Alzheimer Disease-specific Conformation of Hyperphosphorylated Paired Helical Filament-Tau Is Polyubiquitinated through Lys-48, Lys-11, and Lys-6 Ubiquitin Conjugation

Abstract

One of the key pathological hallmarks of Alzheimer disease (AD)is the accumulation of paired helical filaments (PHFs) of hyperphosphorylated microtubule-associated protein Tau. Tandem mass spectrometry was employed to examine PHF-Tau post-translational modifications, in particular protein phosphorylation and ubiquitination, to shed light on their role in the early stages of Alzheimer disease. PHF-Tau from Alzheimer disease brain was affinity-purified by MC1 monoclonal antibody to isolate a soluble fraction of PHF-Tau in a conformation unique to human AD brain. A large number of phosphorylation sites were identified by employing a data-dependent neutral loss algorithm to trigger MS3 scans of phosphopeptides. It was found that soluble PHF-Tau is ubiquitinated at its microtubule-binding domain at residues Lys-254, Lys-311, and Lys-353, suggesting that ubiquitination of PHF-Tau may be an earlier pathological event than previously thought and that ubiquitination could play a regulatory role in modulating the integrity of microtubules during the course of AD. Tandem mass spectrometry data for ubiquitin itself indicate that PHF-Tau is modified by three polyubiquitin linkages, at Lys-6, Lys-11, and Lys-48. Relative quantitative analysis indicates that Lys-48-linked polyubiquitination is the primary form of polyubiquitination with a minor portion of ubiquitin linked at Lys-6 and Lys-11. Because modification by Lys-48-linked polyubiquitin chains is known to serve as the essential means of targeting proteins for degradation by the ubiquitin-proteasome system, and it has been reported that modification at Lys-6 inhibits ubiquitin-dependent protein degradation, a failure of the ubiquitin-proteasome system could play a role in initiating the formation of degradation-resistant PHF tangles.The major pathological hallmarks of Alzheimer disease (AD) 3 are the extracellular formation of senile plaques composed of the amyloid ␤ peptide and the intraneuronal formation of neurofibrillary tangles (NFTs), which have a degradation-resistant core made up of paired helical filaments (PHFs) of the microtubule-associated protein Tau (PHF-Tau). In the AD brain, the degree of NFT formation has been shown to correlate more closely to the loss of neuronal function than the degree of senile plaque accumulation (1). A recent study by SantaCruz et al. (2) has shown, however, that the existence of NFTs alone does not cause neuronal death, implying that a pre-tangle form of Tau may be responsible for the neuronal loss and other pathological symptoms characteristic of disorders involving the Tau protein (tauopathies), including AD.Tau was initially discovered as a phosphoprotein that promotes assembly of microtubules (3); it was later found that hyperphosphorylated Tau is the major protein comprising the PHFs in AD (4, 5). Because hyperphosphorylated Tau is found in all other tauopathies in addition to AD (6), interest then developed in the role that phosphorylation may play in the formation of PHFs and the development of NFTs.The stabilization of ...

Ube2s regulates Sox2 stability and mouse ES cell maintenance

“…Recently, several studies profiled the polyubiquitin signals in neurodegenerative diseases, such as Alzheimer's disease, and found that although the K11 linkage only accounts for a very small percentage of the total ubiquitination content in normal mammalian cells, its accumulation is markedly increased in the neurodegenerative disorder samples. [13][14][15] This correlation highlights a putative role of key enzymatic machineries responsible for K11 polyubiquitin assembly in the process of neurogenesis, which is further supported by the observation that anaphase-promoting complex (APC), an E3 complex governing K11 polyubiquitination, regulates neuronal morphogenesis and differentiation. 16 Of note, Sox2 is highly expressed in the neurogenetic tissues, such as the hippocampus and central canal, and has a critical role in unperturbed neurogenesis.…”

Deletion of the Ubiquitin Ligase CHIP Leads to the Accumulation, But Not the Aggregation, of Both Endogenous Phospho- and Caspase-3-Cleaved Tau Species

“…Previously, our group and others demonstrated that CHIP specifically bound to tau within the microtubule binding region, and other reports revealed that tau is indeed polyubiquitinated within and around this functional domain of the tau protein (Morishima-Kawashima et al, 1993;Cripps et al, 2006). Here, using CHIP (chn-1 in C. elegans) RNAi and CHIP Ϫ/Ϫ mice, we demonstrate that CHIP is essential for the in vivo degradation of phospho-tau species, perhaps indicating that tau must first be released from the microtubule to become a CHIP substrate.…”

“…Very recently, it was demonstrated that aggregated tau species isolated from AD brain tissue by immunoprecipitation with a conformationspecific antibody were, in fact, polyubiquitinated at lysine residues near or within the microtubule binding region (Cripps et al, 2006), suggesting that these sites are not only critical for tau degradation but perhaps have a role in microtubule dynamics. These findings correlate well with the recent finding that CHIP levels were elevated in AD samples relative to control tissue (Sahara et al, 2005), further suggesting that CHIP plays an important role in mature tangle formation.…”

Tau-Based Treatment Strategies in Neurodegenerative Diseases

“…173 PHF-tau has been shown to be ubiquitinated at least at four different sites, and through different lysine linkages on ubiquitin. 175,176 Tau is predominantly monoubiquitinated, and only a minor part of PHF-tau is linked to polyubiquitin chains by Lys42. Conjugation sites for monoubiquitination have been identified within the repeat region.…”