1 research outputs found
Quantitative Proteomics Reveals Extensive Changes in the Ubiquitinome after Perturbation of the Proteasome by Targeted dsRNA-Mediated Subunit Knockdown in <i>Drosophila</i>
The
ubiquitin–proteasome system (UPS), a highly regulated
mechanism including the active marking of proteins by ubiquitin to
be degraded, is critical in regulating proteostasis. Dysfunctioning
of the UPS has been implicated in diseases such as cancer and neurodegenerative
disorders. Here we investigate the effects of proteasome malfunctioning
on global proteome and ubiquitinome dynamics using SILAC proteomics
in <i>Drosophila</i> S2 cells. dsRNA-mediated knockdown
of specific proteasome target subunits is used to inactivate the proteasome.
Upon this perturbation, both the global proteome and the ubiquitinome
become modified to a great extent, with the overall impact on the
ubiquitinome being the most dramatic. The abundances of ∼10%
of all proteins are increased, while the abundances of the far majority
of over 14 000 detected diGly peptides are increased, suggesting
that the pool of ubiquitinated proteins is highly dynamic. Remarkably,
several proteins show heterogeneous ubiquitination dynamics, with
different lysine residues on the same protein showing either increased
or decreased ubiquitination. This suggests the occurrence of simultaneous
and functionally different ubiquitination events. This strategy offers
a powerful tool to study the response of the ubiquitinome upon interruption
of normal UPS activity by targeted interference and opens up new avenues
for the dissection of the mode of action of individual components
of the proteasome. Because this is to our knowledge the first comprehensive
ubiquitinome screen upon proteasome malfunctioning in a fruit fly
cell system, this data set will serve as a valuable repository for
the <i>Drosophila</i> community