The ubiquitin-like modifier FAT10 is degraded by the 20S proteasome in vitro but not in cellulo.

Ubiquitin-independent protein degradation via the 20S proteasome without the 19S regulatory particle has gained increasing attention over the last years. The degradation of the ubiquitin-like modifier FAT10 by the 20S proteasome was investigated in this study. We found that FAT10 was rapidly degraded by purified 20S proteasomes in vitro, which was attributed to the weak folding of FAT10 and the N-terminally disordered tail. To confirm our results in cellulo, we established an inducible RNA interference system in which the AAA-ATPase Rpt2 of the 19S regulatory particle is knocked down to impair the function of the 26S proteasome. Using this system, degradation of FAT10 in cellulo was strongly dependent on functional 26S proteasome. Our data indicate that in vitro degradation studies with purified proteins do not necessarily reflect biological degradation mechanisms occurring in cells and, therefore, cautious data interpretation is required when 20S proteasome function is studied in vitro.

Competitive Metabolite Profiling of Natural Products Reveals Subunit Specific Inhibitors of the 20S Proteasome.

We have developed a syringolin-based chemical probe and explored its utility for the profiling of metabolite extracts as potent inhibitors of the 20S proteasome. Activity-guided fractionation by competitive labeling allowed us to isolate and identify glidobactin A and C as well as luminmycin A from a Burkholderiales strain. The natural products exhibited unique subunit specificities for the proteolytic subunits of human and mouse constitutive and immunoproteasome in the lower nanomolar range. In particular, glidobactin C displayed an unprecedented ß2/ß5 coinhibition profile with single-digit nanomolar potency in combination with sufficiently high cell permeability. These properties render glidobactin C a promising live cell proteasome inhibitor with potent activity against human breast cancer cell lines and comparably low immunotoxicity.