Page 52 - ebook

[A. Biochemistry/Molecular Biology] A-26

The ubiquitination of hyperphosphorylated tau species by

CHIP and its effect on the enhancement of tau fibrils

formation

Won Hoon Choi¹,², LY LE THI HUONG LUU¹,², Insuk Byun¹,², Bokyeong Park¹,², Min Jae Lee¹,²*

¹Department of Biochemistry and Molecular Biology, Seoul National University, College of Medicine, Seoul 03080,

Korea, ²Department of Biomedical Sciences, Seoul National University, College of Medicine, Seoul 03080, Korea

Under pathological conditions, tau undergoes multiple post-translational modifications 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. MS 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.