Caracterización de las proteínas asociadas al microtúbulo conocidas com factor TAU

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Biología Molecular. Fecha de lectura: 10-07-198

In vitro conditions for the self-polymerization of the microtubule-associated protein, tau factor

One of the microtubule associated proteins, tau factor, that appears associated to the paired helical filaments of Alzheimer's disease presents, by itself, after urea treatment, the ability of polymerizing invitro as tested by immunoelectronmicroscopy. These polymers resemble in their width and appearance those of the paired helical filaments. The conditions required for this assembly have been studied and determined the protein concentration needed, the influence of salt concentration and pH as well as the possible modifications (deamination, acylation) which may be implied in such in vitro polymerization.Peer reviewe

Self assembly of microtubule associated protein tau into filaments resembling those found in alzheimer disease

Microtubule associated protein tau factor self-assembles into filamentous structures resembling the paired helical filaments found in Alzheimer disease. Tau polymerization requires of a previous modification; conversion of glutamine into glutamic acid by deamination.Peer reviewe

Altered levels of microtubule proteins in brains of Alzheimer's disease patients

The amount of microtubule protein present in the total soluble protein from brains of Alzheimer's disease patients and from brains of non-Alzheimer age-matched controls, were determined by radioimmunoassay. No differences were found in the amount of tubulin or microtubule-associated protein MAP2 present in either group. However, the amount of tau protein or MAP1 from the brains of Alzheimer's disease patients was about half of that present in their control counterparts.Supported by Grants from the Comisión Asesora para el Desarrollo Tecnológico y Científico and Fondo de Investigaciones Sanitarias.Peer reviewe

The interaction between a Na+-channel toxin and brain microtubule proteins in vitro

The aim of this work was to characterize the interaction of the Na+-channel toxin, purified from venom of the scorpion Leiurus quinquestriatus, with microtubule proteins in vitro. The toxin enhanced microtubule assembly, causing the formation of microtubule ‘bundles’. It interacted with the Na+-channel 270 kDa subunit and was subsequently found to be unrelated to high molecular weight microtubule-associated protein with respect to apparent molecular weight and toxin binding. However, the radiolabelled toxin bound to tubulin, although with a much lower affinity than that published for the reconstituted Na+-channel. This binding appears to occur in the carboxy terminal 4 kDa region of tubulin. These results may reflect a secondary action of the toxin involved in binding to its receptors in the neural plasma membrane.Alan Hargreaves thanks the Wellcome Trust for generous financia! support during the course of most of this work. The project was funded by the Comisión Asesora para el Desarrollo de la Investigación Científica y Técnica. E.M.G. was supported by Fundación 'San Pablo' (CEU).Peer reviewe

Quantitation and characterization of tau factor in porcine tissues

Using a monospecific antibody against brain tau factor purified by affinity chromatography, we have studied the distribution of tau factor or related polypeptides in different cells. The presence of tau in all cell types tested was demonstrated by a radioimmunoassay. Tau factor-related proteins were found in liver, spleen, pancreas, kidney and lung, although at much lower levels than that found in neural cells. In all cases, they copolymerized with tubulin and were heat-resistant. When the distribution of tau factor-related proteins was studied by Western blotting, tau factor antiserum reacted against peptides with an electrophoretic mobility that was similar to those of brain tau factor peptides. Immunofluorescence studies have also been performed with the same antibody to determine the distribution of tau factor-related peptides in PK15 cells. Our results indicated that these peptides were associated to the microtubule network.This work was supported in part by grants from Comision Asesora para el Desarrollo de la Investigacion Cientifica y Tecnica and Fondo de Investigaciones Sanitarias de la Seguridad Social. E.M.G. was supported by Fundacion 'San Pablo' (CEU).Peer reviewe

Characterization of tau protein present in microtubules and paired helical filaments of Alzheimer's disease patient's brain

Two proteins immunologically related to porcine tau protein are found in the brain of Alzheimer's disease patients. One is bound to microtubules and, after isolation by co-polymerization with tubulin, shows a size and tryptic peptide map, similar to the microtubule-associated tau protein, present in the brain of non-demented patients. The other tau-related protein is present as the major protein of a purified fraction of paired helical filaments. The paired helical filament-associated protein shows smaller molecular weight (33,000) than microtubule-associated tau; however, this 33,000 mol. wt protein reacts with a monospecific anti-tau antibody and with an antibody to a 19-amino acid peptide corresponding to amino acids 228–246 of human tau. Furthermore, the 33,000 mol. wt protein and the tau protein have similar tryptic peptide maps. These results suggest that the paired helical filament protein is a modified form of the microtubule-associated tau protein.This work was supported by grants from the Comision Asesora para el Desarrollo Tecnologico y Cientifico, Fondo de lnvestigaciones Sanitarias and Boehringer Ingelheim.Peer reviewe

Collagenous structures present in brain contain epitopes shared by collagen and microtubule-associated protein tau

A novel type of collagenous fibers has been isolated from human brain and characterized by electron microscopy and optical diffraction. It was found that the morphology of the fibers is similar, but not identical, to that of skin collagen. Also, the collagenous fibers show some similarities with the paracrystals that could be assembled in vitro from purified microtubule-associated protein tau. Immunological analyses indicated the presence of epitopes in these collagenous fibers which react with antibodies against collagen and tau.This work was supported by grants from Comision Asesora de Investigacion Cientifica y Tecnica, Spain, and from Boehringer Ingelheim.Peer reviewe

Localization of the tubulin binding site for tau protein

Limited proteolysis of tubulin with subtilisin resulted in the removal of the carboxyl-terminal moiety of tubulin subunits. The remaining peptides from both α and β tubulin lacking the carboxyl terminal did not bind to tau factor nor to MAP2 or MAP1. The carboxyl-terminal fragments bind to tau factor and MAP2 and both compete for the same binding sites in the tubulin molecule. Our results suggest that the carboxyl-terminal region of tubulin is a regulatory domain for the assembly of tubulin and the site for interaction with MAPs.Peer reviewe