IndraLab

"The results revealed a strong interaction between farrerol and UCHL3, and the dissociation constant (Kd) of farrerol/UCHL3 reached a low of 36.73 nM (Fig. 1e and Supplementary Fig. 1d)."

"Farrerol binds to UCHL3 through two amino acid residues, Lys187 and Arg215."

"To elucidate how farrerol specifically binds to UCHL3, we crystallized UCHL3 in the presence of farrerol and determined the structure of the complex at 1."

"The alignment indicated that farrerol did not affect the dynamic structure of UCHL3.Then, we attempted to confirm whether the two amino acid residues were essential for the interaction between UCHL3 and farrerol through the use of CETSAs."

"In contrast to the UCHL3 WT, an improvement in thermal stability was not observed for the two mutants (Fig. 2d–g), indicating that farrerol directly binds to UCHL3 through the two amino acid residues."

"Further SPR assay with the purified recombinant UCHL3–K187A R215A mutant protein and farrerol confirmed that mutating the two amino acid sites abolished the binding of farrerol to UCHL3 (Supplementary Fig. 1f)."

"Since both farrerol and UCHL3 participate in regulating HR by improving the recruitment of RAD51 to DNA damage sites and because we demonstrated that farrerol binds to UCHL3 with a high affinity, we wondered whether farrerol stimulated HR in a UCHL3-dependent fashion."

"Taken together, these data indicated that farrerol promoted DNA repair by HR in a UCHL3-dependent manner.Since the cocrystal structure of the UCHL3–farrerol complex revealed that farrerol directly binds to UCHL3 (Fig. 2a–c), a ubiquitin–AMC assay was then performed to determine whether farrerol activates UCHL3 enzymatic activity, and we found that farrerol affected both K and K , by slightly increasing K from 47.72 to 56.27 nM and significantly increasing K from 11.99 to 19.64 s (Fig. 3i), suggesting that farrerol activates UCHL3 deubiquitinase activity."

"Since we demonstrated that farrerol binds to UCHL3 through two amino acid residues to activate its enzymatic activity, in order to treat HR-overactivated cancer or overcome radio/chemo-resistance, it is likely worthwhile to try generating farrerol derivatives that exhibit an inhibitory effect on UCHL3 while still retaining a high affinity to UCHL3.It was reported that genomic stability is critical in the dynamic process of somatic cells reprogramming into iPSCs and is essential for the pluripotency of ESCs and iPSCs, whereas chromosomal aneuploidy results in a sharp decrease in cell differentiation potentials in vivo ."