ceramide stabilizes beta-site amyloid precursor protein-cleaving enzyme 1 and promotes amyloid beta-peptide biogenesis

The lipid second messenger ceramide regulates several biochemical events that occur during aging. In addition, its level is highly elevated in the amyloid-burdened brains of Alzheimer's disease patients. Here, we analyzed the impact of aberrant ceramide levels on amyloid beta-peptide (Abeta) generation by using a cell-permeable analog of ceramide, C6-ceramide, and several biochemical inhibitors of the sphingomyelin/glycosphingolipid biosynthetic pathway. We found that C6-ceramide increased the biogenesis of Abeta by affecting beta-but not gamma-cleavage of the amyloid precursor protein. Similarly to C6-ceramide, increased levels of endogenous ceramide induced by neutral sphingomyelinase treatment also promoted the biogenesis of Abeta. Conversely, fumonisin B1, which inhibits the biosynthesis of endogenous ceramide, reduced Abeta production. Exogenous C6-ceramide restored both intracellular ceramide levels and Abeta generation in fumonisin B1-treated cells. These events were specific for amyloid precursor protein and were not associated with apoptotic cell death. Pulse-chase and time-course degradation experiments showed that ceramide post-translationally stabilizes the beta-secretase BACE1. Taken together, these data indicate that the lipid second messenger ceramide, which is elevated in the brains of Alzheimer's disease patients, increases the half-life of BACE1 and thereby promotes Abeta biogenesis.     

Maturation and endosomal targeting of beta-site amyloid precursor protein-cleaving enzyme. The Alzheimer's disease beta-secretase

The amyloidogenic Abeta peptide is liberated from the amyloid precursor protein (APP) by two proteolytic activities, beta-secretase and gamma-secretase. Recently, a type I membrane protein termed BACE (beta-site APP cleaving enzyme) with characteristics of an aspartyl protease has been identified as the beta-secretase. We undertook a series of biochemical and morphological investigations designed to characterize the basic properties of this protein. Initial studies indicated that BACE undergoes N-linked glycosylation at three of four potential sites. Metabolic pulse-chase experiments revealed that after core glycosylation, BACE is rapidly and efficiently transported to the Golgi apparatus and distal secretory pathway. BACE was also found to be quite stable, being turned over with a t(12) of approximately 16 h. Retention of BACE in the endoplasmic reticulum by introduction of a C-terminal dilysine motif prevented complex carbohydrate processing and demonstrated that propeptide cleavage occurs after exit from this organelle. BACE exhibited intramolecular disulfide bonding but did not form oligomeric structures by standard SDS-polyacrylamide gel electrophoresis analysis and sedimented as a monomer in sucrose velocity gradients. Immunofluorescence studies showed a largely vesicular staining pattern for BACE that colocalized well with endosomal, but not lysosomal, markers. Measurable levels of BACE were also detected on the plasma membrane by both immunostaining and cell surface biotinylation, and cycling of the protein between the cell membrane and the endosomes was documented. A cytoplasmic dileucine motif was found to be necessary for normal targeting of BACE to the endosomal system and accumulation of the protein in this intracellular site.     

Mechanism of Inhibition of beta-site amyloid precursor protein-cleaving enzyme (BACE) by a statine-based peptide

Inhibition of beta-site amyloid precursor protein-cleaving enzyme by a statine-based inhibitor has been studied using steady state and stopped-flow methods. A slow onset rate of inhibition has been observed under steady state conditions, and a K(i) of 22 nm has been derived using progress curves analysis. Simulation of stopped-flow protein fluorescence transients provided an estimate of the K(d) for initial inhibitor binding of 660 nm. A two-step inhibition mechanism is proposed, wherein slower "tightening up" of the initial encounter complex occurs. Two hypotheses have been proposed in the literature to address the nature of the slow step in the inhibition of aspartic proteases by peptidomimetic inhibitors: a conformational change related to the "flap" movement and displacement of a catalytic water. We compared substrate and inhibitor binding rates under pre-steady-state conditions. Both ligands are likely to cause flap movement, whereas no catalytic water replacement occurs during substrate binding. Our results suggest that both ligands bind to the enzyme at a rate significantly lower than the diffusion limit, but there are additional rate limitations involved in inhibitor binding, resulting in a k(on) of 3.5 x 10(4) m(-)1 s(-)1 for the inhibitor compared with 3.5 x 10(5) m(-)1 s(-)1 for the substrate. Even though specific intermediate formation steps might be different in the productive inhibitor and substrate binding to beta-site amyloid precursor protein-cleaving enzyme, a similar final optimized conformation is achieved in both cases, as judged by the comparable free energy changes (DeltaDeltaG of 2.01 versus 1.97 kcal/mol) going from the initial to the final enzyme-inhibitor or enzyme-substrate complexes.     

Characterization of the ectodomain shedding of the beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1)

Generation of the amyloid peptide through proteolytic processing of the amyloid precursor protein by beta- and gamma-secretases is central to the etiology of Alzheimer's disease. beta-secretase, known more widely as the beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), has been identified as a transmembrane aspartic proteinase, and its ectodomain has been reported to be cleaved and secreted from cells in a soluble form. The extracellular domains of many diverse proteins are known to be cleaved and secreted from cells by a process known as ectodomain shedding. Here we confirm that the ectodomain of BACE1 is secreted from cells and that this processing is up-regulated by agents that activate protein kinase C. A metalloproteinase is involved in the cleavage of BACE1 as hydroxamic acid-based metalloproteinase inhibitors abolish the release of shed BACE1. Using potent and selective inhibitors, we demonstrate that ADAM10 is a strong candidate for the BACE1 sheddase. In addition, we show that the BACE1 sheddase is distinct from alpha-secretase and, importantly, that inhibition of BACE1 shedding does not influence amyloid precursor protein processing at the beta-site.     

Kinetic studies on beta-site amyloid precursor protein-cleaving enzyme (BACE). Confirmation of an iso mechanism

The steady-state kinetic mechanism of beta-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed proteolytic cleavage was evaluated using product and statine- (Stat(V)) or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent kinetic isotope effects, and proton NMR spectroscopy. The noncompetitive inhibition pattern observed for both cleavage products, together with the independence of Stat(V) inhibition on substrate concentration, suggests a uni-bi-iso kinetic mechanism. According to this mechanism, the enzyme undergoes multiple conformation changes during the catalytic cycle. If any of these steps are rate-limiting to turnover, an enzyme form preceding the rate-limiting conformational change should accumulate. An insignificant solvent kinetic isotope effect (SKIE) on k(cat)/K(m), a large inverse solvent kinetic isotope effect on k(cat), and the absence of any SKIE on the inhibition onset by Stat(V) during catalysis together indicate that the rate-limiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at delta 13.0 ppm and phi of 0.6) has been observed by NMR spectroscopy in the enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics the tetrahedral intermediate of catalysis. Collapse of this intermediate, involving multiple steps and interconversion of enzyme forms, has been suggested to impose a rate limitation, which is manifested in a significant SKIE on k(cat). Multiple enzyme forms and their distribution during catalysis were evaluated by measuring the SKIE on the noncompetitive (mixed) inhibition constants for the C-terminal reaction product. Large, normal SKIE values were observed for these inhibition constants, suggesting that both kinetic and thermodynamic components contribute to the K(ii) and K(is) expressions, as has been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in the catalytic reaction of beta-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the enzyme might provide inhibitors of greater pharmacological relevance.     

beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and gamma-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that beta subunits of voltage-gated sodium channels are sequentially processed by BACE1 and gamma-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.     

Generation of the amyloid-beta peptide N terminus in Saccharomyces cerevisiae expressing human Alzheimer's amyloid-beta precursor protein

The Alzheimer's amyloid-beta precursor protein (betaAPP) is a type 1 membrane-spanning protein from which the Alzheimer's disease amyloid-beta peptide (Abeta) is proteolytically derived. To date, attempts to identify the enzymes responsible for Abeta generation have failed. Here we report the accumulation of Abeta-immunoreactive peptides in yeast expressing human betaAPP. Characterization of these peptides by metabolic labeling, immunoprecipitation with Abeta-specific antibodies, and N-terminal radiosequencing indicates that these peptides include the Abeta peptide at their N termini. The Abeta-like peptides generated in yeast were recovered predominantly as 8- and 12-14-kDa species. A 4-kDa species was recovered either when a protease-deficient strain was used to prevent breakdown or when the 8- and 12-14-kDa species were treated with disaggregating agents. The likely existence in yeast of enzymes generating the Abeta N terminus indicates that the molecular identification of yeast beta-secretase-like enzymes may be accomplished using genetic screens or empirical approaches based upon the sequenced genome of Saccharomyces cerevisiae.     

Endoproteolysis of beta-secretase (beta-site amyloid precursor protein-cleaving enzyme) within its catalytic domain. A potential mechanism for regulation

Sequential proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase activities yields the amyloid beta peptide that is widely deposited in the brains of individuals with Alzheimer's disease. The membrane-anchored aspartyl protease beta-site APP-cleaving enzyme (BACE) exhibits all of the characteristics of a beta-secretase and has been shown to cleave APP at its beta-site in vitro and in vivo. We found that BACE undergoes cleavage on a surface-exposed alpha-helix between amino acid residues Leu-228 and Ala-229, generating stable N- and C-terminal fragments that remain covalently associated via a disulfide bond. The efficiency of BACE endoproteolysis was observed to depend heavily on cell and tissue type. In contrast to brain where holoprotein was predominant, BACE was found primarily as endoproteolyzed fragments in pancreas, liver, and muscle. In addition, we observed a marked up-regulation of BACE endoproteolysis in C2 myoblasts upon differentiation into multinucleated myotubes, a well established model system of muscle tissue specification. As in liver, BACE exists as endoproteolyzed fragments in the hepatic cell line, HepG2. We found that HepG2 cells are capable of generating amyloid beta peptide, suggesting that endoproteolyzed BACE retains measurable beta-secretase activity. We also found that BACE endoproteolysis occurs only after export from the endoplasmic reticulum, is enhanced in the trans-Golgi network, and is sensitive to inhibitors of vesicular acidification. The membrane-bound proteases tumor necrosis factor alpha-converting enzyme and furin were not found to be responsible for this cleavage nor was BACE observed to mediate its own endoproteolysis by an autocatalytic mechanism. Thus, we characterize a specific processing event that may serve to regulate the enzymatic activity of BACE on a post-translational level.     

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.     

Methyl dynamics of the amyloid-beta peptides Abeta40 and Abeta42

To probe the role of side chain dynamics in Abeta aggregation, we studied the methyl dynamics of native Abeta40 and Abeta42 by measuring cross relaxation rates with interleaved data collection. The methyl groups in the C-terminus are in general more rigid in Abeta42 than in Abeta40, consistent with previous results from backbone (15)N dynamics. This lends support to the hypothesis that a rigid C-terminus in Abeta42 may serve as an internal aggregation seed. Interestingly, two methyl groups of V18 located in the central hydrophobic cluster are more mobile in Abeta42 than in Abeta40, most likely due to the paucity of V18 intra-molecular interactions in Abeta42. V18 may then be more available for inter-molecular interactions to form Abeta42 aggregates. Thus, the side chain mobility of the central hydrophobic cluster may play an important role in Abeta aggregation and may contribute to the difference in aggregation propensity between Abeta40 and Abeta42.     

Secretion and differential localization of the proteolytic cleavage products Abeta40 and Abeta42 of the Alzheimer amyloid precursor protein in human fetal myogenic cells

Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease. These peptides are proteolytic cleavage products of the amyloid precursor protein (APP) and are generated by beta- and gamma-secretases. Here we show by multiparameter immunofluorescence imaging in muscle cells that localization of the Abeta40 and Abeta42 cleavage products reveals different myocyte types in a three-dimensional culture system. These myocyte types are heterogeneous by selective intracellular concentration of either Abeta40 or Abeta42 in vesicular structures, whilst only the Abeta40 peptide is secreted as indicated by Western blot analysis. This cellular pattern of APP proteolysis and Abeta peptide secretion correlates with lack of L-APP mRNA splice isoforms. Differential secretion and intracellular accumulation of Abeta peptides is characteristic for the early myocyte development and might be related to cell fusion.     

Synthesis and evaluation of curcumin derivatives toward an inhibitor of beta-site amyloid precursor protein cleaving enzyme 1

To research a new non-peptidyl inhibitor of beta-site amyloid precursor protein cleaving enzyme 1, we focused on the curcumin framework, two phenolic groups combined with an sp₂ carbon spacer for low-molecular and high lipophilicity. The structure–activity relationship study of curcumin derivatives is described. Our results indicate that phenolic hydroxy groups and an alkenyl spacer are important structural factors for the inhibition of beta-site amyloid precursor protein cleaving enzyme 1 and, furthermore, non-competitive inhibition of enzyme activity is anticipated from an inhibitory kinetics experiment and docking simulation.     

Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence.

A distinguishing feature of Alzheimer's disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4-amyloid peptide and the intracellular accumulation of potential amyloidogenic and non-amyloidogenic fragments. The relative amount of amyloid-containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. 'Humanizing' the rodent APP sequence by introducing three mutations in the beta A4-region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4-peptide to non-amyloidogenic p3-peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed-back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeting amyloid-beta peptide (Abeta) oligomers by passive immunization with a conformation-selective monoclonal antibody improves learning and memory in Abeta precursor protein (APP) transgenic mice

Passive immunization of murine models of Alzheimer disease amyloidosis reduces amyloid-beta peptide (Abeta) levels and improves cognitive function. To specifically address the role of Abeta oligomers in learning and memory, we generated a novel monoclonal antibody, NAB61, that preferentially recognizes a conformational epitope present in dimeric, small oligomeric, and higher order Abeta structures but not full-length amyloid-beta precursor protein or C-terminal amyloid-beta precursor protein fragments. NAB61 also recognized a subset of brain Abeta deposits, preferentially mature senile plaques, and amyloid angiopathy. Using NAB61 as immunotherapy, we showed that aged Tg2576 transgenic mice treated with NAB61 displayed significant improvements in spatial learning and memory relative to control mice. These data implicated Abeta oligomers as a pathologic substrate for cognitive decline in Alzheimer disease.     

The generation of neurons and oligodendrocytes from a common precursor cell

We have used a recombinant retrovirus carrying the lacZ gene to study the developmental potential of precursor cells from the embryonic rat cerebral cortex in dissociated cell culture. Virus was used to label a small number of cultured cells genetically so that their fate could be determined. Infected clones were detected with an anti-beta-galactosidase serum, and the labeled cells were identified using monoclonal antibodies. The results revealed that most precursor cells generated a single cell type, the majority being either neurons or oligodendrocytes. However, a proportion of the neuronal clones also included oligodendrocytes. This proportion increased until embryonic day 16 when 18% of the neuronal clones were of this type. This suggests that during neurogenesis in the cerebral cortex there exists a cell with the potential to generate these two quite different neural cell types.