Ji Hyeon
Kim‡
a,
Jeeyoung
Lee‡
a,
Won Hoon
Choi‡
a,
Seoyoung
Park‡
b,
Seo Hyeong
Park
a,
Jung Hoon
Lee
b,
Sang Min
Lim
c,
Ji Young
Mun
d,
Hyun-Soo
Cho
e,
Dohyun
Han
f,
Young Ho
Suh
ab and
Min Jae
Lee
*ab
aDepartment of Biomedical Sciences, Seoul National University Graduate School, Seoul 03080, Korea. E-mail: minjlee@snu.ac.k; Fax: +82 2-744-4534; Tel: +82 2-740-8254
bDepartment of Biochemistry & Molecular Biology, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea
cConvergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seoul 02792, Korea
dNeural Circuit Research Group, Korea Brain Research Institute, Daegu 41062, Korea
eDepartment of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Korea
fProteomics Core Facility, Biomedical Research Institute, Seoul National University Hospital, Seoul 03080, Korea
First published on 17th March 2021
The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3β and CHIP, respectively. The resulting phospho–ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one–three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho–ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho–ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a “multiple-hit model,” where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies.
Tau is an extraordinarily hydrophilic (average hydropathicity of −0.87) and basic polypeptide (net pI = 8.2).8 Despite these biophysical features, in pathological states, otherwise soluble tau monomers self-assemble into β-sheet-enriched straight or paired helical filaments.9 Genetic mutations found in autosomal dominant tauopathies, e.g., G272V, P301L, V337M, and E342V, may reduce tau affinity toward microtubules, thereby contributing its discharge, self-assembly and fibrillar aggregation.10 On the other hand, evidence indicates that these mutations alone might not be sufficient to induce substantial conformational changes and that a wide range of post-translational modifications (PTMs) of tau plays a prerequisite part in the autonomous fibrillization propensity.11 Nonetheless, biochemical crosstalk among the PTMs has not yet been clearly characterized. Regarding proteostatic regulation, the primary effect of tau ubiquitylation on proteasomal degradation is a matter of controversy, partly because the disordered structure of tau enables this protein to be recognized and degraded by proteasomes even in the absence of ubiquitin.12,13 Moreover, a recent study by Arakhamia et al. on human CBD and AD brains reveals that polyubiquitin (polyUb) chains on tau are also involved in inter-protofilament packing of tau.14
Phosphorylation has been considered the rate-limiting step of tau fibrillization since hyperphosphorylated tau was identified in neurofibrillary tangles and filamentous inclusions in postmortem brain tissues from patients with AD.15 Despite this notion, pharmacological inhibition of this PTM event has resulted in only a limited benefit in various clinical trials.16 Although these negative outcomes do not necessarily invalidate the strategy of targeting tau kinases, we presumed that the results instead reflect complex consequences of tau phosphorylation coordinated with other PTMs. Here, we report an unforeseen function of tau phosphorylation: promotion of tau ubiquitylation. This sequential enzymatic process generated more readily degradable phospho–ub-tau species. By contrast, when ubiquitylation was prolonged, phospho–ub-tau became hyperubiquitylated and transformed into insoluble filament cores. We also developed a small-molecule inhibitor of tau ubiquitylation, which effectively blocked tau aggregate formation in rat primary cortical and hippocampal neurons. These data for the first time uncover the biochemical crosstalk between tau phosphorylation and ubiquitylation that determines its fates. This study also suggests a more efficient and possibly mechanism-modifying strategy for delaying the clinical onset of tauopathies by targeting tau ubiquitylation instead of phosphorylation.
As the ubiquitylation reaction time following phosphorylation was extended, the resultant tau products possessed significantly longer polyUb chains in a time-dependent manner, thereby generating tau species not entering the resolving gel (hyper-ub-tau) after a 24 h reaction (Fig. 1C). In complementary experiments, we additionally modified tau via acetylation in several combinations with phosphorylation and demonstrated that acetylation by p300 essentially did not affect tau phosphorylation or vice versa (Fig. S2A and B†). Regardless of preceding acetylation, tau phosphorylation/ubiquitylation robustly generated slowly migrating phospho–ub-tau species (Fig. 1D) in vitro. Our results clearly indicated a hitherto unidentified role of tau phosphorylation: conversion of ubiquitylation modes of tau (and its potential degradation and aggregation tendency as described below). We anticipate that tau phosphorylation occurring in the flanking regions of MBDs may lead to its conformational change, which gives CHIP greater access to Lys residues in the MBDs and promotes the covalent conjugation of extra ubiquitin moieties.
To assess the topology of polyUb chains on phospho–ub-tau, we carried out in vitro ubiquitylation reactions with either wild-type (WT) or mutant ubiquitin with a Lys to Arg (K#R) substitution. In the reactions with ubiquitin-K11R and -K63R, similar polyUb chains were generated relative to those of WT, whereas the formation of polyUb chains failed when ubiquitin-K48R was used (Fig. 2C). According to these results, the polyUb chains on phospho–ub-tau seemed to be mainly linked through the canonical degradation signal (Lys48-linked polyUb chain). This notion is consistent with other studies, showing impaired tau degradation in the presence of various proteasome inhibitors.21,22 In addition, the polyUb chains of ub-tau and phospho–ub-tau were both efficiently deubiquitylated by the catalytic activity of ubiquitin-specific protease 2 (USP2; Fig. 2D). Therefore, tau ubiquitylation is a reversible process (counterbalanced by deubiquitylation) before which the ultimate proteolysis by the proteasome is commenced.
Our data collectively indicated that phosphorylation events might be as critical as ubiquitylation for catabolic regulation of tau proteins. Several reports have shown that intrinsically disordered tau could be degraded by both ubiquitin-independent (by 20S proteasomes) and -dependent manners (by 26S).12,13 Considering that the half-life of unmodified tau is longer than that of most of the other unstructured proteins, tau may require a multistep process (phosphorylation-induced ubiquitylation) under stress conditions for more efficient 26S proteasome-mediated proteolysis. The preceding phosphorylation may mask excess positive charges of tau or alleviate structural and physical constraints for the interaction with E3 ubiquitin ligases, such as CHIP.
Fig. 3 The expanded set of ubiquitylation sites in tau after phosphorylation in vitro were identified with mass spectrometry. (A) Phosphorylation and ubiquitylation sites of recombinant tau after in vitro PTM reactions were identified by mass spectrometry (MS). Orange and red circles depict ubiquitylation sites of ub-tau (ubiquitylation only) and phospho–ub-tau (phosphorylation and subsequent ubiquitylation), respectively. Amino acid residues with single letter codes in N-domains (N1 and N2) are depicted in blue, the proline-rich domain (PRD) in purple, and microtubule-binding domain (MBD) repeats 1 to 4 (R1–R4) in red. Phosphorylation and acetylation sites are indicated with green and blue circles, respectively. Residues modified by both acetylation and ubiquitylation are Lys259 and Lys274 (gold and italic). Black underlining highlights four Lys–Ile/Cys–Gly–Ser motifs. (B) PTMs are pointed out on a schematic diagram of tau40. Color schemes are depicted as in (A). Although phosphorylation was abundant on the PRD, ubiquitylation occurred mostly on the MBD. (C) A distinct change in relative abundance and modification sites of tau ubiquitylation following phosphorylation was revealed by quantitative MS. Degrees of upregulation (red) are explained by the color key of the heat map. Pre-existing ubiquitylation sites in ub-tau and de novo sites in phospho–ub-tau are denoted by the orange and red text, respectively. Fold-inductions (from ub-tau to phospho–ub-tau) of ubiquitin signal intensities at the indicated Lys residues are shown. The red lines indicate the core structure comprising the tau fibrils in corticobasal neurodegeneration (CBD; ref. 14). |
We hypothesized that these migration-resistant tau species might represent less soluble tau filaments. To directly assess the effect of tau hyperubiquitylation on tau fibrillization, we first used thioflavin T (ThT), which intercalates into β-sheet structures of self-assembled multimeric tau23 without aggregation inducers such as anionic heparin. Intact tau alone did not generate any ThT-positive signals even after 4 d, but, in contrast, hyper-ub-tau proteins emitted significantly stronger fluorescence signals than those of phospho-tau or ub-tau (Fig. 4B). ThT signals of hyper-ub-tau began to be prominent after ∼24 h ubiquitylation reactions and gradually increased until time point 96 h in the reactions. A possible explanation for this observation is the conversion of hyper-ub-tau to the nucleate for tau self-assembly. When we fractionated tau species depending on molecular weights with size-exclusion chromatography and examined their transmission electron microscopy (TEM) images and in vitro seeding activity, we found that fractionated tau species with >250 kDa-size, but not the fractions containing monomeric tau, have clear fibrillar morphologies and potent transformation ability of the monomeric tau into aggregates (Fig. S5A–C†). These data strongly support the notion that hyper-ub-tau not only acts as a nucleate for pathologic tau aggregates but also undergoes facilitated self-assembly into early intermediates of insoluble aggregates. Consistently, a filter trap assay of diverse tau species revealed that hyper-ub-tau proteins were distinctively less membrane-penetrating, compared with other tau species, even in the absence of aggregation inducers (Fig. 4C). Other components of the PTM reactions, such as UBA1 (E1) and CHIP (E3) enzymes, were not trapped in the cellulose acetate membrane (Fig. S5B†).
We have further explored the role of phosphorylation-induced tau ubiquitylation by modifying the four key Lys resides (K321, K343, K353, and K375; Fig. 3), which were identified from the MS analysis. We found that single, double, or triple substitution of these sites to Arg generated little changes in the biochemical behaviors of tau proteins upon sequential phosphorylation and ubiquitylation (data not shown). However, purified tau variants with quadruple Lys mutations (tau-4KR) showed significant reduced in vitro aggregation propensities, observed by ThT analysis or filter trap assays (Fig. 4D and E). These data strongly suggest that the hyperubiquitylation of tau, rather than phosphorylation, may be critical for its fibrillization.
Complementing the biochemical experiments, we examined intact and modified tau proteins by negative-staining TEM and found that hyperubiquitylated tau formed narrower and shorter filament bundles than those of phospho-tau or ub-tau (Fig. 4F). Intact tau did not fibrillize without aggregation inducers, and phospho-tau formed mainly amorphous aggregates. This observation is similar to the results of coarse-grained modeling, where tau phosphorylation facilitates the formation of amorphous aggregates instead of amyloid fibrils.24 The short fibrils might function as a protofilamental seed for further tau aggregation. Taken together, our in vitro data strongly indicate the mediatory function of tau hyperubiquitylation in the conformational transition of tau from a soluble monomer to oligomers and eventually molecular seeds for the pathological self-assembly. Via crosstalk with other PTMs, tau may be differentially ubiquitylated and aggregated into distinct conformations of a tau filament (tau strains), thereby potentially determining the biochemical or symptomatic phenotype of diverse tauopathies.
CHIP functions as a critical quality controller of cellular proteome implicated in diverse human diseases,29 but, to dates, small-molecule inhibitors suppressing the catalytic activity of CHIP remain essentially unexplored. To validate the role of CHIP in tau hyperubiquitylation and aggregation both in vitro and in vivo, we conducted a biophysical screening of more than 500 curated small-molecule fragments, which was based on the NMR analysis implemented with a saturation transfer difference (STD) protocol (Fig. 5A; see Methods). The fragments that directly bind to purified CHIP were chemically combined and further modified to strengthen the binding affinities. Among them, we have confirmed that compound #153 effectively and specifically blocked the CHIP-mediated ubiquitylation of inhibited proteasomes (IC50 = ∼85 μM; Fig. S6†).30 When their activity was assessed in our standard in vitro tau ubiquitylation reactions, we found that compound #153, but not its structural control #154, had a strong inhibitory effect toward CHIP-mediated tau ubiquitylation (Fig. 5B and C). This CHIP inhibitor also drastically reduced the levels of hyper-ub-tau species that were generated by sequential in vitro phosphorylation/ubiquitylation and were non-migratable to the resolving gel (24 and 48 h; Fig. 5D, lanes 7 and 8). Compound #153 (50 μM) had little effect on tau phosphorylation and manifested no noticeable cytotoxicity even when combined with MG132 (10 μM) for 6 h (Fig. 5E, F, and S7A†).
We then proceeded to apply the small molecule to live cells to determine whether the suppression of tau ubiquitylation by CHIP might delay the process of tau aggregation into the insoluble fractions. After transient overexpression of tau in immortalized mouse hippocampal HT22 cells, they were treated with MG132 and OA in the presence or absence of compound #153, for 6 h. A gel-based analysis and subsequent quantification showed markedly reduced levels of monomeric and oligomeric tau from the insoluble fraction of WCLs with compound #153 (Fig. 6A and B). Similar effects of the CHIP inhibitor were observed in nonneuronal HEK293 cells (Fig. S7B†). Next, we isolated rat primary cortical cells and treated them with compound #153 along with OA and/or MG132. We found that the levels of endogenous tau, both phosphorylated and non-phosphorylated forms, in the insoluble fraction were significantly reduced in the presence of the CHIP inhibitor but not by its structural control #154 (Fig. 6C, S7C and D†). When we enriched overexpressed tau from HT22 WCLs using immunoprecipitation, we observed that compound #153 significantly reduced the extent of polyubiquitination of tau (Fig. 6D and E). The correlation between the aggregation propensity of tau in the cultured cells and its degree of polyubiquitylation may implicate the possible role of tau ubiquitylation in the fibrillization process upon the phosphorylation queue.
Fig. 6 The CHIP inhibitor delays the aggregation propensity of tau in cultured cells. (A) Reduced levels of tau, both monomeric and oligomeric forms, in the detergent-insoluble fraction of HT22 mouse hippocampal cells. Tau40 was transiently overexpressed in HT22 cells in the presence or absence of 30 nM OA, 10 μM MG132, and/or 100 μM compound #153 for 6 h. RIPA-soluble and -insoluble fractions of whole cell lysates (WCLs) were isolated and subjected to SDS-PAGE/IB with indicated antibodies. (B) Relative levels of insoluble monomeric tau (upper) and oligomeric tau (lower) were quantified with normalization to those of soluble total tau and plotted as mean ± SD from three independent experiments as performed in A. N.S, not significant. **, p < 0.01 (one-way ANOVA followed by the Bonferroni post hoc test). (C) Similar to the experiments as in A, except that the assay was performed in rat primary cortical cells with either compound #153 or #154 control (50 μM) to monitor the changes of endogenous tau. Shown is one of the sets of triplicate experiments (Fig. S7C†). (D) Reduced polyubiquitylation of tau in HT22 cells where EGFP-tagged tau was transiently overexpressed and treated with MG132 (10 μM) and/or compound #153 (50 μM) for 6 h. WCLs were subjected to immunoprecipitation with anti-GFP antibodies, and subsequent SDS-PAGE/IB. (E) Co-immunoprecipitation analysis as in D, except that OA (30 nM) was treated instead of MG132. (F) Representative fluorescence images of HT22 cells with AT8 immunostaining, which transiently overexpress tau-EGFP after treated with DMSO, MG132 (10 μM), OA (30 nM), and/or compound #153 (50 μM) for 6 h. Tau-positive signals formed in OA and MG132-treated cells exhibit dense and curvilinear morphologies but the CHIP inhibitor effectively reduced the signal. (G) Reduced tau aggregation tendency in primary rat hippocampal neurons in the presence of 50 μM compound #153. Various combinations of 30 nM OA and 10 μM MG132 were co-treated to the primary neurons in 12-well plates at day 7 in vitro. Immunostaining with AT8 antibodies with DAPI and phalloidin co-counterstaining. Scale bar, 10 μm. |
HT22 cells exhibited only basal fluorescent signals when tau-EGFP was transfected under normal conditions (Fig. 6F). In the presence of OA and MG132, however, the thin and hair-like structures of tau appeared in the cytoplasm with one or two weak EGFP-positive inclusions near the microtubule-organization center. In sharp contrast, we observed that the curvilinear phospho-tau signals detected with AT8 immunostaining were effectively depleted when the cells were treated with compound #153 (Fig. 6F). Consistently, primary hippocampal neurons treated with compound #153 appeared to have reduced AT8-positive punctate signals, which were mainly located in the dendritic branches and neurites (Fig. 6G and S8†). With phalloidin staining of assembled F-actin,31 we also observed visibly restored synaptic networks after treatment with the CHIP inhibitors. Therefore, the small-molecule inhibitor of CHIP not only inhibits tau ubiquitylation in vitro but also effectively delays the tau aggregation process in cultured neurons under stress conditions (for example, in the presence of both MG132 and OA). These data collectively indicate that CHIP may play a critical part in tau aggregation in vivo, which was more eminent when tau dephosphorylation and 26S proteasome-mediated clearance were impaired.
Among the novel ubiquitin-conjugated Lys residues after phosphorylation (Fig. 3), four residues (from K274 to K375) were found in recently identified tau protofilament repeats in human CBD brains.32 The ubiquitin moieties may provide additional hydrogen bonds between the tau backbone and side chains along the axis of the filaments. Therefore, the ubiquitin chains on tau potentially stabilize parallel β-strand stacking, leading to ordered tau fibrils rather than the formation of amorphous aggregates via random stacking. We also found that most phosphorylation sites (>20) had no significant changes after tau ubiquitylation and were located outside the MBDs in our MS analysis (Fig. 3B), consistent with a previous study reporting little changes in the phosphorylation sites during AD progression.33 The competition between acetylation and ubiquitylation on the same Lys residue seems to need further investigation as monitoring the global dynamics of diverse PTMs will provide an insight into tauopathy pathogenesis.
Our findings may clarify many previously incomprehensible results regarding tau phosphorylation. For example, overexpression of constitutively active GSK3β in transgenic mice does not aggravate tau fibrillization-related pathology but rather alleviates many neuropathological symptoms when these mice are crossed with tau transgenic mice.34,35 Moreover, tau phosphorylation inhibitors showed only minimal therapeutic effectiveness in various clinical trials.36–38 Accumulating evidence suggests that a soluble pool of phospho-tau may not necessarily be the direct etiology of tauopathy. Our study revealed that the phosphorylation of tau is a prerequisite signal for adequate tau ubiquitylation, which leads to tau degradation in physiological states but to tau aggregation in pathological states. Therefore, as is the case for cyclins, cyclin-dependent kinases, and SMAD1, the phosphate groups on tau may be a novel class of “phospho-degrons” that are specifically recognized by CHIP.39–41 This accords with the earlier observations showing preferential binding of CHIP to phosphorylated forms of tau,18,42 whilst expanding the role of CHIP-mediated tau ubiquitylation from proteolysis to fibrillization. It is worth noting that increasing evidence suggests that truncated tau (tau-Asp421) generated by caspase-3 may possess elevated fibrillogenic propensities and stronger binding affinities toward CHIP than the full-length tau protein.43–49 Therefore, altered caspase activity during tauopathy progression could be another functional contributor to complex tau homeostasis. Acetylation, ubiquitylation, and methylation are expected to crosstalk with one another for lysine residues of tau and are subjected to highly complex functional regulation. How tau phosphorylation and ubiquitylation affect its truncation (and vice versa) remains to be elucidated.
In addition, heparin-fibrillized tau exhibits polymorphic structures and none of them are consistent with from monomorphic fibrils derived from patients with AD that have larger cores with different repeat compositions.50 Although heparin may participate in neutralizing positive charges of the filament core, its polar and nonpolar interactions to the crosslinked tau molecules are expected to be global and intrinsically nonspecific. Conversely, our data indicate that the influence of polyUb chains are limited to certain Lys residues, especially in the fourth MBD, which is the integral part of tau filament core in AD or CBD brain but, in heparin-fibrillized tau, shows a disordered structure.8 This also raises questions about the relevance of using the four MBD-containing K18 fragments, which lack the key residues to adopt the same tau structures identified from AD brain although they more readily form heparin-induced filaments than full-length tau.
We observed that tau aggregation into the insoluble fraction of live cells was induced synergistically when both phosphorylation and ubiquitylation of tau were promoted but its proteasomal clearance was inhibited. By developing the potent CHIP inhibitor that effectively antagonizes tau ubiquitylation and fibrillization in test tubes and cultured primary neurons, we demonstrated that the CHIP-mediated ubiquitylation of phospho-tau may serve as a critical rate-limiting step of pathological tau transformation in vivo. As tau proteins can slowly be degraded in a ubiquitin-, ATP-, and 26S proteasome-independent manner,12 it is conceivable that the contribution of CHIP to tau ubiquitylation is more critical after tau phosphorylation (i.e., the first pathogenic hit; Fig. 7). Together, these data suggest that the beneficial effects of inhibiting CHIP enzymes are better observed when proteasomes fail to adequately degrade tau proteins (as seen under proteostatic and oxidative stress conditions). Therefore, reduced proteasome activity or elevated polyUb conjugate levels may serve as a good indication for a CHIP-targeting therapeutic strategy. Small nucleates of hyperubiquitylated tau, which is a non-processable substrate for the proteasome and potentially inhibits its activity via the “clogging” effect,51,52 may initiate a vicious cycle producing excessive amounts of tau aggregates. This may relate tauopathy etiology to the decreased proteasomal activity reported in postmortem human AD brains.53,54 In this regard, the ubiquitin molecules abundantly present in insoluble paired helical filaments are not mere undigested remnants but rather reflect the extensive and autonomous modification of tau during the aggregation process.
To summarize, our biochemical experiments uncovered a novel cooperative, multistep regulation of tau, which is based on PTMs and ultimately has either beneficial (through tau proteostasis) or detrimental (tau fibrillization) consequences, depending on the cellular environment (e.g., cellular proteasomal activity). Only with the preceding phosphorylation, tau proteins are adequately ubiquitylated and eliminated by the 26S proteasome. When the tau-proteolytic system is impaired or when excess phospho–ub-tau species clog proteasomes, tau proteins undergo biochemical transformation into insoluble, ThT-positive short fibrils. We still have a limited understanding of the signature PTMs on tau involved in pathological self-assembly and cellular regulation towards the specificity and activity of the enzymes involved. Nonetheless, our findings provide important insights into molecular pathophysiology of diverse tauopathies and will likely point to novel therapeutic approaches targeting tau ubiquitylation. The application of CHIP inhibitors to animal models of tauopathy, along with longitudinal biochemical and neuropathological analyses, will validate this strategy.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1sc00586c |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2021 |