Biomedical subjects

D B Teplow

Publications and source records attributed to D B Teplow.

At least 37 records · Page 2Linked to original sources

PubMed1998

Structural and kinetic features of amyloid beta-protein fibrillogenesis.

Alzheimer's disease (AD) is an archetype of a class of diseases characterized by abnormal protein deposition. In each case, deposition manifests itself in the form of amyloid deposits composed of fibrils of otherwise normal, soluble proteins or peptides. An ever-increasing body of genetic, physiologic, and biochemical data supports the hypothesis that fibrillogenesis of the amyloid beta-protein is a seminal event in Alzheimer's disease. Inhibiting A beta fibrillogenesis is thus an important strategy for AD therapy. However, before this strategy can be implemented, a mechanistic understanding of the fibrillogenesis process must be achieved and appropriate steps selected as therapeutic targets. Following a brief introduction to AD, I review here the current state of knowledge of A beta fibrillogenesis. Special emphasis is placed on the morphologic, structural, and kinetic aspects of this complex process.

Amino Acid Sequence↗

PubMed1997

Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate.

Fibrillogenesis of the amyloid beta-protein (Abeta) is a seminal pathogenetic event in Alzheimer's disease. Inhibiting fibrillogenesis is thus one approach toward disease therapy. Rational design of fibrillogenesis inhibitors requires elucidation of the stages and kinetics of Abeta fibrillogenesis. We report results of studies designed to examine the initial stages of Abeta oligomerization. Size exclusion chromatography, quasielastic light scattering spectroscopy, and electron microscopy were used to characterize fibrillogenesis intermediates. After dissolution in 0.1 M Tris-HCl, pH 7.4, and removal of pre-existent seeds, Abeta chromatographed almost exclusively as a single peak. The molecules composing the peak had average hydrodynamic radii of 1.8 +/- 0.2 nm, consistent with the predicted size of dimeric Abeta. Over time, an additional peak, with a molecular weight >100,000, appeared. This peak contained predominantly curved fibrils, 6-8 nm in diameter and <200 nm in length, which we have termed "protofibrils." The kinetics of protofibril formation and disappearance are consistent with protofibrils being intermediates in the evolution of amyloid fibers. Protofibrils appeared during the polymerization of Abeta-(1-40), Abeta-(1-42), and Abeta-(1-40)-Gln22, peptides associated with both sporadic and inherited forms of Alzheimer's disease, suggesting that protofibril formation may be a general phenomenon in Abeta fibrillogenesis. If so, protofibrils could be attractive targets for fibrillogenesis inhibitors.

Alzheimer Disease↗

PubMed1997

Kinetic theory of fibrillogenesis of amyloid beta-protein.

Prior quasielastic light scattering (QLS) studies of fibrillogenesis of synthetic amyloid beta-protein (Abeta)-(1-40) at low pH have suggested a kinetic model in which: (i) fibrillogenesis requires a nucleation step; (ii) nuclei are produced by Abeta micelles in addition to seeds initially present; and (iii) fibril elongation occurs by irreversible binding of Abeta monomers to the fibril ends. Here we present the full mathematical formulation of this model. We describe the temporal evolution of the concentrations of Abeta monomers and micelles as well as the concentration and size distribution of fibrils. This formulation enables deduction of the fundamental parameters of the model-e.g., the nucleation and elongation rate constants kn and ke-from the time dependency of the apparent diffusion coefficient measured by QLS. The theory accurately represents the experimental observations for Abeta concentrations both below and above c*, the critical concentration for Abeta micelle formation. We suggest that the method of QLS in combination with this theory can serve as a powerful tool for understanding the molecular factors that control Abeta plaque formation.

Amyloid beta-Peptides↗

PubMed1997

Enhanced production and oligomerization of the 42-residue amyloid beta-protein by Chinese hamster ovary cells stably expressing mutant presenilins.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of early onset familial Alzheimer's disease. To elucidate their pathogenic mechanism, wild-type (wt) or mutant (M146L, C410Y) PS1 and wt or mutant (M239V) PS2 genes were stably transfected into Chinese hamster ovary cells that overexpress the beta-amyloid precursor protein (APP). The identity of the 43-45-kDa PS1 holoproteins was confirmed by N-terminal radiosequencing. PS1 was rapidly processed (t1/2 = 40 min) in the endoplasmic reticulum into stable fragments. Wild-type and mutant PS2 holoproteins exhibited similar half lives (1.5 h); however, their endoproteolytic fragments showed both mutation-specific and cell type-specific differences. Mutant PS1 or PS2 consistently induced a 1.4-2.5-fold increase (p < 0.001) in the relative production of the highly amyloidogenic 42-residue form of amyloid beta-protein (Abeta42) as determined by quantitative immunoprecipitation and by enzyme-linked immunosorbent assay. In mutant PS1 and PS2 cell lines with high increases in Abeta42/Abetatotal ratios, spontaneous formation of low molecular weight oligomers of Abeta42 was observed in media, suggesting enhanced Abeta aggregation from the elevation of Abeta42. We conclude that mutant PS1 and PS2 proteins enhance the proteolysis of beta-amyloid precursor protein by the gamma-secretase cleaving at Abeta residue 42, thereby promoting amyloidogenesis.

Amyloid beta-Peptides↗

PubMed1997

Presenilin proteins undergo heterogeneous endoproteolysis between Thr291 and Ala299 and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue.

Humans inheriting missense mutations in the presenilin (PS)1 and -2 genes undergo progressive cerebral deposition of the amyloid beta-protein at an early age and develop a clinically and pathologically severe form of familial Alzheimer's disease (FAD). Because PS1 mutations cause the most aggressive known form of AD, it is important to elucidate the structure and function of this multitransmembrane protein in the brain. Using a panel of region-specific PS antibodies, we characterized the presenilin polypeptides in mammalian tissues, including brains of normal, AD, and PS1-linked FAD subjects, and in transfected and nontransfected cell lines. Very little full-length PS1 or -2 was detected in brain and untransfected cells; instead the protein occurred as a heterogeneous array of stable N- and C-terminal proteolytic fragments that differed subtly among cell types and mammalian tissues. Sequencing of the major C-terminal fragment from PS1-transfected human 293 cells showed that the principal endoproteolytic cleavage occurs at and near Met298 in the proximal portion of the large hydrophilic loop. Full-length PS1 in these cells is quickly turned over (T1/2 approximately 60 min), in part to the two major fragments. The sizes and amounts of the PS fragments were not significantly altered in four FAD brains with the Cys410Tyr PS1 missense mutation. Our results indicate that presenilins are rapidly processed to N- and C-terminal fragments in both neural and nonneural cells and that interference with this processing is not an obligatory feature of FAD-causing mutations.

Alzheimer Disease↗

PubMed1996

Metabolism of the "Swedish" amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the "beta-secretase" site occurs in the golgi apparatus.

The 4-kDa beta-amyloid peptide (Abeta), a principal component of parenchymal amyloid deposits in Alzheimer's disease, is derived from amyloid precursor proteins (APP). To identify potential intracellular compartments involved in Abeta production, we expressed human APP-695 (APPwt) and APP-695 harboring the Swedish double mutation (APPswe) associated with familial early-onset Alzheimer's disease, in mouse N2a cells. We demonstrate that cells expressing APPswe secrete high levels of Abeta peptides and beta-secretase-generated soluble APP derivatives (APP s beta) relative to cells expressing APPwt. In addition, we observed a concomitant diminution in the levels of alpha-secretase-generated soluble APP derivatives (APP s alpha). Our interpretation of these findings is that beta-secretase cleavage occurs in an intracellular compartment and disables those substrates which would normally be cleaved by alpha-secretase. As anticipated, the levels of APPswe are diminished relative to the steady-state levels of surface-bound APPwt; moreover, surface-bound APPswe and APPwt molecules are released from the plasma membrane after cleavage by alpha-secretase, but not by beta-secretase. Finally, by examining the rate of appearance of specific APP metabolites generated by beta-secretase, we now unequivocally demonstrate that beta-secretase cleavage of APPswe occurs within the Golgi apparatus, as early as the medial compartment.

Alzheimer Disease↗

PubMed1996

Degradation of amyloid beta-protein by a serine protease-alpha2-macroglobulin complex.

Progressive cerebral deposition of the amyloid beta-peptide (Abeta) is an early and constant feature of Alzheimer's disease. Abeta is derived by proteolysis from the beta-amyloid precursor protein. beta-Amyloid precursor protein processing and the generation of Abeta have been extensively characterized, but little is known about the mechanisms of degradation of this potentially neurotoxic peptide. We identified and purified a proteolytic activity in culture medium that can degrade secreted Abeta but not larger proteins in the medium. Detection of the activity in conditioned medium required the presence of fetal bovine serum and the passage of the cells with a pancreatic trypsin preparation. Its inhibitor profile showed that the activity was a serine protease other than trypsin or chymotrypsin. The protease occurs as a stable approximately 700-kDa complex with the inhibitor, alpha2-macroglobulin (alpha2M), that retains activity against small substrates such as Abeta. Its NH2-terminal sequence suggests that the protease is previously unidentified. Our results indicate that the Abeta-degrading protease we have detected is a non-trypsin component of a pancreatic trypsin preparation or else derives from a zymogen in serum that is activated by a protease in the latter preparation. Because Abeta-bearing plaques in Alzheimer's disease brain contain both alpha2M and receptors of alpha2M-protease complexes, the same or a similar alpha2M-protease complex could arise in vivo and play a role in Abeta clearance.

Amino Acid Sequence↗

PubMed1996

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Amyloid beta-Peptides↗

PubMed1996

The role of APP processing and trafficking pathways in the formation of amyloid beta-protein.

The amyloid beta-protein (A beta) is a proteolytic fragment of the beta-amyloid precursor protein (beta APP). We previously reported the constitutive secretion of A beta peptides from a variety of cells expressing beta APP under normal culture conditions. These endogenously produced A beta peptides have heterogeneous N- and C-termini that vary as a function of beta APP missense mutations. Treatment of A beta-secreting cells with agents that alter intravesicular pH showed that an acidic compartment is required for proper A beta generation. One such compartment appears to be the endosome. Immunolabeling of cell-surface beta APP in living neurons and non-neuronal cells directly demonstrated the endocytosis of the protein and its rapid recycling (within 5-10 minutes) to the cell surface, as well as the trafficking of some beta APP to lysosomes. Expression of beta APP with various deletions of the cytoplasmic domain, including the NPTY motif, leads to decreased internalization and an associated decrease in the production of A beta peptides that begin at the usual asp1 start site. These and other data suggest that A beta production begins with cleavage of beta APP by a still unknown protease(s) (beta-secretase[s]) at the met-asp bond proceeding the A beta N-terminus and that this occurs in part in early endosomes. To characterize the substrate requirements of beta-secretase, beta APP was mutagenized by placing stop codons within or at the end of the transmembrane domain or substituting other amino acids for the wild-type met and asp at the P1 and P1' positions. These experiments showed that proper beta-secretase cleavage requires the precursor to be membrane-anchored and is highly sequence specific; most substitutions at met or asp substantially decrease A beta production. Analogous mutagenesis experiments around the A beta C-terminus revealed that the unknown protease(s) cleaving here ("gamma-secretase[s]") does not show such specificity. Cells secreting A beta may also be useful for examining the critical issue of the aggregation of A beta into its neurotoxic polymeric form under physiological conditions. In this regard, we have found that beta APP-expressing CHO cells show aggregation of > or = 10-20% of their secreted A beta peptides into SDS-stable dimers, trimers and sometimes tetramers under normal culture conditions. The identity of these small multimers was confirmed by extensive immunochemical characterization and radiosequencing. They are present at approximately 100-500 pM levels in conditioned medium of CHO transfectants. Using this endogenous A beta aggregating system, we have begun to examine variables that influence aggregation and compounds which may retard it. In conclusion, studies of the regulation of A beta production and aggregation in cell culture can provide information under physiological conditions that can complement analyses of these processes in vivo.

Amyloid beta-Peptides↗

PubMed1996

Inhibition of amyloid beta-protein production in neural cells by the serine protease inhibitor AEBSF.

Cerebral deposition of amyloid beta protein (A beta) is an early and critical feature of Alzheimer's disease. A beta production requires the proteolytic release of A beta from the beta-amyloid precursor protein (beta APP). Thus, inhibition of A beta release is a prime therapeutic goal. Here, we show that the broad spectrum, irreversible serine protease inhibitor, AEBSF, inhibits the constitutive production of A beta in five different human cell lines, both neural and nonneural. AEBSF also stabilizes full-length beta APP and enhances alpha-secretion, as shown by an increase in the proteolytic derivative, alpha-APPS. Further, we demonstrate that the inhibitory effect of AEBSF is specific for A beta proteins starting at Aspartate 1, suggesting that AEBSF directly inhibits beta-secretase, the Methionine-Aspartate (Met-Asp)-cleaving enzyme. These results indicate that specific inhibition of this A beta-generating protease is possible in living human neural cells and provide information about the characteristics of this as yet unidentified enzyme.

Amino Acid Sequence↗

PubMed1995

Lysosomal processing of amyloid precursor protein to A beta peptides: a distinct role for cathepsin S.

To investigate the potential contribution of the lysosomal compartment in the processing of amyloid precursor protein (APP) to amyloid beta-peptides (A beta s), we stably overexpressed a series of lysosomal proteases (the cysteine proteases, cathepsins B, L and S, and the aspartic protease, cathepsin D) in a human kidney epithelial cell line (293) transfected to express high levels of beta APP. Preliminary experiments indicated that 293 cells endogenously synthesize cathepsins B, L and D, but not cathepsin S. A beta secretion was assessed by immunoprecipitation and ELISA and found to be increased approximately 2-fold following cathepsin S expression, but to be unchanged (cathepsins B, L) or decreased (cathepsin D) in the other double transfectants. E-64d, an inhibitor of lysosomal cysteine proteases, significantly reduced A beta secretion by the cathepsin S transfectants, but had no effect on cells expressing the other proteases. Radiosequencing of A beta secreted by cathepsin S-expressing cells revealed that a previously unreported variant beginning at Met -1 (relative to the most common A beta N-terminus, Asp -1) accounted for most of the increase in A beta secretion. Immunostaining of human brain sections revealed cathepsin S in cortical neurons and glia in samples of brain from patients with Alzheimer's disease. These results provide evidence in living cells for a pathway in which cathepsin S generates A beta from amyloidogenic fragments of beta APP in the endosomal/lysosomal compartment. This pathway appears to be inducible, distinct from a constitutive pathway used by 293 and other cells to generate A beta, and may be relevant to the pathogenesis of Alzheimer's disease.

Alzheimer Disease↗

PubMed1995

Truncated forms of the human prion protein in normal brain and in prion diseases.

The cellular form of the prion protein (PrPc) is a glycoprotein anchored to the cell membrane by a glycosylphosphatidylinositol moiety. An aberrant form of PrPc that is partially resistant to proteases, PrPres, is a hallmark of prion diseases, which in humans include Cruetzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia. We have characterized the major forms of PrP in normal and pathological human brains. A COOH-terminal fragment of PrPc, designated C1, is abundant in normal and CJD brains as well as in human neuroblastoma cells. Sequence analysis revealed that C1 contains alternative NH2 termini starting at His-111 or Met-112. Like PrPc, C1 is glycosylated, anchored to the cell membrane, and is heat-stable. Consistent with the lack of the NH2-terminal region of PrPc, C1 is more acidic than PrPc and does not bind heparin. An additional fragment longer than C1, designated C2, is present in substantial amounts in CJD brains. Like PrPres, C2 is resistant to proteases and is detergent-insoluble. Our data indicate that C1 is a major product of normal PrPc metabolism, generated by a cleavage that disrupts the neurotoxic and amyloidogenic region of PrP comprising residues 106-126. This region remains intact in C2, suggesting a role for C2 in prion diseases.

Adult↗

PubMed1995

Aggregation of secreted amyloid beta-protein into sodium dodecyl sulfate-stable oligomers in cell culture.

Filamentous aggregates of the 40-42-residue amyloid beta-protein (A beta) accumulate progressively in the limbic and cerebral cortex in Alzheimer's disease, where they are intimately associated with neuronal and glial cytopathology. Attempts to model this cytotoxicity in vitro using synthetic peptides have shown that monomeric A beta is relatively inert, whereas aggregated A beta reproducibly exerts a variety of neurotoxic effects. The processes that mediate the conversion of monomeric A beta into a toxic aggregated state are thus of great interest. Previous studies of this conversion have employed high concentrations (10(-5)-10(-3) M) of synthetic A beta peptides under nonbiological conditions. We report here the detection of small amounts (< 10(-9) M) of SDS-stable A beta oligomers in the culture media of Chinese hamster ovary cells expressing endogenous or transfected amyloid beta-protein precursor genes. The identity of these oligomers (primarily dimers and trimers) was established by immunoprecipitation with a panel of A beta antibodies, by electrophoretic comigration with synthetic A beta oligomers, and by amino acid sequencing. The oligomeric A beta species comprised approximately 10-20% of the total immunoprecipitable A beta in these cultures. A truncated A beta species beginning at Arg 5 was enriched in the oligomers, suggesting that amino-terminal heterogeneity can influence A beta oligomerization in this system. Addition of Congo red (10 microM) during metabolic labeling of the cells led to increased monomeric and decreased oligomeric A beta. The ability to detect and quantitate oligomers of secreted A beta peptides in cell culture should facilitate dynamic studies of the critical process of initial A beta aggregation under physiological conditions.

Amyloid beta-Peptides↗

PubMed1995

The vacuolar H(+)-ATPase inhibitor bafilomycin A1 differentially affects proteolytic processing of mutant and wild-type beta-amyloid precursor protein.

We analyzed the effect of the vacuolar H(+)-ATPase inhibitor bafilomycin A1 (bafA1) on the processing of beta-amyloid precursor protein (beta APP). In kidney 293 cells stably transfected with the wild-type beta APP cDNA, bafA1 caused a stabilization of mature beta APP and its 10-kDa COOH-terminal fragment. Moreover, it caused a 2-3-fold increase in secretion of soluble APP and amyloid-beta protein (A beta). Interestingly, bafA1 treatment of cells transfected with a mutant beta APP isoform that occurs in a Swedish kindred with familial Alzheimer's disease resulted in a decrease of A beta production and no increase of soluble APP secretion. Identical results were obtained when the effect of bafA1 was analyzed on fibroblasts derived from affected versus unaffected members of the Swedish family. These data demonstrate a differential effect of bafA1 on the production of A beta derived from wild-type or Swedish mutant beta APP. Radiosequencing of A beta derived from bafA1-treated cells expressing wild-type beta APP revealed a marked increase of A beta peptides starting at amino acids phenylalanine 4 and valine -3 and a relative decrease of A beta molecules beginning at the typical NH2 terminus of aspartate 1. Cells transfected with the Swedish mutation and treated with bafA1 did not produce these alternative A beta peptides, so that bafA1 treatment resulted in a decrease of A beta starting at aspartate 1. Our data indicate that multiple proteases are able to cleave A beta at or near its NH2 terminus. Inhibition of the protease cleaving at aspartate 1 by bafA1 and perhaps other similar agents can result in an increase of alternatively cleaved peptides.

Alzheimer Disease↗

PubMed1995

Identity of PSA purified from seminal fluid by different methods: comparison by amino acid analysis and assigned extinction coefficients.

To determine the true extinction coefficient of prostate specific antigen (PSA) and to measure any differences in PSA when isolated from seminal fluid by four different published methods, we studied 10 different lots of PSA by quantitative amino acid analysis. Despite an expected PSA concentration of 1 mg/ml based on gravimetric analysis at an average optical density of 1.45 at 280 nm, we recovered only 0.79 mg/ml by quantitative amino acid analysis (range 0.752 to 0.820 mg/ml with a coefficient of variation [C.V.] of 3.3% among the 10 lots). The concentration of 0.79 mg/ml was based on a molecular weight of 28,430 daltons for glycosylated PSA determined by ion spray mass spectroscopy [Bélanger et al: Prostate, 27:187-197, 1995]. From these 10 amino acid analyses, we calculated the extinction coefficient of PSA at 280 nm as 1.84 +/- 0.04 ml x mg-1 x cm-1 (range 1.78 to 1.90 with a C.V. of 2.2%). Similar concentrations of PSA were obtained by amino acid analysis regardless of the method of purification. These observations support the presence of a single form of PSA in seminal fluid and are consistent with the molecular evidence that PSA is transcribed from a single gene locus on the long arm of chromosome 19 [Riegman et al.: Genomics 14:6-11, 1992]. They do not support the recent contention by the Roswell Park group that the PSA they isolated in 1979 [Wang et al.: Invest Urol 17:159-163, 1979; Wang et al.: Prostate 24:107-108, 1994] is different from p30 reported a year earlier [Sensabaugh: J Forensic Sci 23:106-115, 1978].

Amino Acids↗

PubMed1995

Generation of amyloid beta protein from its precursor is sequence specific.

Cerebral deposition of amyloid beta protein (A beta) is an early and critical feature of Alzheimer's disease. Here we analyze the substrate requirements of proteases ("beta-secretases") that cleave the beta-amyloid precursor protein (beta APP) at the N-terminus of A beta (Asp-597 of beta APP695) in intact human cells. The cleavage requires a membrane-bound substrate but tolerates shifts in the distance of the hydrolyzed bond from the membrane. The major protease has a minimum recognition region of Val-594 to Ala-598; most substitutions in this sequence strongly decrease or eliminate A beta production. Only the Swedish familial Alzheimer's disease mutation (K595N/M596L) strongly increases A beta production. Moreover, in this mutant but not in the wild type, the entire cytoplasmic tail with its reinternalization signals can be deleted without affecting A beta N-terminal cleavage, consistent with the concept that cleavage of this mutant occurs in a different cellular compartment than that of wild-type molecules. Our results have important implications for current intensive approaches to develop assays for and identify enzymes with beta-secretase activity.

Alzheimer Disease↗

PubMed1995

Polarized sorting of beta-amyloid precursor protein and its proteolytic products in MDCK cells is regulated by two independent signals.

Progressive cerebral deposition of the amyloid (A beta) beta-protein is an early and invariant feature of Alzheimer's disease. A beta is derived by proteolysis from the membrane-spanning beta-amyloid precursor protein (beta APP). beta APP is processed into various secreted products, including soluble beta APP (APPs), the 4-kD A beta peptide, and a related 3-kD peptide (p3). We analyzed the mechanisms regulating the polarized basolateral sorting of beta APP and its proteolytic derivatives in MDCK cells. Deletion of the last 32 amino acids (residues 664-695) of the beta APP cytoplasmic tail had no influence on either the constitutive approximately 90% level of basolateral sorting of surface beta APP, or the strong basolateral secretion of APPs, A beta, and p3. However, deleting the last 42 amino acids (residues 654-695) or changing tyrosine 653 to alanine altered the distribution of cell surface beta APP so that approximately 40-50% of the molecules were inserted apically. In parallel, A beta was now secreted from both surfaces. Surprisingly, this change in surface beta APP had no influence on the basolateral secretion of APPs and p3. This result suggests that most beta APP molecules which give rise to APPs in MDCK cells are cleaved intracellularly before reaching the surface. Consistent with this conclusion, we readily detected intracellular APPs in carbonate extracts of isolated membrane vesicles. Moreover, ammonium chloride treatment resulted in the equal secretion of APPs into both compartments, as occurs with other non-membranous, basolaterally secreted proteins, but it did not influence the polarity of cell surface beta APP. These results demonstrate that in epithelial cells two independent mechanisms mediate the polarized trafficking of beta APP holoprotein and its major secreted derivative (APPs) and that A beta peptides are derived in part from beta APP holoprotein targeted to the cell surface by a signal that includes tyrosine 653.

Amino Acid Sequence↗

PubMed1995

beta-Amyloid, protein processing and Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder resulting in the deposition of amyloid beta-peptide (A beta) in senile plaques in cerebral and limbic corteces and the walls of meningeal and cerebral blood vessels. A beta is a proteolytic break-down product of a membrane bound precursor, the beta-amyloid precursor protein (beta APP). Conventional secretory processing of beta APP prevents A beta formation. An additional processing pathway of beta APP involving endosomal/lysosomal targeting is described. Within isolated lysosomes amygloidogenic fragments are found which might serve as precursors for A beta production. From such precursors A beta might be proteolytically processed. Indeed, secreted A beta was identified in the media of cultured cells. A beta is also secreted in vivo and can be detected in human plasma and cerebral spinal fluid. These findings provide a cellular system to analyze the molecular mechanism and the biological regulation of A beta generation. Furthermore, the effect of inherited mutations within the beta APP gene in some cases of familial AD can now be analyzed in such tissue culture cells transfected with the mutant cDNA constructs. A model will be presented proposing that A beta generation might occur during reinternalization of the full-length molecule.

Alzheimer Disease↗