Oxidation of cysteine-322 in the repeat domain of microtubule-associated protein tau controls the in vitro assembly of paired helical filaments.

Abstract

One of the hallmarks of Alzheimer disease is the pathological aggregation of 7 protein into paired helical filaments (PHFs) Several diseases are characterized by the pathological aggregation of a protein into fibers. This includes Alzheimer disease (AD), an age-related dementia. Brains of AD patients contain two types of deposits, the amyloid plaques and the neurofibrillary aggregates [tangles, paired helical filaments (PHFs)]. The former consists mainly of the proteins A,B (the derivative of a membrane protein, APP); the latter consists of the microtubule-associated protein T (reviewed in refs. 1 and 2). Analyzing the disease requires an understanding of the aggregation processes of these proteins. In the case of amyloid fibers the A,B peptide self-associates by forming intermolecular interactions between ,3-pleated sheets. The fibers show a typical cross-,B structure (3, 4), and they can be stained with dyes that are thought to interact preferentially with (3-stranded proteins, such as Congo red or thioflavin S (5, 6).The second hallmark of AD is the neurofibrillary tangles (NFTs) that consist of PHFs; they also stain with thioflavin S and (to a lesser extent) Congo red, thus suggesting a 1B substructure (6-8). In a previous study it was therefore unexpected to find that neither the fibers nor the subunit protein, T, reveals secondary structure but rather behaves like a random-coil denatured protein (9). The observations suggest that the aggregation of X-is based on some principle other than ,3-strand interactions. In addition to aggregation, T protein in AD is also extensively phosphorylated, especially at Ser-Pro motifs (review, ref. 10), and it has been suspected that this modification was related to the assembly into PHFs. Thus another unexpected finding was that PHFs can be reassembled in vitro from fragments of T protein that lacked most of the phosphorylation sites and aggregated independently of phosphorylation (11). These studies also showed that the "repeat" domain of Tcan form antiparallel dimers and that synthetic PHFs assemble from dimers that are chemically crosslinked with N,N'-phenylenedimaleimide (PDM) via sulfhydryls, implying that these links can be important in generating AD PHFs.With these observations in mind, we initiated a study on the role of i-s sulfhydryl groups in PHF assembly and show here that the formation of synthetic PHFs depends on the intermolecular crossbridging of Cys-322. The results support the notion that an increase in the redox potential of aging neurons is responsible for the aggregation of PHFs, independently or in addition to the disequilibrium between protein kinases and phosphatases that leads to the concomitant hyperphosphorylation. Thus, the increase in redox potential and an excess protein oxidation found in aging brain (12) would provide a link for the aggregation of both types of AD fibers since the amyloid fibers also appear to be related to increased oxidation (13,14).

Neuronal expression of myeloperoxidase is increased in Alzheimer's disease

“…For example, oxidation of the methionine residue in b-amyloid has been shown to alter aggregation kinetics of the peptide (Hou et al 2002;Palmblad et al 2002). Similarly, oxidative modification of tau protein can increase in vitro assembly into paired helical filaments (Schweers et al 1995;Gamblin et al 2000). Increased levels of several oxidation products have been found in AD brain including protein carbonyls (Smith et al 1998), o,o¢-dityrosine (Hensley et al 1998), AGEs (Vitek et al 1994;Yan et al 1994), 3-nitrotyrosine , lipid oxidation products Pratico et al 1998), and oxidized DNA (Mecocci et al 1994;Gabbita et al 1998).…”

Phosphorylation of Microtubule-associated Proteins MAP2 and MAP4 by the Protein Kinase p110mark

“…This might explain the predominant role of the phosphorylation in repeat 1 of tau, compared with the cooperation between 1 and 3 in MAP2 or between 1 and 4 in MAP4. There is increasing evidence for a folded structure in the repeats: the two cysteines in repeats 2 and 3 of tau are in close proximity (Schweers et al, 1995), and the reaction of certain antibodies can only be explained by discontinuous epitopes involving a folded repeat domain . Interestingly, the accessibility of the core of Alzheimer PHFs to proteases is also explained if one assumes that the repeats of tau are folded up; the result is that the resistant core is formed by peptides roughly equivalent to three repeats, but in different combinations (end of 1 plus 3, 4, 4a, or end of 1 plus 2, 3, 4, Jakes et al (1991)).…”

Cysteine Oxidation of Tau and Microtubule-associated Protein-2 by Peroxynitrite

“…Sulfhydryl oxidation of tau has been suggested as a possible early step in the formation of paired helical filaments (16,17). For example, cysteine 322 in the microtubule-binding domain of human tau has been identified as a residue capable of forming disulfide cross-links (16). In a model suggested by Schweers et al (16), tau-tau disulfide-linked dimers form first, and then the dimers serve as a template for noncovalent paired helical filament formation.…”

“…Tau isolated from Alzheimer disease brains no longer binds to microtubules; thus, microtubule stability in neurons is compromised. Sulfhydryl oxidation of tau has been suggested as a possible early step in the formation of paired helical filaments (16,17). For example, cysteine 322 in the microtubule-binding domain of human tau has been identified as a residue capable of forming disulfide cross-links (16).…”