The Core of Tau-Paired Helical Filaments Studied by Scanning Transmission Electron Microscopy and Limited Proteolysis

Abstract

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of fulllength molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of ∼60 kDa/nm and filaments from full-length tau (htau40∆K280 mutant) have ∼160 kDa/nm, compared with ∼130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.Alzheimer's disease is characterized by the coexistence of two different amyloid deposits in the brain, the extracellular senile plaques composed of fibrils of A peptides and the intracellular paired helical filaments (PHFs) 1 consisting mostly of the microtubule-associated protein tau. Tau is expressed in the adult brain in six isoforms which differ by the presence of two inserts in the N-terminal part (exons 2 and 3) and the second repeat R2 (exon 10) in the repeat domain (Figure 1). The physiological function of tau is to bind and stabilize axonal microtubules. The soluble form of tau has a mostly random coil conformation (1, 2) and thus belongs to the emerging group of natively unfolded proteins (3)(4)(5). Whereas the aggregation of the A peptide can be explained in part by its hydrophobic character, this cannot by applied to tau, which has an overall hydrophilic character and is unusually well soluble. However, tau contains short motifs in the repeat domain which are weakly hydrophobic.

Identification of the Core Structure of Lysozyme Amyloid Fibrils by Proteolysis

“…This interpretation is similar to that given in the recent analysis of the fibril morphology of fragments of tau protein as compared to that of the whole protein molecule. 71 The results of this study are in line with similar observations indicating that the entire sequence of a protein molecule need not be incorporated in the amyloid core structure. 31,33,36,37,49,50,72 For example, only the central region 31-109 of the 140 amino acid residue chain of α-synuclein is involved in the fibrillar β-sheet core, as deduced from proteolysis experiments with proteinase K, 50 and in the case of the Aβ(1-40) peptide, some 12 to 16 residues of the N-terminal region of the peptide appear to be excluded from the fibril structure and therefore prone to proteolysis.…”

Self-assembly of Bacteriophage-associated Hyaluronate Lyase (HYLP2) into an Enzymatically Active Fibrillar Film

“…7). It also suggests that the entire sequence of the protein molecule is not incorpo- rated in the fibrillar core structure and is well supported by earlier studies made on Tau protein and ␣-synuclein (35,43).…”

Understanding the Kinetic Roles of the Inducer Heparin and of Rod-like Protofibrils during Amyloid Fibril Formation by Tau Protein

“…There has been some debate about whether heparin is a major constituent of fibrils (30,43,44). The observation made in this study that the final fibrils contain less than one heparin molecule for every 20 protein molecules (see "Results") is the strongest evidence so far that heparin is only a minor constituent of fibrils.…”

“…8), fibril growth occurs by monomer addition to the aggregation-competent dimer, and heparin is not assigned any role in fibril elongation. On the other hand, if elongation were to occur by linear addition of either PHP or PH, then the observations here (see below) and elsewhere (30,43) that heparin is not an integral part of the fibril structure would require that heparin dissociate from the growing fibril once its role in elongation is over. This would happen, for example, if growth of the fibril is accompanied by conformational changes in the assembled Tau molecules, which lead to the dissociation of heparin from the fibrils.…”