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Introduction
Turnover of cellular proteins was discovered in the 1930s in studies of
Rudolf Schoenheimer, but it was in the 1960s that is became apparent that this was not
just turnover, but a highly selective process. By the end of the 1970s two independent
groups were working on two different topics: in the lab of Avram Hershko in Haifa,
Israel, Hersko and Ciechanover were working on the ATP dependent degradation of
the tyrosine aminotransferase. They isolated a protein and named it ATP-dependent
proteolysis factor 1 (APF-1). On the other side of the Atlantic Ocean, Alexander
Varshavsky, settled down in Boston and became interested in a DNA binding protein
containing one C-terminus and interestingly two N-termini! The DNA binding protein
was histone 2a and the other protein was identified as ubiquitin, a 76-residue
ubiquitously (hence its name) expressed protein of unknown function that was
described (as a free protein) by Gideon Goldstein and colleagues in 1975. In 1980,
Keith Wilkinson, Michael Urban and Arthur Haas showed that APF-1 and ubiquitin
were the same protein.
Ubiquitin-Proteasome System (UPS)
Ubiquitin conjugation to substrate proteins is one of
the best-known post-translational modifications in the cell. It can not only destine its
target proteins for degradation, but is also involved in gene transcription, endocytosis
and DNA damage repair. The most well-known function is of course protein
breakdown. The addition of four or more ubiquitins to a substrate target it to the
proteasome (see figure). This mega-Dalton complex is responsible for unfolding and
degrading proteins back to aminoacids.
Ubiquitination is a consequence of ubiquitin transfer
between different proteins: ubiquitin is first activated in an ATP dependent manner by
an ubiquitin-activating enzyme, called E1 (see figure). Activated ubiquitin is then
transferred via a thioester intermediate to an ubiquitin-conjugating enzyme, called E2.
This activated E2 then acts in concert with an ubiquitin-ligase, called E3, to transfer
the ubiquitin to a target substrate, forming an isopeptide bond between the ε-amino
group of the Lysine residue of the substrate and the C-terminal Glycine residue of
ubiquitin. To study the behaviour of ubiquitin in living cells, we fused the Green
Fluorescent Protein (GFP) to the N-terminus of ubiquitin as was described before by
Yewdell and Dantuma. In this system we are able to compare the ubiquitin and the
proteasome in stably transfected living cells, because we already had a β1i-GFP
(LMP2-GFP) cell line with a tagged proteasome subunit.
Approach
As mentioned above, we coupled GFP to the Nterminus of ubiquitin and generated stable cell lines expressing this fusion protein. We
have done several tests to ensure that the construct does what it is supposed to do
(binding covalently to target proteins) and does not do what it is not supposed to do
(changing MHC class I expression or the cell cycle). We have tried to gain more
insight in ubiquitin localization and mobility with the help of the fluorescence
techniques Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence
Loss In Photobleaching (FLIP). For very fast FRAP measurements we have used a
confocal equipped with an external bleaching laser to avoid changing mirrors, laser
intensities etc. Diffusion and localization of the proteasome is not affected by
proteasome inhibition, and FLIP experiments show that no exchange between the
nucleus and the cytoplasm is possible (see example trace).
Reference
T. Groothuis and E. Reits, Manuscript submitted
Fatal error
Problems in the ubiquitin-proteasome system are thought to be
involved in several diseases, mainly aggregation diseases like Huntington's, Alzheimer
and Parkinson's disease. Extended CAG repeats in the involved proteins aggregate in
the cytoplasm and the nucleus, and cannot be degraded by the proteasome although
both ubiquitin and proteasome are present in the aggregates. Aggregates are formed by
the cell to better cope with the misfolded proteins.
On the horizon
Therapies targeted at the proteasome may also help to treat cancers.
Currently, potential therapeutic applications associated with inhibiting this enzyme are
evaluated in clinical trials in a variety of solid and hematologic malignancies.
Millennium is currently developing VELCADE™ (bortezomib) for injection, the first
proteasome inhibitor to be studied in human clinical trials for any disease. The use of
of this drug to treat refractory multiple myeloma has been granted fast-track status by
the U.S. Food and Drug Administration (FDA), and it has also been granted Orphan
Product Designation for the treatment of multiple myeloma.
Fast Recovery
Fluorescence Recovery After Photobleaching , or FRAP, is a non-invasive technique,
which uses photobleaching (also termed fading). It occurs when a fluorophore
permanently loses the ability to fluoresce due to photon-induced chemical damage and
covalent modification of the fluorophore. Diffusing proteins in a living cell are
partially bleached, after which recovery of fluorescence in the bleached spot is
recorded. Read more about FRAP and its applications on the following page:
More on FRAP >>
Technicalities
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Molecular cloning techniques were used to construct the fluorescently labeled
ubiquitin chimera
Westernblot analysis and confocal microcopy confirmed expression of the
recombinant protein
FRAP with confocal microscopy was applied to investigate the localization and
mobility of the proteasome and ubiquitin
Metabolic labelling with radioactive 35-S Methionine/Cystein for protein
turnover
Electron microscopy generated ultrastructural insight