title: Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains.
abstract: Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.
authors.forename: Shahri
authors.initials: S
authors.lastname: Raasi
authors.forename: Cecile M
authors.initials: CM
authors.lastname: Pickart
mesh_terms: D000818:Animals; D002384:Catalysis; D002417:Cattle; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry*; D025821:Polyubiquitin / Q000737:chemistry* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; D025801:Ubiquitin; D025821:Polyubiquitin; C086985:RAD23A protein, human; D007768:Lactalbumin; D005982:Glutathione Transferase; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D045643:DNA Repair Enzymes; D008239:Lysine