Ubiquitin-ovomucoid fusion proteins as model substrates for monitoring degradation and deubiquitination by proteasomes.

摘要: Abstract Protein degradation by 26S proteasomes requires the coordinated action of multiple binding and catalytic activities to process ubiquitinated protein substrates. For purpose studying conjugate independently substrate targeting unfolding steps, we have developed substrates based on an N‐terminal fusion ubiquitin irreversibly unfolded protein, 83 amino acid HA epitope‐tagged first domain chicken ovomucoid. Fluorescent labeling six cysteines in ovomucoid moiety (OM) with Lucifer Yellow iodoacetamide yields UbOMLY; can be extended addition a K48‐linked polyubiquitin chain form UbnOMLY. UbOMLY derivatives provide versatile monitor both deubiquitination vitro. Comparisons conjugates OMLY folded dihydrofolate reductase (DHFR) assays help resolve identify rate‐limiting steps proteasome degradation.