labeling of specific enzyme classes in a covalent fashion.

three different elements with partially opposing,
but complementary roles; the ubiquitin conjugating cascade, the
deubiquitylating enzymes and the proteasome complex (Fig. 1).

>> The alkylation of nucleophilic residues (Cys, Ser, Thr) present in protease active sites
by reactive electrophiles has been a very successful realization of this
method. The

>> deconjugating enzymes
that usually contain strong nucleophiles at their active sites
when compared to the respective ligases/transferases.

>> probes bearing a C-terminal
electrophile (Fig. 2).

>>A complex proteome such as a cell or tissue lysate is labeled by
incubation with ubiquitin probe, followed by SDS-PAGE separation
and immunoblotting against the epitope tag genetically encoded
in the probe construct. Thereby a read-out, which is dependent
on DUB abundance and activity, is obtained.

>> competition assays
between an inhibitor candidate compound and the activitybased
probe can be performed in cell lysates. In this type of assay
format the competition between the small molecule inhibitor and
the probe leads to a reduced labeling profile for the activity-based
probe.

>>specificity of inhibition,

The proteasome complex, composed of at least
three different proteolytically active subunits β1, β2 and β5, degrades
the majority of soluble proteins within the cell following
polyubiquitylation [4]. The UPS not only enables the targeted destruction
of proteins no longer required by the cell, but also enables
the selective deactivation of signaling molecules, removal of pathogenic
proteins and production of peptides for presentation to the
immune system. The

>> is highly conserved
among eukaryotes (Kerscher et al., 2006). Ub adopts a globular
structure in which all seven lysine residues and the N terminus
are solvent exposed and can therefore form linkages with the
C terminus of another Ub to form poly-Ub chains

>>The Ub linkage type and the chain length encode the
fate of a ubiquitinated substrate.

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