title: Homogeneous, bioluminescent proteasome assays.
abstract: Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.
authors.affiliation: Promega Corporation, 2800 Woods Hollow Road, Madison, WI, 53711, USA, martha.obrien@promega.com.
authors.forename: Martha A
authors.initials: MA
authors.lastname: O'Brien
authors.forename: Richard A
authors.initials: RA
authors.lastname: Moravec
authors.forename: Terry L
authors.initials: TL
authors.lastname: Riss
authors.forename: Robert F
authors.initials: RF
authors.lastname: Bulleit
mesh_terms: D000818:Animals; D020169:Caspases / Q000378:metabolism; D002457:Cell Extracts; D006801:Humans; D008156:Luciferases / Q000737:chemistry; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D046988:Proteasome Endopeptidase Complex / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D008156:Luciferases; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease