RESUMO
The genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) were identified as the genes that harbour mutations that cause more than 60% of early onset familial Alzheimer's disease cases (FAD) (1-3). So far, more than 40 missense mutations have been described for presenilin-1 and two have been found in the gene coding for presenilin-2 (reviewed in refs. 4 and 5). Carriers of mutated presenilin genes develop in their brain neuropathological changes characteristic of Alzheimer's disease including the deposition of amyloid Aß peptide. The latter is released from its cognate amyloid precursor protein (APP) by a two-step proteolytic conversion: first, proteolysis of APP by ß-secretase, which releases the N-terminus of Aß, and second, conversion of the remaining fragment by γ-secretase, which cleaves within the predicted transmembrane region of APP. This releases the C-terminus of Aß, which may end either at position 40 or, to a lesser extent, at position 42 (reviewed in ref. 6). The latter species, Aß(1-42), is more prone to aggregation and deposition than Aß(1-40) and is produced at higher levels in the brains and primary fibroblasts of FAD patients carrying PS missense mutations (7). The same result was obtained when cultured cells transfected with mutated PS1 orPS2, or transgenic mice harboring missense PS1 were analyzed for the production of Aß(1-42): in every case increased amounts of the longer Aß(1-42) species were observed (8-10). The mechanisms by which mutations in the PS genes affect the proteolytic processing of APP by γ-secretase have not been resolved in detail. There are two possibilities by which the normal processing of APP may be disturbed: either mutations in the presenilins affect APP metabolism in an indirect way by modulation of proteases or interaction with proteins involved in APP intracellular routing, or presenilins may modulate APP processing directly through physical interactions with APP. Such a direct interaction between presenilins and APP was first demonstrated by us for PS2 (11). Later on, formation of stable complexes with APP was reported not only for PS2 but also for PS1 (12,13,13a).
RESUMO
Alzheimer's disease is characterized by neurodegeneration and deposition of betaA4, a peptide that is proteolytically released from the amyloid precursor protein (APP). Missense mutations in the genes coding for APP and for the polytopic membrane proteins presenilin (PS) 1 and PS2 have been linked to familial forms of early-onset Alzheimer's disease. Overexpression of presenilins, especially that of PS2, induces increased susceptibility for apoptosis that is even more pronounced in cells expressing presenilin mutants. Additionally, presenilins themselves are targets for activated caspases in apoptotic cells. When we analyzed APP in COS-7 cells overexpressing PS2, we observed proteolytic processing close to the APP carboxyl terminus. Proteolytic conversion was increased in the presence of PS2-I, which encodes one of the known PS2 pathogenic mutations. The same proteolytic processing occurred in cells treated with chemical inducers of apoptosis, suggesting a participation of activated caspases in the carboxyl-terminal truncation of APP. This was confirmed by showing that specific caspase inhibitors blocked the apoptotic conversion of APP. Sequence analysis of the APP cytosolic domain revealed a consensus motif for group III caspases ((IVL)ExD). Mutation of the corresponding Asp664 residue abolished cleavage, thereby identifying APP as a target molecule for caspase-like proteases in the pathways of programmed cellular death.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Caspases/metabolismo , Citoplasma/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animais , Apoptose/efeitos dos fármacos , Células COS , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Humanos , Hidrólise , Células Jurkat , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismoRESUMO
Mutations in the presenilin genes are associated with early onset familial Alzheimer's disease and lead to increased accumulation of beta A4 peptide, the proteolytic product of the amyloid precursor protein (APP). To test whether presenilins interfere with APP metabolism, presenilin-2 (PS2) was coexpressed with APP in mammalian cells. Analysis of PS2 immunoprecipitates revealed that a fraction of APP was associated with the PS2 immunocomplexes. This non-covalent association was specific for the APP family of proteins and restricted to immature forms, occurring probably during transit through the endoplasmic reticulum. Additionally, coexpression with PS2 resulted in a decrease of APP secretion, suggesting a direct participation of presenilins in the intracellular sorting, trafficking and processing of APP molecules.
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Proteínas de Membrana/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Presenilina-2 , Ligação Proteica , TransfecçãoRESUMO
Amyloid precursor-like protein 1 (APLP1) represents an integral membrane type 1 protein of unknown function which was originally cloned from a mouse cDNA library on the basis of sequence similarity with the Alzheimer's amyloid precursor protein (APP). Here we report on the molecular cloning and expression of the human APLP1 (hAPLP1). hAPLP1 consists of 650 amino acids, displays 89% identity on the amino acid level to its mouse homologue and has a calculated molecular mass of 72 kDa. hAPLP1 synthesized in a cell-free system displays an apparent molecular mass of approximately 80 kDa in SDS-containing gels and becomes N-glycosylated when the in vitro translation is performed in the presence of microsomes. The hAPLP1 cDNA was also expressed ectopically in COS-7 cells and the protein expression was analyzed by immunoprecipitation and western blotting. We have demonstrated that hAPLP1 represents a novel glycoprotein which carries both N- and O-linked glycans. Moreover, hAPLP1 undergoes limited proteolysis which results in the secretion of the carboxy-terminal truncated molecule into the cells conditioned medium. Examination of cells transfected with hAPLP1 cDNA by confocal laser microscopy reveals an intense perinuclear and Golgi staining, a pattern resembling the subcellular distribution of APP. Using a novel hAPLP1-specific antiserum, we identified soluble hAPLP1 in the human cerebrospinal fluid, which suggests that secretion of hAPLP1 from brain cells also takes place in vivo.