Functional studies of the ubiquitin-proteasome system using GFP-based reporters

Sammanfattning: Ubiquitin-dependent proteasomal degradation is of paramount importance for cellular processes such as cell cycle progression, transcriptional regulation, apoptosis and disposal of misfolded and aberrant proteins. Moreover the ubiquitin-proteasome system is the main producer of peptides used by major histocompatibility complex class 1 for antigen presentation. Considering its critical involvement in these cellular processes, it is not surprising that aberrant ubiquitin-proteasome- dependent degradation is implicated in human disorders. The aim of this thesis has been to develop reporter systems for quantitative and functional analysis of this system in cells and to use these reporters to gain insight into the interactions between viral and cellular proteins and the ubiquitin- proteasome system. The green fluorescent protein (GFP) was chosen as a reporter for degradation and was in the initial studies modified by the addition of degradation signals that transformed the GFP into a substrate of the proteasome. Upon inhibition of the system the GFP reporters accumulate and the level of obstruction can be monitored by the fluorescence. The GFP reporters were initially characterised and used for studies in cellular systems but more recently we developed a transgenic mouse model constitutively expressing one of the reporters. We used the GFP reporters and new site-specific inhibitors to elucidate the individual contribution of the different proteasomal active sites. Our study demonstrated that the catalytic activities are of unequal importance for degradation. Furthermore this study indicated the presence of a non-catalytic modifier site that regulates the activity of the proteolytic sites through binding of peptides. The role of a cellular and a viral repetitive sequence on proteolysis was also investigated using the GFP reporters. The Epstein-Barr virus derived Gly-Ala repeat functions as a stabilising domain. We concluded that it protects a protein from degradation in a lengthdependent manner. Another repetitive sequence with a length-dependent effect is the poly-GIn repeat, which is expanded in several proteins involved in neurodegeneration. We showed that in contrast to the Gly- Ala repeat this repeat stabilises proteins through the formation of insoluble aggregates. Another protein associated with neurodegeneration, and that also colocalises with different aggregates involved in such diseases, is the transcript mutant of ubiquitin, UBB+1. With the use of the GFP reporters we demonstrated that UBB+1 is a substrate of the proteasome and is ubiquitinated in a specific manner and this causes all inhibition of the ubiquitin-proteasome system. It remains to be resolved if this inhibitory activity contributes to neuropathogenesis. Together these studies shed some light on the roles of the ubiquitin-proteasome system in normal and pathologic conditions. The cellular and transgenic animal models presented in this thesis will be important tools for ftirther studies on this intriguing proteolytic system.