RESUMEN
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.
Asunto(s)
Péptidos beta-Amiloides/genética , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide , Sitios de Unión , Western Blotting , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Fibroblastos/metabolismo , Sustancias de Crecimiento/farmacología , Datos de Secuencia Molecular , Plásmidos , Inhibidores de Proteasas/metabolismo , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacología , Mapeo RestrictivoAsunto(s)
Enfermedades del Sistema Nervioso/enzimología , Proteínas Quinasas/metabolismo , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Supervivencia Celular/fisiología , Humanos , Isquemia/enzimología , Isquemia/patología , Excitación Neurológica/patología , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/fisiopatología , Fosforilación , Proteínas Quinasas/fisiologíaRESUMEN
The activity of protein tyrosine kinase was determined in extracts from Alzheimer's disease brains and age- and postmortem time-matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p less than 0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine-positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismo , Anciano , Enfermedad de Alzheimer/patología , Western Blotting , Humanos , Inmunohistoquímica , Fosforilación , Fosfotirosina , Tirosina/análogos & derivadosRESUMEN
Human platelets can be stimulated by thrombin or ionomycin to secrete soluble truncated amyloid beta-protein precursor and particulate membrane fragments which contain C-terminal and N-terminal immunoreactive amyloid beta-protein precursor. This suggests a possible circulating source of beta-protein in serum which may play a role in the formation of amyloid deposits. The release of soluble amyloid beta-protein precursor could be involved in normal platelet physiology.