ABSTRACT
Most cases of early-onset familial Alzheimer's disease (FAD) are caused by mutations in the genes encoding the presenilin 1 (PS1) and PS2 proteins, both of which undergo regulated endoproteolytic processing. During apoptosis, PS1 and PS2 were shown to be cleaved at sites distal to their normal cleavage sites by a caspase-3 family protease. In cells expressing PS2 containing the asparagine-141 FAD mutant, the ratio of alternative to normal PS2 cleavage fragments was increased relative to wild-type PS2-expressing cells, suggesting a potential role for apoptosis-associated cleavage of presenilins in the pathogenesis of Alzheimer's disease.
Subject(s)
Alzheimer Disease/genetics , Apoptosis , Caspases , Cysteine Endopeptidases/metabolism , Membrane Proteins/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amino Acid Chloromethyl Ketones/pharmacology , Amino Acid Substitution , Animals , Caspase 3 , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Activation , Etoposide/pharmacology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Oligopeptides/pharmacology , Phosphorylation , Presenilin-1 , Presenilin-2 , Rats , Staurosporine/pharmacology , Tumor Cells, CulturedABSTRACT
Ferritin mRNAs are translationally regulated by the binding of either of two cytosolic proteins, iron regulatory protein 1 (IRP1) or IRP2, to the iron responsive element (IRE) located in their 5' untranslated region (UTR). Rat liver IRP1 was purified by anion exchange, gel filtration, and affinity chromatography using a concatemerized version of the IRE. Two bands with M(r) of 95,000 and 100,000 were observed by reducing SDS-PAGE. A single protein was responsible for both bands since: (1) [32P]IRE RNA specifically cross-linked to both components; (2) alkylation with iodoacetamide resulted in formation of a single species with M(r) of 95,000; and (3) they possessed identical peptide patterns after digestion with cyanogen bromide. The N-terminal sequence of rat liver IRP1 was MKNPFAHLAEPLDPAQPGKKFNLNKLEDSRYGRLPFXIRVLLEAAV which is identical to the sequence deduced from the cDNA. Rat liver IRP1 has an amino acid composition similar to that of bovine liver caconitase. Several species of IRP1 were observed by two-dimensional gel electrophoresis with pIs ranging from 7.5 to 8.0. Rat liver IRP1 bound the IRE with high affinity (K(D) = 0.04 nM) and repressed translation of ferritin mRNA in vitro. IRP1 bound 100-fold less well to an IRE variant and failed to significantly repress translation of a ferritin mRNA containing the mutated IRE. We conclude that decreases in the affinity of interaction between IRP1 and the IRE, of a magnitude similar to that observed when the binding protein in converted to c-aconitase, are sufficient to significantly enhance translation of ferritin mRNA in vitro.
Subject(s)
Iron-Sulfur Proteins/isolation & purification , Liver/chemistry , RNA-Binding Proteins/isolation & purification , Amino Acid Sequence , Animals , Cattle , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Ferritins/genetics , Ferritins/metabolism , Iron Regulatory Protein 1 , Iron Regulatory Protein 2 , Iron-Regulatory Proteins , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Isoelectric Point , Molecular Sequence Data , Protein Binding , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RatsABSTRACT
Mutations in the presenilin genes, PS1 and PS2, cause a major portion of early onset familial Alzheimer's disease (FAD). The biological roles of the presenilins and how their pathological mutations confer FAD are unknown. In this study, we set out to examine the processing and degradation pathways of PS2. For regulated expression of PS2, we have established inducible cell lines expressing PS2 under the tight control of the tetracycline-responsive transactivator. Western blot analysis revealed that PS2 was detected as an approximately 53-55-kDa polypeptide (54-kDa PS2) as well as a high molecular mass form (HMW-PS2). Using a stably transfected, inducible cell system, we have found that PS2 is proteolytically cleaved into two stable cellular polypeptides including an approximately 20-kDa C-terminal fragment and an approximately 34-kDa N-terminal fragment. PS2 is polyubiquitinated in vivo, and the degradation of PS2 is inhibited by proteasome inhibitors, N-acetyl-L-leucinal-L-norleucinal and lactacystin. Our studies suggest that PS2 normally undergoes endoproteolytic cleavage and is degraded via the proteasome pathway.
Subject(s)
Cysteine Endopeptidases/metabolism , Membrane Proteins/metabolism , Multienzyme Complexes/metabolism , Protein Processing, Post-Translational , Alzheimer Disease/etiology , Biopolymers/metabolism , Humans , Membrane Proteins/genetics , Octoxynol , Peptide Fragments/metabolism , Polyubiquitin , Presenilin-2 , Proteasome Endopeptidase Complex , Recombinant Proteins/metabolism , Solubility , Tumor Cells, Cultured , Ubiquitins/metabolismABSTRACT
A candidate gene for the chromosome 1 Alzheimer's disease (AD) locus was identified (STM2). The predicted amino acid sequence for STM2 is homologous to that of the recently cloned chromosome 14 AD gene (S182). A point mutation in STM2, resulting in the substitution of an isoleucine for an asparagine (N141l), was identified in affected people from Volga German AD kindreds. This N141l mutation occurs at an amino acid residue that is conserved in human S182 and in the mouse S182 homolog. The presence of missense mutations in AD subjects in two highly similar genes strongly supports the hypothesis that mutations in both are pathogenic.
Subject(s)
Alzheimer Disease/genetics , Chromosomes, Human, Pair 1/genetics , Membrane Proteins/genetics , Adult , Aged , Alzheimer Disease/ethnology , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Cloning, Molecular , DNA, Complementary/genetics , Female , Gene Expression , Germany/ethnology , Humans , Lod Score , Male , Membrane Proteins/chemistry , Middle Aged , Molecular Sequence Data , Mutation , Pedigree , Point Mutation , Presenilin-2ABSTRACT
The Alzheimer beta-amyloid precursor protein (APP) contains an ectodomain zinc binding site that has been reported to modulate the heparin affinity and protease-inhibitory properties of the molecule. This motif, GVEFVCCP, is highly conserved in amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2), as well as in the Drosophila and Caenorhabditis elegans APP-like proteins (APPL and APL-1). To determine whether the function of this domain is preserved in the human APP-like proteins, the effect of zinc in modulating the elution profile of these proteins upon heparin-Sepharose chromatography was studied. Both APLP1 and APLP2 bound heparin-Sepharose and had NaCl elution profiles similar to that of APP. As previously reported for APP, zinc increased the recovery of APLP1 and APLP2 upon heparin-Sepharose chromatography. APP, APLP1, and APLP2 all bind zinc-chelating Sepharose, indicating that the zinc binding motif may be functionally conserved in these proteins. Additionally, APP, APLP1, and APLP2 migrate at higher molecular sizes (approximately 40 kDa) on SDS-polyacrylamide gel electrophoresis than their predicted molecular sizes. We report data that compare the physicochemical properties of APP to its novel APLP homologues and indicate that these molecules behave as a family of zinc-modulated, heparin-binding proteins.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Caenorhabditis elegans Proteins , Drosophila Proteins , Helminth Proteins/metabolism , Membrane Proteins , Nerve Tissue Proteins/metabolism , Zinc/metabolism , Amino Acid Sequence , Amyloid beta-Protein Precursor/genetics , Animals , Binding Sites , Caenorhabditis elegans , Chromatography , Conserved Sequence , Drosophila , Electrophoresis, Polyacrylamide Gel , Humans , Mice , Molecular Sequence Data , Multigene Family , Sepharose/analogs & derivatives , Sepharose/metabolism , Sequence Homology, Amino AcidABSTRACT
A beta 1-40, a major component of Alzheimer's disease cerebral amyloid, is present in the cerebrospinal fluid and remains relatively soluble at high concentrations (less than or equal to 3.7 mM). Thus, physiological factors which induce A beta amyloid formation could provide clues to the pathogenesis of the disease. It has been shown that human A beta specifically and saturably binds zinc. Here, concentrations of zinc above 300 nM rapidly destabilized human A beta 1-40 solutions, inducing tinctorial amyloid formation. However, rat A beta 1-40 binds zinc less avidly and is immune to these effects, perhaps explaining the scarcity with which these animals form cerebral A beta amyloid. These data suggest a role for cerebral zinc metabolism in the neuropathogenesis of Alzheimer's disease.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Peptide Fragments/metabolism , Zinc/metabolism , Alzheimer Disease/etiology , Amyloid beta-Peptides/chemistry , Animals , Brain/metabolism , Edetic Acid/pharmacology , Humans , Kinetics , Mice , Peptide Fragments/chemistry , Rats , Solubility , Zinc/pharmacologyABSTRACT
Abnormalities of zinc homeostasis occur in Alzheimer's disease (AD), a dementia characterized by the aggregation of A beta in the brain, and in Down syndrome, a condition characterized by premature AD. We studied the binding of Zn2+ to a synthetic peptide representing residues 1-40 (A beta 1-40), as well as other domains of A beta. Two classes of Zn2+ binding were identified by 65Zn2+ labeling: highly specific pH-dependent high affinity (K(a) = 107 nM) binding, and lower affinity (K(a) = 5.2 microM) binding. Gel filtration chromatography identified monomeric, dimeric, and polymeric A beta species. Zinc induced a marked loss of A beta solubility upon chromatographic analysis. This was attributed to precipitation onto the column glass, which contains aluminosilicate, and was confirmed by the observation of zinc-accelerated precipitation of A beta by kaolin, a hydrated aluminum silicate suspension. Zinc binding also increased A beta resistance to tryptic cleavage at the secretase site, indicating that a small (<3 microM) increase in brain Zn2+ concentration could significantly alter A beta metabolism. We propose that elevated brain interstitial zinc levels may increase A beta adhesiveness and interfere with A beta catabolism. Consequently, abnormalities of regional zinc concentrations in the brains of patients with AD or Down syndrome may contribute to A beta amyloidosis in these disorders.