title: Optimization of in vitro measurement of proteasome activity in mammalian cells using fluorogenic substrates.
abstract: The proteasome is the major multi-catalytic machinery responsible for protein degradation and maintenance of the proteome. The 26S proteasome is an ATP-dependent proteolytic complex, dedicated to the degradation of poly-ubiquitinated proteins. It consists of a 20S proteolytic core and one or two flanking 19S regulatory complexes. The three catalytic subunits harboring chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L; also termed PGPH) activities respectively reside in the 20S proteasome that can also exist in a free form and degrade oxidized and unfolded proteins. Impaired proteasome function has been implicated in the pathogenesis of a number of diseases including Alzheimer's disease, diabetes, cancer and aging. The emerging interest in proteasome function as diagnostic marker of various human pathologies and therapeutic target necessitates the development of accurate, sensitive and reliable methodologies for the assessment of proteasome activity. Herein, we describe an optimization procedure for the measurement of CT-L, T-L and C-L activities in cell lysates of fibroblasts (HFL-1), melanocytes (B16F10) and peripheral blood mononuclear cells (PBMCs) using fluorogenic peptide substrates in a mid-throughput 96-well plate format. Optimization involves the composition of cell lysis and assay buffers, and the determination of the concentrations of specific fluorogenic substrates and protein content in the reaction to attain appropriate linear catalytic response during measurement. Additional parameters assessed include the concentration of the cell lysate and of ATP in the cell lysis and assay buffers. Our methodological analysis provides useful guidelines for the accurate and rapid determination of proteasome activity in various cell types.
authors.affiliation: National Hellenic Research Foundation (Institute of Biology, Medicinal Chemistry & Biotechnology), Greece. Electronic address: kgeorgila@gmail.com.
authors.forename: Konstantina
authors.initials: K
authors.lastname: Georgila
authors.affiliation: National Hellenic Research Foundation (Institute of Biology, Medicinal Chemistry & Biotechnology), Greece.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Voutetakis
authors.affiliation: National Hellenic Research Foundation (Institute of Biology, Medicinal Chemistry & Biotechnology), Greece.
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Delitsikou
authors.affiliation: National Hellenic Research Foundation (Institute of Biology, Medicinal Chemistry & Biotechnology), Greece.
authors.forename: Niki
authors.initials: N
authors.lastname: Chondrogianni
authors.affiliation: National Hellenic Research Foundation (Institute of Biology, Medicinal Chemistry & Biotechnology), Greece.
authors.forename: Efstathios S
authors.initials: ES
authors.lastname: Gonos