Page 53 - ebook

The ubiquitination of hyperphosphorylated
tau species by CHIP and its effects on the enhancement of tau

fibrils formation

1,2
1,2
1,2
1,2
Won Hoon Choi , Ly Thi Huong Luu Le , Bokyeong Park , Insuk Byun , and Min Jae Lee 1,2
1 Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Korea,
2 Department of Biomedical Sciences, Seoul National University College of Medicine, Kore
Abstract
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 GSK3b 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.

A B A B

C C
Figure 1: Tau phosphorylation is required for adequate Figure 2: Adequately ubiquitinated phosphor-
tau ubiquitination (A) Recombinat tau-FL was ub-tau species are degraded by 26S
phosphorylated using GSK3β in vitro, and then proteasome in vitro (A) The polyUb chains on
subjected to ubiquitination reaction. Ubiquitination of phospho-ub-tau are K48-linked polyUb chain
phosphor-tau generated smeared bands of higher- (B) In vitro deubiquitylation of polyUb
molecular-weight tau species. (B) Similar with panel A moieties conjugated with tau or phosphor-tau
except that phosphorylated tau is ubiquitinated for (C) Phospho–ub-tau was completely degraded
longer period up to 24 h. (C) Acetylated tau is by 26S proteasomes in less than 2 h but was
ubiquitination in vitro. However, acetylation did not virtually unaffected by 20S proteasomes
affect tau ubiquitination patern.
A B C A

B
Figure 4: New ubiquitination sites in
phpospho-tau were identified by mass
spectrometry (A) Orange and red circles
depict ubiquitination sites of ub-tau and
Figure 3: Phosphor-ub-tau more readily transform to fibrils after phosphor-ub-tau (B) Quadruple Lys
prolonged incubation (A) Hyper-ub-tau were observed on top of mutations (tau-4KR, K321, K343, K353,
separating gel after 4 days ubiquitination reaction of phosphorylated tau K375) showed significant reduced in
(B) Moreover, thioflavin T signal also significantly increased with vitro aggregation propensities, observed
prolonged incubation (C) Trapped tau oligomer were detected by IB. by ThT analysis, further emphazing the
important of these residues.
A B C Conclusion Conclusion
Under physiological conditions, the tau protein is decorated with a relatively
small number of ubiquitin moieties, which are incapable of directly
interacting with proteasomes. At an early stage of tauopathies, tau proteins
are modified by phosphorylation, and the resulting structural changes may
allow tau to be conjugated with adequate numbers of polyUb for
proteasomal degradation (dashed arrows). When the clearance of phospho–
ub-tau is inefficient, possibly due to a combination of proteasome
inactivation and excessive amounts of loads on the proteasome (as observed
in late-stage tauopathies), tau progressively becomes hyperubiquitylated and
self-polymerizes into insoluble fibrils (solid arrows). Clinical significance of
this in vivo PTM-mediated tau proteostatic pathway remains to be
D determined.
Figure 5: CHIP inhibitor compound #153 delay
insoluble tau formation in vitro and in cultured cells
(A) Ubiqitination of tau was reduced by compound
#153 (B) Hyper-ub-tau was significantly decreased in
the presence of compound #153 (C) Reduced level of
oligomeric form of tau in HT22 cells after treatment
of CHIP inhibitor (D) Reduced tau aggregation
tendency in primary rat hippocampal neurons in the
presence of 50 mM compound #153.
References
(1) JH Kim, JY Lee, WH Choi, “CHIP-mediated hyperubiquitination of tau promotes its self-assembly into the insoluble tau filament”, Chem. Sci., 2021, 12, 5599
(2) Park S, Lee JH, Jeon JH, Lee MJ. Degradation or aggregation: the ramifications of post-translational modifications on tau. BMB Rep. 2018 Jun;51(6):265-273