The Alzheimer's amyloid precursor is cleaved intracellularly in the trans-Golgi network or in a post-Golgi compartment.
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Alzheimer Disease↗
Biomedical subjects
L M Refolo
Publications and source records attributed to L M Refolo.
Bacterial expression, purification, and functional mapping of the amyloid beta/A4 protein precursor.
The secreted form of Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in cell growth regulation (Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T., and Schenk, D.B. (1989) Cell 58, 615-622). Using a strong prokaryotic expression system, we expressed, in Escherichia coli, peptide fragments covering different regions of the secreted form of APP-695. The longest of these fragments (KB75, 572 amino acids from Val-20 to Ile-591), which contained neither the Kunitz-type protease inhibitor (KPI) domain nor the amyloid beta/A4-protein domain, was purified and shown to be biologically active in terms of growth regulation. Two other APP fragments (KB48, 316 amino acids from Val-20 to Met-335; and RB17, 150 amino acids from Thr-296 to Pro-445), overlapping by only 40 amino acids at a close site C-terminal to the KPI insertion site, were also active. Furthermore, a chemically synthesized 40-residue peptide corresponding to this region of overlap also stimulated the growth of A-1 fibroblasts. These results establish the presence of growth-promoting activity in the secreted form of APP-695 and suggest that the site of this activity of APP-695 lies within a 40-amino acid domain next to the KPI insertion site.
Amino Acid Sequence↗
Expression of the Alzheimer amyloid precursor in brain tissue and effects of NGF and EGF on its metabolism.
Alzheimer's disease (AD) is the major cause of serious dementia and memory loss in several million elderly Americans. The most prominent lesions in the brains of these patients are the depositions of two types of abnormal filaments: the predominantly intraneuronal neurofibrillary tangles (NFT), which consist of paired helical filaments (PHF), and the extracellular amyloid fibers. These changes are characteristic of AD, and a final diagnosis of this disease is based on the presence of large numbers of these abnormal filamentous structures in the patient's brain. The amyloid fibers consist of a peptide subunit termed beta-protein or A4 peptide, which derives from a larger precursor protein. In this study we review the structural characteristics, regulation of expression, and metabolism of the Alzheimer amyloid precursor in brain tissue and cell cultures.
Aged↗
The Alzheimer amyloid precursor is associated with the detergent-insoluble cytoskeleton.
The amyloid beta-protein (A beta P), the main component of neuritic plaques in Alzheimer's disease (AD), is derived by unknown mechanisms from a family of amyloid precursor proteins (APPs). Using a detergent extraction procedure, we have found that in brain and in neural cell lines, 50-90% of APP is bound to detergent-insoluble cytoskeleton. Labeling experiments performed in a C6 glioma cell line indicated that both cell surface and intracellular APPs are associated with the cytoskeleton. This association requires intact microtubules and is modulated by protein phosphorylation and by cell density. These findings suggest that the function of cellular APP, presently unknown, involves the cytoskeleton and particularly microtubules. The dynamic nature of the binding and its dependence on microtubules and protein phosphorylation suggest it as a possible target in AD, where abnormal cytoskeletal structures and protein phosphorylation have been reported. Altered cytoskeletal binding of APP might lead to its aberrant proteolysis and generation of the A beta P.
Alzheimer Disease↗
Human retina D2 receptor cDNAs have multiple polyadenylation sites and differ from a pituitary clone at the 5' non-coding region.
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Amino Acid Sequence↗
The Alzheimer's amyloid precursor protein is produced by type I astrocytes in primary cultures of rat neuroglia.
The production of beta-amyloid precursor protein (beta APP) by primary mixed glial cell cultures from cerebral cortex of 2-3 day postnatal rats was examined by Northern and Western blotting and by immunocytochemistry. A single 3.5 kb beta APP RNA transcript was detected using probes recognizing all forms of beta APP messenger RNA. No signal was detected with a probe specific for the alternately spliced Kunitz protease inhibitor (KPI) region. In Western blot analysis of protein extracts, antisera specific for either the cytoplasmic or extracellular parts of beta APP detected several proteins ranging from 105 to 140 kDa. None of these were recognized by an antiserum specific to the KPI insert of the beta APP. Multicolor immunofluorescence showed beta APP immunoreactivity in type I (GFAP+ A2B5-) astrocytes, distributed in a fibrillar pattern like that of glial fibrillary acidic protein (GFAP). No beta APP immunoreactivity was detected in oligodendrocytes (GC+) or in A2B5+ progenitor cells. Moreover, no cultured cells showed immunostaining with an antiserum specific for the KPI sequence of beta APP. We conclude that type I astrocytes in primary culture produce amyloid precursor protein, but that oligodendrocytes and their precursors do not. Moreover, type I astrocytes produce predominantly the beta APP subtypes which lacks the KPI sequence.
Amyloid↗
Differential brain expression of the Alzheimer's amyloid precursor protein.
The expression of the Alzheimer amyloid protein precursor (AAPP) was examined in human, monkey, dog and rat brains. Two proteins, one identified as AAPP695 and the other as AAPP751, were immunoprecipitated from the in vitro translation of human, dog and rat brain polysomes. The AAPP751 to AAPP695 ratio was highest in human, intermediate in dog and lowest in rat brain polysomes. Human cerebral cortex contained higher levels of the AAPP751 mRNA than either dog or rat cortex. AAPP695 was detected in both cerebral cortex and cerebellum of all species examined. In contrast, AAPP751 was detected predominantly in the cortex of human, monkey and to a lesser extent dog brains while it was not detected in rat brain. These findings indicate that the amyloid precursors are differentially expressed in different mammalian brains and suggest that AAPP751 is mainly expressed in the brain regions involved in plaque formation.
Aged↗
Nerve and epidermal growth factors induce the release of the Alzheimer amyloid precursor from PC 12 cell cultures.
Antisera against specific sites of the Alzheimer beta Amyloid protein precursor (beta APP) were used to study the effects of nerve and epidermal growth factors on the expression and processing of this protein in PC12 cell cultures. Two major beta APP proteins (140 and 105 kDa) both containing the Kunitz-protease inhibitor insert (KPI), were detected in cell extracts of naive PC12 cells. Treatment of these cultures with nerve growth factor (NGF) induced the release of two beta APP species 125 and 120 kDa, both of which contained the KPI domain and lacked the carboxy-terminal portion of the precursor. The released beta APP contained O-linked sugars. Only one of the released beta APP proteins bound to the lectin Concanavalin A indicating that they differ in their glycosylation. Epidermal growth factor (EGF) also induced the release of beta APP proteins into the culture medium with similar electrophoretic mobilities as those released by NGF.
Adrenal Gland Neoplasms↗
Effects of SOS and MucAB functions on reactivation and mutagenesis of M13 replicative form DNA bearing bulky lesions.
We have previously determined the specificity of -1 frameshifts induced by aflatoxin-B1-2,3-dichloride (AFB1C12) in phage M13 double-strand replicative form (RF) DNA. The system consists of: (i) in vitro adduction of RF DNA of BK8, a lacZ + 1 frameshift derivative of phage M13mp8; (ii) transfection into unirradiated or UV-irradiated bacterial host cells; (iii) scoring and sequencing of revertants (i.e., -1 frameshifts). The previous data had shown that induction of SOS functions enhanced mutagenesis. However, this increase in mutagenesis is not accompanied by enhanced survival in a majority of the strains tested. Here, we present evidence to show that the lack of SOS reactivation is a specific property of the RF DNA system rather than a specific property of the lesion. A model mechanism based on the replicative strategy of transfected RF DNA can account for these observations. In addition, we have calculated individual Weigle mutagenesis factors at 8 major mutagen induced sites reported previously. Analysis of these data indicates that, within a restricted subset of possible mutational events (i.e., -1 frameshifts), Weigle mutagenesis is affected by both the DNA sequence environment of the mutation site as well as the repair phenotype of the cell.
Aflatoxin B1↗
Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride.
In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives. BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing. The -1 frameshift (BK8) results suggest the following. (1) The E. coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required. The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis. The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage. (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution. Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage. The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct. The frequency of these complex mutations is significantly enhanced by mucAB activity. In this system, recA activity is required for generation of significant levels of complex mutations. An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts. Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution. All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites. Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2. To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.
Aflatoxin B1↗
DNA replication-blocking properties of adducts formed by aflatoxin B1-2,3-dichloride and aflatoxin B1-2,3-oxide.
The carcinogen aflatoxin B1 (AFB1), upon activation to a hypothesized AFB1-2,3-oxide (AFB1-oxide), reacts with DNA guanines. Aflatoxin B1-2,3-dichloride (AFB1-Cl2) was originally synthesized as an electronic analog for the putative AFB1-oxide, which has never been isolated due to presumed reactivity. We have previously shown that AFB1-oxide reacts with base-paired DNA guanines in a sequence-specific manner, as revealed by an alkali-degradation analysis. On the basis of a replication-block analysis, we have shown that AFB1-Cl2 reacts with single-stranded DNA preferentially at inverted repeat sequences, which were suggested to be capable of forming intrastrand base-paired structures. Here, we present data to show the following. Both AFB1-oxide and AFB1-Cl2 react with guanines in double-stranded DNA to induce similar sequence-specific, alkali-labile sites. Reactivity with partial DNA duplexes as well as the use of single-strand specific chemical probes directly demonstrates that AFB1-Cl2, like AFB1-oxide, prefers base-paired guanines over non-base-paired guanines. DNA replication block patterns induced by AFB1-oxide are essentially similar to those induced by AFB1-Cl2. Unexpectedly, and unlike other tested DNA lesions, Mn2+ does not appear to affect the template blocking properties of the adduct formed by AFB1-Cl2 or AFB1-oxide. The sites for replication stoppage as well as the lack of a Mn2+ effect on adducted templates have implications for the mechanisms of mutagenesis by activated AFB1.
Aflatoxin B1↗
Sequence context effects in DNA replication blocks induced by aflatoxin B1.
The genotoxic effects of the potent mutagenic carcinogen aflatoxin B1 (AFB1) are believed to be mediated by its reaction with the N-7 atom of guanine residues in DNA. We have analyzed the effect of AFB1-induced chemical modification on the template function of single-stranded DNA in vitro. The experimental strategy involves the elongation of a primer on a modified template by Escherichia coli DNA polymerase I (large fragment) and analysis of the products by high-resolution gel electrophoresis. Our data show that (i) AFB1 induces specific replication blocks one nucleotide 3' to the sites of occurrence of guanine residues on template DNA; (ii) AFB1-induced replication blocks occur predominantly at sequences capable of participation in intrastrand base pairing; (iii) within the intrastrand base-paired regions there are strong sequence context effects, in accordance with the previously described [Muench, K. F., Misra, R. P. & Humayun, M. Z. (1983) Proc. Natl. Acad. Sci. USA 80, 6-10] specificity "rules" that apply to the reaction of AFB1 with guanine residues in double-stranded DNA; (iv) there is evidence that the (7-guanyl)-AFB1 adducts as well as secondary derivatives such as the formamidopyrimidine-AFB1 act as replication blocks. In summary, these data suggest that previously observed inhibition of DNA replication and transcription by AFB1 is directly attributable to (7-guanyl)-AFB1 adducts or their secondary reaction products.
Aflatoxin B1↗