Determination of Proteasomal Activities - Protein Misfolding and Cellular Stress in Disease and Aging - page 175

Chapter 12
Determination of Proteasomal Activities
Cristovao F. Lima and Suresh I.S. Rattan
Abstract
The proteasome is an important component of the intracellular system for the turnover of proteins.
Themammalian proteasome is engaged to degrade a bulky fraction of soluble intracellular proteins both
in an ubiquitin-dependent and independent manner. The proteasome is composed by a central catalytic
core - the 20S proteasome - where three different proteases are located, whose activities can be
measured. A detailed protocol for measuring accurately the three activities of the 20S proteasome in cell
and tissue homogenates, using specific fluorogenic substrates and a microplate reader fluorometer, are
described. Successful applications of this method include determining changes in the proteasomal activities
during aging, anti-aging interventions, cell cycle analysis, and in various disease states including
neurodegenerative diseases and cancers.
Key words: 20S Proteasome, Turnover of proteins, Chymotrypsin-like activity, Trypsin-like activity,
Caspase-like activity, Microplate reader fluorometer
1. Introduction
In order to maintain cellular homeodynamics, macromolecules
within a cell are submitted to constant repair and turnover. In the
case of proteins, cells have several mechanisms of turnover, which
include the ubiquitin and non-ubiquitin proteasome-mediated
pathways, calpains or calcium-dependent proteases, and lysosome-
mediated pathways, such as macroautophagy, microautophagy,
and chaperone-mediated autophagy. The proteasome system rep-
resents about 1% of the total cellular protein, is present both in the
cytosol and the nucleus of mammalian cells, and is responsible for
the degradation of a large portion of soluble intracellular proteins
( 1, 2 ). It is a multicomponent enzymatic system incorporating
different regulators and the catalytic core - the 20S proteasome.