EURON and THEME joint PhD meeting

50
EURON and THEME joint meeting 2011
Hydrolysis of the mutant ubiquitin (UBB+1)
accumulating in neurodegenerative disorders by
UCH-L3
F.J.A. Dennissen 1 , N. Kholod 1 , D.J.H.P. Hermes 1 , N. Kemmerling 1 , H.W.M
Steinbusch 1 , N.P. Dantuma 2 and F.W. van Leeuwen 1
1 Department of Neuroscience, Faculty of Health, Medicine and Life Sciences (FHML), Maastricht University,
Maastricht, the Netherlands; 2 Department of Cell and Molecular Biology (CMB), Karolinska Institutet,
Stockholm, Sweden.
The ubiqutitin proteasome system (UPS) is of paramount importance for protein
quality control since misfolded proteins are usually degraded by the UPS. The
mutant ubiquitin UBB +1 accumulates selectively in the hallmarks of tauopathies
and polyglutamine diseases. The UBB +1 protein is generated due to a frameshift
at the mRNA level resulting in a loss of the C-terminal glycine and an addition
of 20 amino acids to the C-terminus of this protein (Van Leeuwen et al., Science
279, 242-247,1998). As a result, UBB +1 cannot be used for ubiquitination and
impairs the UPS when being ubiquitinated. Furthermore, ubiquitinated UBB +1
is refractory to deubiquitination by isopeptidase T. Studies in yeast and human
cells showed that expression of UBB +1 gives rise to an additional C-terminally
truncated product that corresponds in size with ubiquitin (Verhoef et al.,FASEB J.
23, 123-33, 2009). In the transgenic UBB +1 mouse impaired contextual behaviour
is observed ( Fischer et al.,Neurobiol.of Aging, 30, 847-863 2009)
In order to identify the peptidase(s) responsible for the C-terminal truncation of
UBB +1 we performed a systematic screen with 175 yeast deletion strains. After
identifying the responsible enzyme for C-terminal truncation in yeast we cloned
the mouse and human homologues and co-expressed them with UBB +1 in HEK293
cells. We determined the effect of the individual enzymes on truncation of UBB +1
using SDS-PAGE as was done for yeast.
For yeast, we found the deubiquitylation enzyme YUH1 to be responsible for
hydrolysis of the C-terminal extension of UBB +1 . Human and mouse homologue
of YUH1, UCH-L3, were also able to hydrolyse the C-terminus of UBB +1 . Other
members of the family, UCH-L1, -L4, -L5 or the naturally occurring human CRA_f
isoform of UCH-L3 could not induce any truncation.
Human and mouse UCH-L3 are able to hydrolyse the C-terminal extension of
UBB +1 . Hydrolysis of UBB +1 s C-terminal tail prevents detection with the antibodies
specific for this extension. Consequently, we hypothesize that UBB +1 which is
detected in post-mortem tissue may be detectable due to lack of truncation by
C-terminal hydrolyses (e.g. UCH-L3).