The E3 Ubiquitin Ligase, CHIP/STUB1, Inhibits Aggregation of Phosphorylated Proteoforms of Microtubule-associated Protein Tau (MAPT)

Abstract

Hyper-phosphorylated tau accumulates as insoluble fibrils in Alzheimer's disease (AD) and related dementias. The strong correlation between phosphorylated tau and disease has led to an interest in understanding how cellular factors discriminate it from normal tau. Here, we screen a panel of chaperones containing tetratricopeptide repeat (TPR) domains to identify those that might selectively interact with phosphorylated tau. We find that the E3 ubiquitin ligase, CHIP/STUB1, binds 10-fold more strongly to phosphorylated tau than unmodified tau. The presence of even sub-stoichiometric concentrations of CHIP strongly suppresses aggregation and seeding of phosphorylated tau. We also find that CHIP promotes rapid ubiquitination of phosphorylated tau, but not unmodified tau, in vitro. Binding to phosphorylated tau requires CHIP's TPR domain, but the binding mode is partially distinct from the canonical one. In cells, CHIP restricts seeding by phosphorylated tau, suggesting that it could be an important barrier in cell-to-cell spreading. Together, these findings show that CHIP recognizes a phosphorylation-dependent degron on tau, establishing a pathway for regulating the solubility and turnover of this pathological proteoform.

Keywords: intrinsically disordered protein; phospho-degrons; protein aggregation; protein–protein interactions; tauopathy.

Figure 1:. Amongst the TPR chaperones, CHIP preferentially suppresses p.tau aggregation.

(A) ThT curves of unmodified tau aggregation in the presence of TPR cochaperones. Data is shown as mean ± SEM (n=3). Tau concentration is 10 μM and TPR protein concentrations are 10 μM. (B) Lag time for unmodified tau aggregation in the presence of TPR cochaperones derived from (A). Data is normalized to tau alone and represented as mean ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test (**p<0.01, ****p<0.0001). (C) ThT curves of phosphorylated tau (p.tau) aggregation in the presence of TPR cochaperones. Data is shown as mean ± SEM (n=3). Tau concentration is 10 μM and TPR protein concentrations are 10 μM (D) Lag time for unmodified tau aggregation in the presence of TPR cochaperones derived from (C). Data is shown relative to tau alone and represented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc test (***p<0.001). (E) TEM micrographs of tau or p.tau samples (10 μM) following 24-hour fibrillization in the presence of TPR co-chaperones (10 μM). Scale bar represents 100 nm. Data shown is representative of two replicate experiments.

Figure 2:. CHIP preferentially inhibits aggregation and seeding of phosphorylated tau.

(A) Affinity comparison of CHIP interaction with unmodified or phosphorylated tau. Increasing concentrations of tau proteoforms were incubated with immobilized CHIP (see Methods) and binding was analyzed by ELISA. Data is shown as mean ± SD (n=3). (B) Equilibrium binding constants for CHIP.tau interactions as determined in (A). Data is shown as mean ± SD (n=3). Statistical significance was determined by student’s unpaired t-test (****p<0.0001). (C) Ubiquitination of tau variants by CHIP in vitro. Unmodified or phosphorylated tau (4 μM) was incubated with CHIP (4 μM), ubiquitination machinery, and ATP/MgCl₂ for the indicated times and analyzed by immunoblotting. Samples lacking CHIP were used as controls. Ub’n = ubiquitinated. Data shown is representative of two replicate experiments. (D) ThT curves for tau (10 μM) aggregation in the presence of varying molar ratios of CHIP. Data is shown as mean ± SEM (n=3). (E) Lag time for unmodified tau aggregation in the presence of varying molar ratios of CHIP derived from (D). Data is shown relative to tau alone and represented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc test (*p<0.05). (F) ThT curves for p.tau (10 μM) aggregation in the presence of varying molar ratios of CHIP. Data is shown as mean ± SEM (n=3). (G) Lag time for p.tau aggregation in the presence of varying molar ratios of CHIP derived from (F). Data is shown relative to tau alone and represented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Dunnet’s post-hoc test (**p<0.01). (H) Model for tau seeding assay workflow. Tau seeding samples were prepared by incubating tau or p.tau (10 μM) with CHIP (10 μM) for 24 hrs, as above. (I) Representative fluorescence microscopy images showing seeded aggregation of tauRD-mClover in HEK293T biosensor cells after 72-hour incubation. (J) Quantitation of tau aggregation in HEK293T biosensor cells after seeding with tau or p.tau fibrils formed in the absence or presence of CHIP. Lipofectamine 2000 (L2K CTRL) without tau was used as a negative control. Data is shown as mean ± SD (n=6). Statistical significance was determined by two-way ANOVA with Bonferroni’s post-hoc test (****p<0.0001, ns = p>0.05).

Figure 3:. CHIP restricts aggregation and seeding of disease-associated P301S mutant phospho-tau

(A) ThT curves for p.tau P301S aggregation in the presence of various TPR cochaperones. Data is shown as mean ± SEM (n=3). Tau concentration is 10 μM and TPR protein concentrations were 10 μM. (B) Lag time for P301S p.tau aggregation in the presence of various cochaperones derived from (A). Data is shown relative to p.tau alone and represented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc test (*p<0.05). (C) Affinity comparison of CHIP interaction with WT (purple) or P301S (blue) unmodified (solid) tau or p.tau (dashed). Increasing concentrations of tau proteoforms were incubated with immobilized CHIP and analyzed by ELISA. Data is shown as mean ± SD (n=3). (D) Equilibrium binding constants for CHIP.tau interactions as determined in (D). Data is shown as mean ± SD (n=3). Statistical significance was determined by one-way ANOVA with Bonferroni’s post-hoc test (***p<0.001, ns = p>0.05). (E) ThT curve of p.tau P301S (10 μM) aggregation in the presence of varying molar ratios of CHIP. Data is shown as mean ± SEM (n=3). (F) Quantitation of tau aggregation in HEK293T biosensor cells after seeding with WT or P301S p.tau fibrils formed in the absence or presence of CHIP. Data is shown as mean ± SEM (n=6). Statistical significance was determined by one-way ANOVA with Bonferroni’s post-hoc test (****p<0.0001).

Figure 4:. CHIP recognizes p.tau through a distinct TPR binding mode

(A) Workflow of the fluorescence polarization (FP) assay used to measure binding to CHIP’s TPR domain. Briefly, CHIP (blue, PDB: 2C2L) is bound to a fluorescent tracer (orange) and displacement by tau or p.tau (purple) leads to a decrease in FP signal (mP). 5-FAM = 5-carboxyfluorescein; Ahx = 6-aminohexanoic acid. (B) Competition curves for CHIP-tracer interactions in the presence of tau or p.tau. Data is shown as mean ± SD (n=4). (C) ThT curves of tau (solid lines) or p.tau (dashed lines) aggregation in the absence or presence of WT or K30A mutant CHIP. Data is shown as mean ± SEM (n=3). Tau concentration is 10 μM and CHIP WT and CHIP K30A concentrations are 10 μM. (D) Max ThT fluorescence after 18 hours of tau aggregation in the presence of WT or mutant CHIP derived from (C). Data is shown as mean ± SD (n=3). Statistical significance was determined compared to tau only by one-way ANOVA with Dunnett’s post-hoc test (***p<0.001, ns = p>0.05).

Figure 5:. The CHIP TPR domain is required for anti-aggregation activity

(A) Structural depiction of CHIPopt peptide (yellow) binding to CHIP’s TPR domain (grey) (PDB 6NSV), highlighting the interactions between the aspartate side chain and C-terminal carboxylate of CHIPopt, which are are coordinated by CHIP’s residues K30 and K95. (B) Ubiquitination of tau variants (4 μM) by CHIP (4 μM) in vitro in the presence of CHIPopt. Tau or p.tau was incubated with CHIP, ubiquitination machinery, ATP/MgCl₂, and the indicated concentration of CHIPopt for 10 minutes and analyzed by immunoblotting. Samples lacking CHIPopt were used as controls. Ub’n = ubiquitinated. Data shown is representative of two replicate experiments. (C) ThT curves of tau (10 μM) aggregation in the presence of CHIP (10 μM) and the indicated molar ratio of CHIPopt. Data is shown as mean ± SEM (n=3). (D) Lag time for of tau aggregation in the presence of CHIP and the indicated molar ratio of CHIPopt derived from (C). Data is normalized to tau alone and represented as mean ± SD (n=3). Statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc test (*p<0.05, ns = p>0.05). (E) ThT curves of p.tau (10 μM) aggregation in the presence of CHIP (10 μM) and the indicated molar ratio of CHIPopt. Data is shown as mean ± SEM (n=3). (F) Lag time for of p.tau (10 μM) aggregation in the presence of CHIP (10 μM) and the indicated molar ratio of CHIPopt derived from (E). Data is shown relative to tau alone and represented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc test (*p<0.05, ns = p>0.05). (G) ThT curves of p.tau P301S (10 μM) aggregation in the presence of CHIP (10 μM) and the indicated molar ratio of CHIPopt. Data is shown as mean ± SEM (n=3). (H) Lag time for p.tau P301S aggregation in the presence of CHIP and the indicated molar ratio of CHIPopt derived from (G). Data is shown relative to tau alone and represented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc test (*p<0.05, ns = p>0.05).

Figure 6:. Endogenous CHIP restricts seeded aggregation of phospho-tau

(A) Co-immunoprecipitation (co-IP) of tauRD complexes from HEK293T biosensor cells. TauRD-mClover was immunoprecipitated, and the resulting amount of CHIP and Hsp70 in the sample was analyzed by immunoblotting (see Methods). WT HEK293T cells not expressing the tau biosensor were used as control, and protein loading was confirmed by stain-free gel. Data shown is representative of two replicate experiments. (B) Quantitation of tau aggregation in HEK293T biosensor cell lines after seeding with tau or p.tau fibrils. The seeding samples were prepared as above (tau (10 μM), 24 hrs). Data is shown as mean ± SD (n=6). Statistical significance was determined by two-way ANOVA with Bonferroni’s post-hoc test (**p<0.01,****p<0.0001, ns = p>0.05). (C) Model for CHIP regulation of p.tau aggregation and seeding. Tau is pathologically phosphorylated amidst the progression of neurodegenerative diseases, which promotes the formation of fibrillar species that seed aggregation in a transcellular manner. In the donor cell (left), CHIP directly interacts with monomeric p.tau to inhibit the formation of mature fibrils or seeding aggregates. In the recipient cell (right), CHIP selectively blocks the templated aggregation of phosphorylated tau by stabilizing a ternary complex of CHIP, p.tau, and Hsp70.