Determination of Proteasomal Activities - Protein Misfolding and Cellular Stress in Disease and Aging - page 177

produces peptides with about 8-10 amino acids that are suitable to
be presented as antigen by the class I major histocompatibility com-
plex (MHC-I) ( 3 ). The immunoproteasome is mainly located in the
cytosol, but there is a special nuclear 11S regulator that is not induced
by interferon-g, which is particularly abundant in the brain ( 5, 8 ).
It was shown that the different proteasome forms might be
distributed in the cells, and also a hybrid proteasome (11S-20S-
19S) was identified ( 9 ). These studies have also shown that the
free 20S proteasome levels exceed all other forms by two- to
threefold. All forms with a regulator attached to the a-rings,
where the 20S proteasome core and the regulators are superim-
posed opening a channel into the proteolytic chamber, result in
increased activity ( 3 ).
In the eukaryotic 20S proteasome, only three b-subunits in
each of the inner rings possess catalytic activities: b1 - a peptidyl-
glutamyl-peptide-hydrolase or caspase-like activity that cleaves
after acid residues such as glutamic acid; b2 - a trypsin-like activ-
ity that cleaves after basic residues like arginine or lysine; and b5
- a chymotrypsin-like activity that cleaves after large hydrophobic
residues such as tyrosine or phenylalanine ( 4, 5, 10 ). The active
center of each of these subunits is a threonine residue at the
N -terminus of their protein chain, where the hydroxyl group acts
as nucleophile defining a new type of protease as compared with
serine and cystein proteases ( 10 ).
As stated above, the composition of b-subunits in the inner
ring can be modified to form the immunoproteasome by incor-
porating inducible forms by de novo synthesis replacing their
constitutive counterparts. These modify proteasome peptidase
activities by increasing chymotrypsin-like and trypsin-like activi-
ties and decreasing caspase-like activity, which would be impor-
tant to produce peptides with higher affinity for MHC class I
complex ( 5 ).
Several specific proteasome inhibitors have been developed.
Some widely used inhibitors are natural compounds such as epoxo-
mycin and lactacystin, which act as irreversible inhibitors ( 5 ). Also,
peptide aldehydes have been synthesized, such as Cbz-Leu-Leu-
Leucinal (MG132), which are competitive inhibitors, and are able
to enter cells. It was shown that MG132 is a more potent inhibitor
against the activated 20S core than against the 26S proteasome.
Nevertheless, it reduces the degradation of ubiquitin-conjugated
proteins by the 26S complex without affecting its ATPase or iso-
peptidase activities ( 4 ). However, MG132 has also some affinity to
inhibit lysosomal and calcium-activated proteases ( 4, 11 ).

1.1. Method-
Background
for Estimating
Proteasome Activity

The three peptidase activities of the proteasome can be assayed
using fluorogenic synthetic peptide substrates, which provide a
convenient and sensitive way to monitor the proteasome activity
in crude homogenates and purified fractions. These substrates