Allantopyrone A promotes cross-linking of ubiquitin-specific protease 5 (USP5) and inhibits its deubiquitinase activity by targeting distinct domains.

Allantopyrone A is an α-pyrone metabolite produced by the plant endophytic fungus Allantophomopsis lycopodina KS-97. We previously demonstrated that allantopyrone A decreased the abundance of many components of 26S proteasome fractions while inducing the appearance of an additional protein with a higher apparent molecular mass than known proteasome components. In the present study, we identified this protein as ubiquitin-specific protease 5 (USP5) using mass spectrometry. In cell-based assays, allantopyrone A promoted the cross-linked form of USP5, which was shown by immunoprecipitation to represent a USP5 dimer. The cross-linked form of USP5 was markedly reduced when the C-terminal zinc finger ubiquitin-binding domain (cUBP; residues 175-283) was deleted or when Cys195 was substituted with alanine. Allantopyrone A was also found to reduce the activity of multiple deubiquitinases. USP5 labeling by ubiquitin-vinyl methyl ester was markedly reduced by allantopyrone A or by a Cys335 substitution. Consistent with these biological effects of allantopyrone A on USP5, in silico docking studies suggested that allantopyrone A interacts with Cys195 on the surface of the cUBP domain and is positioned near Cys335 within the catalytic center of the USP domain. These results demonstrate that allantopyrone A promotes USP5 cross-linking and inhibits its deubiquitinase activity through interactions with distinct domains.