טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDaria Krutauz
SubjectExtended Ubiquitin Species are Protein-Based
DeUbiquitination Inhibitors
DepartmentDepartment of Biology
Supervisor Full Professor Glickman Michael
Full Thesis textFull thesis text - English Version


Abstract

Ubiquitination of proteins is an abundant reversible post-translational modification process. Numerous ubiquitin specific proteases regulate specificity of the ubiquitination process and maintain the pool of free ubiquitin in cells. The outcome is a modular signal that can be recognized by designated receptors in a variety of cellular pathways including proteasomal degradation, cellular trafficking, DNA transcription and more. Involvement of ubiquitin in a wide spectrum of cellular processes explains how aberrations in components of the ubiquitin system are associated with many diseases. An example is a frameshift mutation in the ubiquitin transcript called UBB. The translated UBB protein is a C-terminally extended ubiquitin that accumulates in the brain under certain neuropathological conditions such as Alzheimer’s disease. UBB is thought to be an inhibitor of the proteasome and as such, the underlying cause for the buildup of toxic proteins correlated with these pathologies. Trying to shed a light on the interrelation between the UBB protein and proteasome machinery, we complemented previously reported observations by showing that such extended ubiquitin species accumulate also in yeast cells. In fact, these species are substrates for the ubiquitination machinery and are ubiquitinated by one to three ubiquitin moieties rendering them into substrates for proteasome degradation, but only if the C-terminal extension is at least 25 amino acids long. Shorter carboxyl-terminal tails result in stable ubiquitin moieties that accumulate imposing mild stress on the ubiquitin-proteasome system, but this does not inhibit yeast cell growth and does not leads to growth defects. Expanding our studies with isolated components, we were unable to detect an inhibitory effect on 26S proteasomes, whereas 20S proteasome complexes were able to trim the unstructured “tails” from UBB molecule. The 26S proteasome holoenzyme is the predominant form of proteasomes in rapidly dividing cells, thereby potentially explaining why extended ubiquitin appears inert and is able to accumulate in vivo. However, we found that extended ubiquitin molecules were resistant to deubiquitinating enzymes and served as broad-specificity inhibitors of deubiquitinases. Based on our observations we attribute inhibitory effect of the UBB protein to the deubiquitinating enzymes upstream to the proteasome. Moreover this inhibitory effect is produced by disability of the oligopeptide extension to be cleaved and does not depend on the extension length. The observations described herein may shift the attention of research efforts on protein accumulation in neurodegenerative process from proteolysis to deubiquitination and to the potential damage that UBB accumulation may have on UPS efficiency.